# # Creator RegulonDB Database # schemeURI http://regulondb.ccg.unam.mx/ # affiliation Center for Genomics Science # Title Dataset of sentences with Regulatory Interactions (RIs) # titleType Title # Publisher Program of Computational Genomics, CCG # PublicationYear 2018 # ResourceType Genomics data # resourceTypeGeneral Dataset # Rights http://regulondb.ccg.unam.mx/menu/download/full_version/terms_and_conditions.jsp # Format text # Version v1.0 # Language en # keywords regulatory interaction, automatic classification, passage detection, information extraction, bionlp # relatedIdentifier https://www.ncbi.nlm.nih.gov/pubmed/?term=24903516 # relatedIdentifierType URL # relationType References # Contributor RegulonDB Team # ContributorType ContactPerson # ContactPersonURI http://regulondb.ccg.unam.mx/menu/about_regulondb/contact_us/index.jsp # ContactPersonEmail regulondb@ccg.unam.mx # Contributor Carlos-Francisco Méndez-Cruz # ContributorType Researcher # Contributor Julio Collado Vides # ContributorType Researcher # Contributor Cecilia Ishida-Gutiérrez # ContributorType Curator # Description # # The file contains a list of validated sentences where Regulatory Interactions (RIs) can be found. # Each line is one sentence with at least one RI, and more than one sentence can be found in the same reference. # Tokenization, and sentence split was performed by OntoGene text mining system (http://www.ontogene.org/) # # Columns: # Column 1: PMID reference used to get the sentence with Regulatory Interaction (RI). # Column 2: ID of sentence assigned by OntoGene. # Column 3: Manual validated sentence where the information about the RI is found. # Column 4: Class, RI = with Regulatory Interaction. # 9409145 66 It accepts a methyl group from methylphosphotriesters and then acts as a transcriptional activator of the ada operon AdiY Escherichia coli SP : P33234 Transcriptional activator of the adiA gene for 253 32 , 237 biodegradative acid - induced arginine decarboxylase AfrR Escherichia coli TE : Q07681 Probable transcriptional activator of the 272 258 afrABRS operon for expression of AF / R1 fimbria in E . coli RDEC - 1 , a rabbit pathogen AggR Escherichia coli SP : P43464 Transcriptional activator of the aggA gene for 265 183 aggregative adherence fimbria I ( AAF / I ) expression in enteroaggregative E . coli strains AppY Escherichia coli SP : P05052 Transcriptional activator of the cyxAB , hyaAB - 243 12 , 131 CDEF and appA operons during the deceleration phase of growth AraC Citrobacter freundii SP : P11765 Regulator of several operons involved in the 281 30 transport and catabolism of L - arabinose ( similar to E . coli AraC ) AraC Escherichia coli SP : P03021 Activator of the expression of the araBAD , 292 174 , 238 , 248 , 266 araFGH and araE operons , which are involved in the transport and catabolism of L - arabinose . RI 9409145 110 ( ii ) Mutations within residues in this region in AraC ( Cys204 3 Tyr , Ser208 3 Ala , Arg210 3 Cys , and His212 3 Tyr or Ala ) reduced binding to and decreased transcriptional activation from the P araBAD promoter ( 26 – 28 , 39 , 78 ) . RI 9409145 133 The AraC protein , which regulates the L - arabinose operons in E . coli , consists of two domains that function in chimeric proteins . RI 9409145 211 Model of AraC induction by L - arabinose at the araBAD promoter and relevant sites at the divergent araC promoter . RI 9409145 223 It was shown that to activate transcription in P araBAD , the AraC protein binding site must overlap the 35 region of the promoter by 4 bp ( 212 ) . RI 9409145 300 RhaS activates transcription from rhaBAD , and transcription from the rhaS gene is controlled by RhaR ( 63 , 244 ) . RI 9409145 341 In addition to the specific regulator , transcription from certain promoters regulated by members of this family requires other proteins for maximal activity ( e . g . , the CRP in the P rhaBAD promoter ) , or histone - like proteins that act as negative regulators ( e . g . , YomA in the VirF - regulated P yop promoters ) ( 52 , 124 , 242 ) . RI 9409145 1009 The plasmid - encoded urease gene cluster of the family Enterobacteriaceae is positively regulated by UreR , a member of the AraC family of transcriptional activators . RI 9409145 1498 Upstream repression and CRP stimulation of the Escherichia coli L - arabinose operon . RI 9409145 2754 Dual regulation of inaA by the multiple antibiotic resistance ( Mar ) and superoxide ( SoxRS ) stress response systems in Escherichia coli . RI 9409145 3168 HrpXv , and AraC - type regulator , activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv . vesicatoria . RI 24272778 53 Together with a bioinformatic analysis of other Enterobacteriaceae species , these data identify a conserved AraC regulon that includes 7 previously described AraC - regulated genes ( araB , araA , araD , araE , araF , araG , and araH ) as well as three novel targets identified in this work ( ytfQ , araT , and araU ) . RI 24272778 170 As expected , expression of known AraC - regulated genes , i . e . , araB , araA , araD , araE , araF , February 2014 Volume 196 Number 3 jb . asm . org 663Stringer et al . FIG 1 ( A ) Validation of putative E . coli AraC target regions identified by ChIP - chip . RI 24272778 229 We did not detect any significant differences in RNAP association , suggesting that under these growth conditions , AraC does not regulate expression of a transcript that initiates within dcp . RI 24272778 320 With the exception of xylA , the last AraC - regulated gene to be identified was araJ , more than 30 years ago ( 27 ) . RI 24272778 329 We also identified AraC - regulated genes that are transcribed due to read - through of inefficient Rho - independent terminators ( ygeA and polB ) . RI 24272778 341 AraC has previously been shown to repress its own transcription by binding to a region overlapping the araC promoter elements ( 32 ) . RI 24272778 343 The location of the AraC binding site upstream of ytfQ is too far upstream of the transcription start site to repress transcription by directly occluding RNAP . RI 24272778 345 GalR has been shown to regulate ytfQ ( 44 ) ( Fig . S1 in the supplemental material ) . RI 24272778 375 Only one other novel AraC - regulated gene identified in this work , E . coli ytfQ , has a known connection to arabinose metabolism ( 39 ) . RI 24272778 394 Hence , polB regulation by AraC may be widely conserved . RI 21890697 9 In this study , we have examined promoter - lac fusions in strains that were argP or argP or that were carrying dominant argP mutations in order to identify several new genes that are ArgP - regulated in vivo , including lysP , lysC , lysA , dapD , and asd ( in addition to argO , dapB , and gdhA ) . RI 21890697 74 At the time that these experiments were done , the genes known to be ArgP regulated in vivo included the following ( gene functions are indicated in parentheses ) : argO ( Arg export ) , dapB ( dihydrodipicolinate reductase in the diaminopimelate - Lys biosynthetic pathway ) , and K . pneumoniae gdhA ( glutamate dehydrogenase for NH 4 assimilation and glutamate biosynthesis ) . RI 21890697 116 Under identical experimental conditions as for argO , the regulatory regions of the other ArgP - regulated genes ( asd , dapB , dapD , gdhA , lysA , lysC , and lysP ) were also bound by ArgP , with apparent K d s ranging from 55 nM to 170 nM ; in all these cases ( unlike the situation with argO ) , the addition of Lys was associated with an increase in the apparent K d , indicating that ArgP binding in these instances is Lys sensitive . RI 21890697 122 The results described above indicated that ArgP binds the lysP regulatory region to mediate its nearly 35 - fold transcriptional activation and that the absence of such ArgP binding either in argP mutants or upon Lys supplementation in argP strains results in very low levels of lysP expression . RI 21890697 130 ArgP and cadBA regulation . RI 21890697 134 Since our findings indicated that ArgP is needed for activation of lysP transcription , we tested whether cadBA expression would be rendered Lys independent even in the argP mutant . RI 21890697 145 Since ArgP is required for transcription of the Arg exporter ArgO , we also tested the promoters of other genes known to be involved in Arg transport ( artP , artJ , argT , and the hisJQMP operon ) as candidates for regulation by ArgP . RI 21890697 158 In its description as IciA , ArgP has been reported to regulate dnaA and nrdA transcription ( 20 , 27 – 29 ) . RI 21890697 160 Our conclusion runs contrary to that of Han et al . ( 20 ) , who overproduced IciA ( ArgP ) and employed an RNase protection assay to show that nrdA transcription is ArgP regulated in vivo . RI 21890697 168 In the case of argO , ArgP mediates its transcriptional activation by Arg , as well as its repression by Lys ( 7 , 26 , 32 , 37 ) ; the latter is achieved by a mechanism involving the active trapping at the argO promoter by ArgP of RNA polymerase , which is then prevented from being released to engage in productive transcription ( 26 ) . RI 21890697 198 LysR also mediates Lys repression ( of lysA ) , but in this case it has been suggested that the coeffector ligand is diaminopimelic acid ( which is the substrate for lysA ) and that the latter ’ s binding to LysR converts it into its activator conformation ( 30 , 36 ) . RI 21890697 204 In vitro , ArgP binds the lysP regulatory region with a K d of around 55 nM and the binding affinity is diminished upon the addition of Lys ( to a K d of 140 nM ) , suggesting that Lys represses lysP in vivo by engendering the loss of ArgP binding to the lysP operator region . RI 21890697 228 At the same time , it is noteworthy that two of the genes that are most prominently regulated by ArgP , argO and lysP , are both also regulated by the leucineresponsive general transcriptional regulator Lrp ( 37 , 41 ) . RI 21890697 317 Lysine represses transcription of the Escherichia coli dapB gene by preventing its activation by the ArgP activator . RI 9537375 6 In a gntR deletion mutant , the expression of a chromosomal gntT : : lacZ fusion is both high and constitutive , confirming that GntR is the negative regulator of gntT . RI 9537375 12 Transcription of gntT is activated by binding of the cyclic AMP ( cAMP ) - cAMP receptor protein ( CRP ) complex to a CRP binding site positioned at 71 upstream of the gntT transcription start site . RI 9537375 36 The results in this report also demonstrate that gntT is regulated by the cyclic AMP ( cAMP ) - cAMP receptor protein ( CRP ) complex , which was found be essential for full induction . RI 9537375 104 To investigate the role of GntR in repression of the gntT gene , the expression of a single - copy chromosomal gntT : : lacZ fusion was measured in a gntR mutant . RI 9537375 125 In summary , these results demonstrate that gntT is positively regulated by CRP . RI 9537375 153 In summary , the results clearly demonstrate that both operators are required for repression of gntT by GntR . RI 9537375 179 During the course of this work , it became apparent that GntR might affect gntT expression by a mechanism that is independent of negative control by operator binding as well as activation by cAMP - CRP . RI 9537375 274 However , it is possible that the closely related YjgS protein , which is 46 % identical to GntR , is involved in the positive regulation of gntT . RI 9537375 282 These results confirm that regulation of the gntT gene by GntR is similar to other negatively regulated systems . RI 9537375 296 We favor the idea that the repression of gntT is the result of DNA looping through interaction between the two GntR molecules as shown for many other operons in E . coli , such as the ara , gal , lac , and deo operons ( 29 ) . RI 9537375 297 A greater extent of derepression results from mutation of the external operator site by comparison to the internal operator site mutants , suggesting that binding of GntR to the external site results in a stronger repression of the gntT gene . RI 8655507 227 It is interesting to speculate that this is the element responsible for binding GntR and repression of gntKU expression . RI 12618441 6 GntR represses the GntI genes gntKU and gntT , whereas GntH was previously suggested to be an activator for the GntII genes gntV and idnDO - gntWH . RI 12618441 30 Both gntT and gntKU are regulated positively by the cyclic AMP ( cAMP ) - cAMP receptor protein ( CRP ) complex and negatively by GntR ; one GntR - binding element for gntK has been proposed ( 12 , 34 ) , and two GntR - binding elements for gntT were experimentally defined ( 26 ) . RI 12618441 48 In this study we have shown the negative regulation mechanism of the GntI system by GntR and also by GntH , an activator for the GntII system . RI 12618441 193 These observations led us to conclude that GntH might bind to GntR elements and might repress the GntI genes . RI 12618441 264 These results suggest that in the presence of gluconate , the expression of the GntI genes was induced at early logarithmic phase and was then repressed by a GntH - dependent mechanism . RI 12618441 279 These results suggest that both GntRbinding sequences , R1 and R2 , are responsible for the repression of gntKU expression , although the affinity of GntR or GntH for R1 seems to be different from that for R2 . RI 12618441 304 These results appear to be consistent with those from the experiments with lacZ operon fusions , as shown in Table 4 , and it is likely that the binding of GntR to a single element ( the R1 sequence ) is capable of decreasing the transcription of the gntKU genes . RI 12618441 311 Significant primary sequence homology between GntR and GntH , especially in their HTH motifs for DNA binding , led us to hypothesize that GntH , an activator for the GntII genes , represses the GntI genes via GntR - binding elements . RI 12618441 329 At the same time , GntH may induce the expression of the GntII genes ; this is supported by evidence showing that the GntII genes have an expression peak at the late logarithmic or stationary phase ( unpublished data ) . RI 12618441 330 We postulate that GntH is responsible for repression of the GntI genes when GntR was released from the operator by interaction with gluconate , and for induction of the GntII genes . RI 8636021 19 Thus , transcription of the marRAB operon is autorepressed by MarR and autoactivated by MarA at a site that also can be activated by SoxS . RI 8636021 39 We report here the existence of a " mar / soxbox " upstream of the - 35 hexamer which binds MarA and enables activation of mar transcription by MarA and SoxS both in vitro and in vivo . RI 8636021 40 Thus , the mar operon has the remarkable property that its first gene product , MarR , represses its transcription whereas the second gene product , MarA , stimulates its transcription . RI 8636021 148 The results , summarized as follows , show that mar can be activated in vivo via either MarA or SoxS acting on the marbox promoter region . RI 8636021 178 Thus , maximum activation of the mar regulon promoter inaA depends on the autostimulatory effect of MarA on the mar operon . RI 8636021 188 We surmise , from the distance of this marbox ( centered at - 61.5 ) to the putative - 35 transcriptional signal ( 17 , 18 ) , that activation of mar by MarA requires interaction with the carboxy - terminal domain of the alpha subunit of RNA polymerase , i . e . , that MarA is a class I activator ( 16 ) of mar as it is for zwf . RI 7836283 19 Expression of the operon , consisting of marR , marA , and marB , is normally repressed by MarR but can be induced by diverse compounds such as tetracycline , chloramphenicol , menadione , and salicylates ( 6 , 16 , 28 ) . RI 7836283 159 Such is the path by which OmpF expression is decreased by mar operon - directed increased expression of micF ( 8 ) . RI 19376854 22 MarA activates the expression of diverse genes , including acrAB , micF , mlr - 1 , - 2 , and - 3 , slp , and inaA , which endow cells with resistance ( 3 ) . RI 19376854 185 In the previous regulatory model , the expression of waaP and waaY was regulated by RfaH ( SfrB ) , an antiterminator protein , and this regulation was achieved over the whole transcript from the top promoter in front of waaQ ( 29 ) . RI 14701822 12 We found that purified MarA was sufficient in vitro to repress transcription of both purA and hdeA . RI 14701822 25 To determine whether the repression by MarA was direct or occurred by an indirect action , we focused on two genes , purA and hdeA . RI 14701822 26 In the intact cell , transcription of purA was decreased in cells constitutively producing MarA ( 4 ) . RI 14701822 27 Expression of hdeA was repressed by induced expression of MarA ( see Ref . 5 , Supplemental Material ) . RI 14701822 32 We show here that MarA directly represses both purA and hdeA . RI 14701822 85 In the same experiment we also examined the expression of hdeA , which had been observed in another macroarray study to be repressed upon MarA induction in cells ( 5 ) . RI 14701822 90 These results show that the decrease in purA and hdeA expression was caused , directly or indirectly , by transiently synthesized MarA rather than by a stress reaction due to constitutively overabundant MarA . RI 14701822 92 This protein , engineered to facilitate purification and hereafter referred to as MarA , had been shown previously to activate nfnB transcription in vitro ( 20 ) . RI 14701822 96 The promoter for the MarA - activated gene nfnB ( 20 ) , also mixed with gnd DNA , was tested in parallel as a positive control for MarA activity . RI 14701822 97 In the presence of 200 nM MarA , there was a 5 – 6 - fold activation in nfnB transcription , as expected ( Fig . 2 ) . RI 14701822 99 The same concentration and batch of MarA protein , on the other hand , repressed both purA and hdeA ( Fig . 2 ) . RI 14701822 100 The average repression ratio was 0.44 for purA ( see Fig . 5 , WT ) and 0.60 for hdeA ( see Fig . 6 , WT ) relative to controls without MarA . RI 14701822 101 These results showed that MarA by itself could directly repress purA and hdeA transcription . RI 14701822 136 These results together indicated that marbox 3 , and particularly its RE2 , was critical for repression of hdeA by MarA . RI 14701822 163 The physiological significance of the repression of purA and hdeA by MarA is not known . RI 14701822 168 The repression of purA by MarA was quantitatively similar in vivo and in vitro , whereas that of hdeA was greater in vivo ( ~ 6 – 25 - fold ) than in vitro ( ~ 1.7 - fold ) . RI 12100559 12 Using in vitro assays and mutagenesis of promoter fusions in whole cells , we identified the cis regulatory element involved in MarA upregulation of the oxygeninsensitive nitroreductase nfnB gene . RI 12100559 42 We show here that MarA directly mediates upregulation of nfnB expression through a cis - recognition marbox element in the nfnB promoter . RI 12100559 45 Transcription of nfnB is greatly increased by induced or constitutive chromosomal expression of MarA but is unaltered by constitutive expression of chromosomal SoxS and Rob . RI 12100559 49 MarA upregulates expression of nfnB Both oxygen - insensitive nitroreductases nfsA and nfnB were shown to be upregulated by constitutive expression of MarA from plasmid pAS10 ( Barbosa and Levy , 2000 ) . RI 12100559 53 To confirm that the nfnB activation observed in the array study was specifically mediated by MarA rather than by a cell stress response to the constitutive expression of the protein from a multicopy number plasmid , Northern blot analysis was performed on cells in which MarA production was inducible . RI 12100559 64 The strong upregulation of nfnB by the plasmidspecified MarA observed in this study contrasts with the reported weaker upregulation of NfnB activity and nfnB expression by SoxS ( Liochev et al . , 1999 ; Pomposiello et al . , 2001 ) . RI 12100559 70 These results show that constitutive and inducible expression of the chromosomal MarA was able to upregulate nfnB expression in contrast with the lack of activation mediated by constitutive expression of the chromosomal SoxS and Rob proteins . RI 12100559 74 Although marbox 1 and marbox 4 were closely related to the degenerate consensus sequence ( 3 nt differences out of 14 specified nt ) ( Fig . 3B ) , their location , downstream from the - 10 hexamer and 83 nt upstream from the - 35 hexamer , respectively , suggested that these marboxes were probably not functional in the MarA activation of nfnB . RI 12100559 78 When reactions were carried out in the presence of RNAP alone as compared with 200 nM purified His 6 - MarA , we detected MarA upregulation of nfnB transcription ( Fig . 4 ) . RI 12100559 136 An important observation was that purified MarA was able to activate transcription in vitro of nfnB . RI 12100559 143 For instance , Rob has been shown to bind and activate the zwf promoter in vitro but whole cell zwf regulation cannot be activated by Rob , although the gene responds to SoxS and MarA ( Ariza et al . , 1995 ; Jair et al . , 1995 1996a ; b ) . RI 12100559 150 However , this idea was contradicted when a single mutation at position 16 ( C to G ) of RE 2 , which has been shown to establish direct contacts with the second of two HTH motifs of MarA ( Rhee et al . , 1998 ; Dangi et al . , 2001 ) , eliminated MarA - mediated activation of the nfnB gene . RI 12067348 21 marA expression is repressed by MarR and is derepressed by the interaction of MarR with various phenolic compounds such as salicylate . RI 12067348 25 The three activators may have different quantitative effects on particular promoters , for example SoxS activates fpr to a much greater extent than does MarA . RI 12067348 69 mdaA ( also known as nfsA ) but not ybjC had previously been shown to be regulated by SoxS although no binding site had been identified ( Liochev et al . , 1999 ) . RI 12067348 87 Although not tested directly , the ybjC - mdaA operon may contain a third gene , rimK , as : ( i ) there does not appear to be either an RNA chain termination signal nor a promoter between mdaA and rimK ( they are separated by 172 bp ) ; ( ii ) Barbosa and Levy ( Barbosa and Levy 2000 ) observed the induction of rimK in their microarray analysis for MarA - induced genes ; and ( iii ) the nearest potential MarA binding site upstream of rimK is that identified for ybjC . RI 12067348 90 They demonstrated that acnA has two promoters , P1 and P2 , the latter being regulated by SoxS , FNR , CRP and ArcA . RI 12067348 111 The – 111 to – 91 site overlaps sequences identified as binding the anaerobic regulators , ArcA and FNR , and the catabolite regulator CRP which have been shown to regulate the acnA P2 promoter ( Cunningham et al . , 1997 ) . RI 12067348 128 Thus , stimulation of b1452 was dependent on MarA and a region ( – 131 to – 111 ) identified as a potential binding site for the activators . map and mdaB . RI 12067348 141 Unlike most other promoters , yhbW seemed to require additional sequence upstream of the – 76 to – 57 site for maximum activation by MarA , SoxS and Rob . RI 12067348 152 Curiously , Barbosa and Levy ( Barbosa and Levy 2000 ) found elevated gatA and gatC transcription due to MarA expression , and Pomposiello and colleagues ( Pomposiello et al . , 2001 ) found elevated gatA , gatB and gatD upon treating cells with paraquat , and elevated gatZ , gatA , gatB , gatC and gatD upon treating cells with salicylate . RI 12067348 269 ( 2001 ) Systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters of Escherichia coli : identifying nucleotides required for DNA binding and transcription activation . RI 11601842 10 The micF gene encodes a non - translated 93 nt antisense RNA that binds its target ompF mRNA and regulates ompF expression by inhibiting translation and inducing degradation of the message . RI 11601842 61 micF RNA is a sufficient factor for functional inactivation of ompF mRNA ; however , it is essential but not sufficient for chemical inactivation of the message ; additional factor ( s ) are involved in mRNA destabilization . RI 11601842 109 Furthermore , OmpRphosphate has been shown to negatively regulate the master flagella regulatory complex , flhDC . RI 11601842 112 Mutations in the tolC locus show increased expression of micF and a concomitant decrease in OmpF . RI 11601842 161 A common phenotype associated with transcriptional activation of micF by Rob , SoxS and MarA is multiple antibiotic resistance . RI 11601842 182 Other unknowns are how OmpR activates micF transcription while binding at far upstream sequences , i . e . in the - 156 to - 216 region ( Figure 4 ( a ) ) . RI 11601842 185 However , in vitro experiments show IHF to repress ompC transcription from two of its three promoters . RI 11601842 239 The mechanism of transcriptional activation of micF and ompC by OmpR as well as a role of IHF in this activation need to be better understood . RI 11601842 854 Identification of the DNA - binding domain of the OmpR protein required for transcriptional activation of the ompF and ompC genes of Escherichia coli by in vivo DNA footprinting . RI 11601842 862 Evidence for multiple OmpR - binding sites in the upstream activation sequence of the ompC promoter in Escherichia coli : a single OmpR - binding site is capable of activating the promoter . RI 2543976 55 Similar to the results of metE expression , MetR greatly stimulates the synthesis of MetH using plasmid pQN1011 as template ( lane 5 vs . lane 7 ) . RI 2543976 58 It should be noted ( lanes 1 - 4 ) that although plasmid pRSE562 directs the synthesis of the MetR protein there is still a large stimulation of metE expression by added MetR protein . RI 2543976 73 If the MetR protein synthesized from pRSE562 was responsible for the observed increase in MetH synthesis , the MetJ protein and AdoMet , if added to the first incubation , should inhibit the increase by preventing MetR synthesis . RI 2543976 128 The results of the present study extend these observations and show that the MetR protein also stimulates the in vitro expression of metH and that both the MetE and MetH proteins synthesized in vitro are enzymatically active . RI 2543976 146 It should be mentioned that Plamann and Stauffer ( 35 ) noted that a deletion in this area resulted in a loss of MetR activation of the in vivo synthesis of a metE - lac fusion protein . RI 2543976 147 The finding of only one MetR binding site suggests that the binding of MetR to this region may be responsible for both the activation of expression of metE as well as the autoregulation of metR ( 20 , 36 ) . RI 2543976 161 Thus , binding of MetJ to this area could serve to simultaneously repress both metR and metE expression . RI 2543976 163 These latter results , together with the experiments described here showing that MetJ binds to this region of DNA , strongly suggest that the - 8 to + 27 region is involved in metE repression . RI 11139621 3 NagC represses nagE , encoding the N - acetylglucosamine - specific transporter , while Mlc represses three PTS operons , ptsG , manXYZ and ptsHIcrr , involved in the uptake of glucose . RI 11139621 27 Recent experiments on the mlc - regulated genes showed that although NagC binding to Mlc operators could be detected in vitro , there was no or little regulation in vivo ( 6,17 , unpublished results ) . RI 11139621 176 Binding of NagC and Mlc to nagE operators in vivo As Mlc showed a stronger affinity for the nagE operator than NagC , it seemed possible that Mlc might be able to regulate the nagE promoter in the absence of NagC in vivo . ThreenagE – lacZ fusions were tested : the standard ‘ loop - forming ’ nagBE – lacZ fusion , whose expression is repressed by NagC binding co - operatively to the nagE and nagB operators ; and two single operator nagE – lacZ fusions , missing the nagB promoter / operator , but carrying the same nagE – lacZ junction . RI 11139621 188 To verify that Mlc does bind preferentially to the nagE operator in vivo , the effect of plasmids overproducing either Mlc or NagC was tested in a strain mutated for both mlc and nagC . Plasmids expressing either NagC or Mlc produce considerable repression of the nagBE – lacZ looped fusion , reducing expression 88 - and 56 - fold ( Table 2 ) . RI 11139621 190 The preferential repression by NagC at the looped nagBE – lacZ fusion , compared with the nagE – lacZ fusion with a single operator site , emphasises that regulation by NagC is normally dependent upon DNA loop formation involving the higher - affinity nagB operator . RI 11139621 197 The expression of ptsG is controlled by Mlc binding to two operators upstream of two promoters , both of which are subject to Mlc repression . RI 11139621 274 It should be remembered that nagE encodes the GlcNAc - specific PTS transporter and the genes for two other PTS transporters ( ptsG and manXYZ ) plus the genes for the central components of the PTS ( ptsHIcrr ) have been shown to be regulated by Mlc ( 6 – 11 ) . RI 11139621 277 However the much stronger repression of this fusion by overproduced Mlc , compared with overproduced NagC , shows that the isolated nagE operator site has a higher affinity for Mlc than NagC . RI 11139621 283 The glmUS operon , which is regulated positively by NagC , also relies upon co - operative binding of NagC to two sites ( 2 ) . RI 7601827 217 For example , nitrate induction of the narGHJI operon ( 21 ) and the narK gene ( 12 ) is controlled exclusively by NarL , while induction of the fdnGHI operon is controlled principally by NarL , but NarP also effects a degree of control ( observed with a narL mutant ) ( 21 ) . RI 7601827 329 Upstream sequence elements required for NarL - mediated activation of transcription from the narGHJI promoter of Escherichia coli . RI 7854119 23 For example , the expression of nitrate reductase is stimulated by NarL whilst fumarate reductase is repressed , thus ensuring that nitrate is used as an electron acceptor in preference to fumarate . RI 8437517 25 For exampie , whiist NarL is responsibie for maximum induction of nitrate reductase during anaerobic growth in the presence of nitrate , FNR - dependent expression of fumarate reductase is repressed by NarL , Thus , NarL ensures that nitrate , rather than energeticaily less rewarding substrates , is used as a terminal electron acceptor ( reviewed by iuchiandLin , 1991 ) . RI 10537212 120 This suggested that the hyb operon is subject to catabolite repression mediated by the cAMP - CRP system ( Busby & Kolb , 1996 ) . RI 11004182 153 Finally , either NarL or NarP was able to activate nrfA - lacZ expression independently of the other , while only NarL could repress expression . RI 11004182 182 This indicates that ( i ) the 70 NarL heptamer site is essential for NarL to function as an activator of nrfA expression and that ( ii ) other sites are used for NarL to repress expression independently of the 70 site , in contrast to prior interpretations ( see Discussion ) . RI 11004182 184 While NarL was still able to activate and to repress nrfA gene expression , the pattern differed from the wild - type nrfA - lacZ reporter fusion ( Fig . 6B ) . RI 11004182 193 Therefore , the 22 heptamer site is a critical site for mediating NarL repression of nrfA gene expression . RI 11004182 237 One of the more striking points in each model is that the NarP protein is nonessential ( i . e . , not a major player ) and that it can function as an antagonist of NarL in its ability to activate nirB or nrfA operon expression . RI 11004182 254 NarX and / or NarQ sensors , both NarL and NarP are phosphorylated where either response regulator can then activate nrfA gene expression . RI 11004182 264 A revised model for NarL - dependent activation of nirB gene expression is shown in Fig . 9 . RI 12079504 24 Mutagenesis of the Fnr recognition sequence at the dmsA promoter region confirmed the contribution of Fnr in the 100 - fold activation of dmsABC expression and explored the DNA sequence and spacing requirements for the Fnr recognition site at the P1 promoter . RI 12079504 32 The enhanced ability of Fnr to activate dmsABC gene expression is possibly , by analogy to Crp , due to increased affinity of Fnr at the consensus versus the native non - consensus Fnr recognition sequence at dmsA . RI 12079504 35 A single base substitution in the left half - site when combined with the Fnr consensus sequence at the right half - site exhibited a 138 - fold activation of dmsA - lacZ expression ( Figure 1 , λJA450 ) , an induction equivalent to the wild - type dmsA promoter that has the symmetrical deviation in the right half - site ( ca . 114 - fold ) . RI 12079504 36 Thus , the left and right half - sites are equivalent in their ability to confer Fnr - dependent activation of dmsA gene expression by RNA polymerase . RI 12079504 37 This is in contrast to the requirement for the left half - site of the Fnr1 regulatory site needed for Fnr - dependent repression at the cydA P1 promoter [ 15 ] . RI 12079504 43 Thus , the dmsA promoter exhibits a preference for - 41.5 target sites like other Fnr - regulated class II promoters [ 16 ] . RI 12079504 52 The effect of cis mutations on Fnr - dependent activation of dmsA - lacZ expression is shown in the lower right portion of the figure . β - galactosidase activity was measured from the cells containing the indicated fusion inserted in single copy at the att site . RI 12079504 139 Also , both half - sites of the Fnr recognition sequence at dmsA are required for Fnrdependent expression and are similar in their ability to activate dmsA transcription . RI 12079504 234 Dong X , Li S , DeMoss J : Upstream sequence elements required for NarL - mediated activation of transcription from the narGHJI promoter of Escherichia coli . RI 12923080 155 At low to intermediate levels of nitrate ( 2 to 6 mM ) NarP appeared to antagonize the ability of NarL to activate fdnG operon expression . RI 12923080 226 NarP thus performs a unique role to fine - tune fdnG operon expression by delaying the ability of NarL to activate when nitrate levels are low . RI 13129959 14 These results establish the unconventional napF operon control region architecture , in which the major promoter P1 is activated by the Fnr protein bound to a site centered at 64.5 with respect to the transcription initiation site , working in conjunction with the phospho - NarP protein bound to a site centered at 44.5 . RI 13129959 200 DISCUSSION The napF operon exhibits a unique pattern of expression and a unique control region architecture in comparison to other characterized E . coli Fnr - and Nar - regulated operons . RI 13129959 211 One obvious difference between the synthetic promoter and the napF operon promoter is that transcription from the latter is also activated by the NarP protein bound to a site centered at position 44.5 . RI 15667305 44 Nitrate - and nitrite - mediated expression of the hcp is dependent on NarL and NarP Sequence analysis of the hcp regulatory region suggested that it contains a binding site for FNR and also binding sites for nitrite - and nitrate - activated regulators , NarL and NarP ( Figure 1 ) . RI 15667305 52 This demonstrated that either NarL or NarP is required to activate transcription from the hcp promoter . RI 15667305 53 Concluding remarks The hcp gene has been shown to be regulated by FNR , NarL and NarP proteins . RI 15978080 11 Summary Expression from the Escherichia coli nrf operon promoter is activated by the anaerobically triggered transcription factor , FNR , and by the nitrate / nitrite ioncontrolled response regulators , NarL or NarP , but is repressed by the IHF and Fis proteins . RI 15978080 18 Hence , Fis protein is a major factor responsible for catabolite repression at the nrf promoter , and Fis can override activation by FNR and NarL or NarP . RI 15978080 32 We previously found that two other DNA - binding proteins , Fis ( factor for inversion stimulation ) and IHF ( integration host factor ) , bind to the nrf promoter and downregulate its activity ( Browning et al . , 2002 ) . RI 15978080 46 Our results show that Fis plays a key role in catabolite repression at the nrf promoter and that its binding overrides the actions of the other regulators . RI 15978080 67 As the DNA binding site for FNR overlaps the acsP1 - 35 element , it is easy to understand how FNR represses acsP1 at the same time as it activates the nrf promoter . RI 15978080 68 Other results in Fig . 2A show that FNR - dependent activation of the nrf promoter is repressed by Fis or IHF proteins . RI 15978080 76 Results in Fig . 2B ( lanes 4 – 6 , 10 – 12 ) show that NarL can enhance FNR - dependent activation of the nrf promoter . RI 15978080 167 Fis and FNR moderate catabolite repression at the nrf promoter The locations of the different mutations that relieve catabolite repression at the nrf promoter suggest the involvement of Fis , FNR and , possibly , IHF ( Fig . 6 ) . RI 15978080 205 To investigate further the involvement of Fis in catabolite repression , the expression of an nrf : : lac fusion was compared in cells with and without a functional fis gene . RI 15978080 260 We suspect that this may account for the downregulation of FNR - dependent activation of the nrf promoter by IHF , but this is unproven . RI 15978080 290 As at the nrf promoter , the nir promoter can be activated by FNR alone but FNRdependent activation is repressed by IHF and Fis ( Wu et al . , 1998 ; Browning et al . , 2000 ) . RI 15978080 294 The binding of NarL or NarP to the nir promoter displaces IHF and overrides the repression of FNR - dependent activation by IHF and Fis ( Browning et al . , 2004 ) . RI 15978080 311 Binding of Fis and IHF to sites centred at positions - 15 and - 54 , respectively , represses FNR - dependent transcription at the nrf promoter . RI 7643383 20 Apparently , only the NarP protein is competent to activate transcription of the aeg - 46.5 operon when bound to the 44.5 region . RI 7643383 31 Some of these operons are regulated by the NarL protein alone , such as the narG and frdA operons , whereas expression of the nirB ( encoding NADH - dependent nitrite reductase ) , nrfA and fdnG operons is controlled by both the NarL and NarP proteins . RI 7643383 34 These binding sites are located upstream of the Fnr - binding site in the control regions of the NarL - activated narG , fdnG and narK operons . RI 7643383 37 Mutational analysis of the nirB and nrfA operon control regions has identified cis - acting regions that are required for both NarL and NarP - dependent induction ( Tyson et al . , 1994 ) . RI 7643383 74 These observations indicate that the region that is essential for normal Fnr - dependent induction of aeg - 46.5 operon expression is located downstream of position 85 . RI 7643383 106 These observations confirm the importance of the 64.5 sequence in the Fnr - dependent induction of aeg - 46.5 operon expression . RI 7643383 136 The results suggest that the NarP protein specifically interacts with the 44.5 region in order to activate aeg - 46.5 operon expression . RI 7643383 225 This unusual spacing in relation to the RNA polymerase binding site may reflect a distinct mechanism by which Fnr ( and NarP ) activates aeg - 46.5 operon expression . RI 7643383 243 However , narG and frdA operon expression is regulated by the NarL protein alone ( Rabin & Stewart , 1993 ) , suggesting that the NarL binding sites in these control regions are not efficiently recognized by the NarP protein . RI 7643383 247 The NarL and NarP proteins are each able to activate expression of the nirB and nrfA operons by interaction with common DNA - binding sites . RI 7643383 249 The NarL / NarP protein binding sites required for activation of the nirB and nrfA operons are located further upstream ( 69.5 and 74.5 , respectively ) than that of the aeg - 46.5 operon ( 44.5 ) . RI 7643383 258 Negative regulation of aeg - 46.5 operon expression by the NarL protein In addition to its function as a transcriptional activator , the NarL protein negatively regulates expression of the frdA , dmsA and nrfA operons in response to nitrate availability . RI 7643383 261 aeg - 46.5 operon expression is also negatively regulated by the NarL protein but in this case NarL does not appear to act as a classical repressor . RI 7643383 268 The NarL and NarP proteins compete for the 44.5 binding site ; binding of NarP further induces aeg - 46.5 operon expression , whereas binding of NarL has no significant effect on the basal anaerobic ( Fnr - dependent ) level of expression . RI 7643383 280 Since only the NarP protein can activate aeg - 46.5 operon expression ( again , presumably by interacting with RNA polymerase ) , the competition from NarL for the binding sites would lead to an antagonization of NarP activation . RI 7643383 284 Thus , antagonization of NarP - independent activation of aeg - 46.5 operon expression by the NarL protein should be more pronounced in the presence of nitrate than nitrate . RI 7643383 477 Upstream sequence elements required for NarL - mediated activation of transcription from the narGHJI promoter of Escherichia coli . RI 7643383 718 NarL - phosphate must bind to multiple upstream sites to activate transcription from the narG promoter of Escherichia coli . RI 8501030 29 3259 This shows that the NarL protein represses aeg - 46 . RI 8501030 154 These results show that the NarP protein activates F ( aeg - 46.5 - lacZ ) expression in response to nitrate and nitrite and that the NarL protein antagonizes this activation . RI 8631699 257 The presence of FNR and CRP consensus sequences upstream of caiF is consistent with the activation of transcription of a caiF - lacZ fusion by these two proteins ( Table 3 ) . RI 8631699 270 When the CaiF protein was overproduced , the pattern of induction of carnitine dehydratase activity was shown to escape from the control of the histone - like protein H - NS and the alternate sigma transcription factor S and to rely primarily on the stimulatory effect of CaiF . RI 8631699 619 DNAprotein recognition : demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABC promoters by the DeoR repressor . RI 9335308 156 For example , the nitrate reductase genes narGHJI are regulated exclusively by NarL and are not responsive to NarP as observed here for cydD , while nitrate induction of the fdnGHI operon is regulated predominantly by NarL but is also responsive in part to NarP ( 33 , 39 ) . RI 9852003 59 The results show that the dcuB gene is expressed exclusively under anaerobic conditions in a manner that is FNR dependent and that it is repressed by NarL in the presence of nitrate and is subject to CRP - mediated catabolite repression . RI 9852003 170 Both the aerobic and anaerobic expression levels of dcuB were only ca . twofold lower in the arcA mutant ( JRG3841 ) , indicating that ArcA plays no more than a minor role in regulating dcuB expression in response to oxygen ( Fig . 5B ) and that ArcA is not responsible for the FNR - independent mechanism of anaerobic activation of dcuB transcription . RI 9852003 261 The organization of the putative NarL sites in the dcuB operator - promoter region clearly does not conform to the preferred 7 - 2 - 7 arrangement , which is consistent with the observation that NarL , but not NarP , represses dcuB expression . RI 9852003 736 Transcriptional co - activation at the ansB promoters : involvement of the activating regions of CRP and FNR when bound in tandem . RI 15995204 53 The phospho - DcuR protein is known to activate the transcription of three operons : the dcuB - fumB operon encoding the DcuB carrier and fumarase B , the frd - ABCD operon encoding fumarate reductase ( Fig . 1C ) , and the dctA gene encoding the aerobic C 4 - dicarboxylate transporter ( 2 , 17 , 18 , 67 ) . RI 15995204 177 We also monitored expression from a previously constructed ( frdA - lacZ ) gene fusion for which both Dcumediated activation and Nar - mediated repression are well documented . RI 15995204 239 Thus , the dcuSR operon control region contains an authenticated 7 - 2 - 7 heptamer pair for phospho - NarL binding overlapping the likely 35 region of the promoter ( Fig . 2B ) and therefore positioned appropriately to effect the observed repression ( Table 2 ) . RI 16261196 164 NorR is a r 54 - dependent transcriptional activator that regulates expression of the NO reductase operons , norVW in E . coli and norAB in R . eutropha [ 21,22 ] . RI 16261196 239 NorR usually regulates cytoplasmic NO reductase norVW and sometimes another membrane - bound NO reductase ( norB ) and the NO dioxygenase hmp . RI 16261196 288 Moreover , a common inverted repeat sequence coincided with the defined here NsrR recognition motif was shown to be involved in NsrR - mediated repression of the ytfE gene . RI 17449618 9 Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655 , including the hmpA , ytfE , and ygbA genes that were previously shown to be regulated by NsrR . RI 17449618 15 As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron - sulfur centers damaged by nitric oxide , the demonstration that hcp - hcr , hmpA , and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein , HCP , might also be part of a defense mechanism against reactive nitrogen stress . RI 17449618 29 The fourth promoter predicted to be regulated by NsrR was P hcp , the promoter of a two - gene operon encoding the hybrid cluster protein , HCP , and its reductase , HCR . RI 17449618 105 Operons observed to be differentially expressed include not only all four of the transcripts , hmpA , ytfE , ygbA , and hcp - hcr , known or predicted to be NsrR - regulated ( 4 , 40 ) , but also other transcripts predicted to be regulated by nitrite or RNS generated from nitrite , for example , nitric oxide ( 11 ) . RI 17449618 110 In the case of the ydbC promoter , the differences in transcript levels between the two transformants were at least as great as those for the NsrRrepressed genes , making P ydbE a prime candidate for direct activation by NsrR . RI 17449618 112 The microarray data for the other promoters that are apparently activated by NsrR must be evaluated cautiously , however , because few of these transcripts encode proteins known or suspected to be involved in the response to RNS , and most of the differences were much smaller ( typically two - to threefold apparent activation ) than the 30 - fold repression of ytfE , the 14 - fold repression of hmpA , and the up - to - 15 - fold repression of hcp . RI 17449618 129 Genes differentially regulated in strains carrying pGIT1 and pGIT8 a Transcript expression ratio in c : Coregulation by d : Gene Product pGIT8 pGIT8 2 2 with NO 2 vs pGIT1 2 vs pGIT8 pGIT8 2 vs pGIT1 with NO 2 Operon structure FNR NarL NarP NsrR site e Reference NsrR repressed hcr NADH oxidoreductase 12.20 4.47 13.09 hcp - hcr A f , g A g 6 wrt mapped TSS hcp Hybrid cluster protein 15.65 5.28 9.93 yccM Predicted 4Fe - 4S membrane protein 4.89 3.19 4.18 yccM A f 69 wrt predicted TSS uspF Nucleotide binding protein 2.09 1.63 1.78 uspF yeaR Hypothetical protein 2.57 3.57 3.12 yeaR - yoaG R A A 20 wrt predicted TSS A f , g R g A g 30 wrt mapped TSS ccmG Cytochrome c biogenesis system 2.75 2.17 2.64 napFDAGHBCccmABCDEFGH ccmF Cytochrome c biogenesis system 2.08 1.73 2.34 napB Periplasmic nitrate reductase 2.07 3.49 3.75 napH Periplasmic nitrate reductase 2.13 3.39 3.86 napD Periplasmic nitrate reductase 2.02 3.49 2.80 napF Periplasmic nitrate reductase 2.41 3.99 3.00 hmpA b Flavohemoglobin 13.07 8.50 14.44 hmpA R f , g A 1 wrt mapped TSS ygbA b Hypothetical protein 4.58 3.20 3.86 ygbA R f 7 wrt mapped TSS nrfA Periplasmic nitrite reductase 5.28 7.37 6.66 nrfABCDEFG A f , g R g A g 63 wrt mapped TSS nrfB Periplasmic nitrite reductase 3.54 4.31 4.67 nrfC Periplasmic nitrite reductase 4.51 5.30 6.19 nrfD Periplasmic nitrite reductase 3.68 3.98 3.99 nrfE Periplasmic nitrite reductase 4.72 5.15 6.44 nrfF Periplasmic nitrite reductase 2.65 3.29 2.47 ytfE b RNS - induced conserved protein 31.91 7.84 33.18 ytfE R g A 12 wrt mapped TSS 17 13 12 6 25 , 26 NsrR activated insB _ 1 IS1 protein InsB 0.40 1.29 0.60 yafE Predicted S - adenosylmethioninedependent 0.36 0.36 0.35 yafDE methyltransferase yafU Predicted inner membrane protein 0.23 0.44 0.16 yafU ykfJ Hypothetical protein 0.43 0.52 0.68 ykfJ [ prfH ] mmuP Predicted ABC transporter 0.37 0.57 0.27 mmuPM A insB _ 2 IS1 protein InsB 0.41 1.46 0.63 yagA Hypothetical protein 0.23 0.56 0.18 yagA insB _ 3 IS1 protein InsB 0.40 1.07 0.59 ykgF Predicted amino acid dehydrogenase 0.40 0.42 0.49 ykgEFG R A betB Betaine aldehyde dehydrogenase 0.47 0.70 0.54 betIBA R A cyoE Cytochrome bo terminal oxidase 0.44 0.94 0.47 cyoABCDE R f A cyoC Cytochrome bo terminal oxidase 0.36 0.96 0.51 cyoB Cytochrome bo terminal oxidase 0.29 0.67 0.38 cyoA Cytochrome bo terminal oxidase 0.26 0.79 0.35 ompF Outer membrane porin 0.43 0.63 0.40 ompF insB _ 4 IS1 protein InsB 0.35 1.18 0.54 dadA D - Amino acid dehydrogenase 0.49 0.76 0.49 dadAX ydbC Putative oxidoreductase 0.03 0.03 0.01 ydbC insB _ 5 IS1 protein InsB 0.40 1.25 0.61 ygeF Hypothetical protein 0.43 0.94 0.49 ygeF insB _ 6 IS1 protein InsB 0.38 1.30 0.61 yiiL L - Rhamnose mutarotase 0.17 0.23 0.16 rhaBAD - yiiL A yjbB Putative - helix protein 0.35 0.42 0.23 yjbB A yjiV Hypothetical protein 0.30 0.26 0.45 yjiU a This table lists genes that display at least a twofold difference in transcript abundance between anaerobically grown cultures of E . coli strain MG1655 harboring pGIT1 , a multicopy plasmid with a copy of the ytfE promoter to which NsrR binds , and pGIT8 , the same plasmid with a mutation which abolishes NsrR binding . RI 17449618 152 These data clearly indicate that the hcp promoter is activated by FNR and repressed by NsrR . RI 17449618 153 In the case of the ytfE promoter , repression by NsrR is the dominant regulatory mechanism , though there is also some evidence for anaerobic repression by FNR , as reported previously ( 25 ) . RI 17449618 157 NsrR : sixth protein to regulate transcription factor at P nrfA . RI 17449618 162 Two approaches were used to confirm that NsrR is this sixth factor that regulates P nrfA . RI 17449618 180 The data from independent duplicate cultures were completely consistent with NsrR repression of P nrfA [ 1,200 and 1,270 nmol of o - nitrophenyl -- D - galactopyranoside hydrolyzed min 1 ( mg bacterial dry mass ) 1 for the pGIT1 transformant compared with 180 and 230 units for the pGIT8 transformant ] . RI 17449618 207 This suggestion was based upon observations that HCP is oxidized by hydrogen peroxide , that an hcp mutant is more sensitive to hydrogen peroxide than its parent , and that hcp transcription is regulated by OxyR . RI 19245365 3 ( 2009 ) 420 , 249 – 257 ( Printed in Great Britain ) doi : 10.1042 / BJ20090183 249 Competition between NarL - dependent activation and Fis - dependent repression controls expression from the Escherichia coli yeaR and ogt promoters Derrick J . RI 19245365 10 Although most NarLdependent promoters are also co - dependent on a second transcription factor , FNR protein , two targets , the yeaR and ogt promoters , are activated by NarL alone with no involvement of FNR . RI 19245365 13 DNase I footprinting studies identified Fisbinding sites that overlap the yeaR promoter NarL site at position 43.5 , and the ogt promoter NarL site at position 78.5 , and suggest that Fis represses both promoters by displacing NarL . RI 19245365 23 In the second part of the present study , we selected another E . coli promoter , the ogt promoter , that transcriptome analysis [ 11 ] had predicted to be activated by NarL alone , and we have used biochemical and genetic analysis to compare its expression and organization with the yeaR promoter . RI 19245365 71 c The Authors Journal compilation c 2009 Biochemical SocietyTranscription activation by Escherichia coli NarL protein 251 Figure 1 NarL regulates the E . coli yeaR promoter ( A ) This Figure shows the E . coli K - 12 yeaR promoter sequence from positions 64 to + 86 with respect to the transcription start site ( + 1 ) ( 12 ) . RI 19245365 135 Fis represses the ogt promoter by displacing upstream - bound NarL To investigate a role of Fis at the ogt promoter , expression of the ogt : : lac fusion was compared in strain JCB387 and in the fis mutant derivative , JCB3871 . RI 19245365 174 β - Galactosidase activity Promoter Mutated site minus NO 3 plus NO 3 Fold induction ogt100 93 + 1 439 + 12 4.7 ogt102 NarL I 92 + 5 107 + 6 1.2 ogt104 NarL II 56 + 2 87 + 3 1.6 the yeaR promoter is repressed by the binding of NsrR to a target that overlaps the 10 hexamer element ( Figure 1A ) [ 12,31,32 ] . RI 19245365 189 In the second part of the present study , we investigated the E . coli ogt promoter , which Constantinidou et al . [ 11 ] had also found to be induced by NarL independently of FNR . RI 19245365 209 Although there is no simple rationale for why the yeaR and ogt promoters are also repressed by Fis , we suggest that rapidly growing cells may opt out of certain stress responses , and we speculate that RNS may be a small risk in these conditions compared with other stresses . RI 19245365 232 As at the yeaR promoter , nitrate - dependent induction can be repressed by Fis that binds to a single site overlapping the upstream DNA site for NarL . RI 10850996 13 We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene ( inaA , marR , aslB , ybaO , mdlA , yfhD , and ybiS ) . RI 10850996 14 One gene , galT , was repressed by Rob . RI 10850996 39 The Rob homologs MarA and SoxS can activate transcription of a broad range of genes in vivo ( reviewed in references 2 and 14 ) , including sodA , fumC , micF , zwf , and inaA , suggesting broadly overlapping activities of these three regulators . RI 10850996 155 Both inaA and marRAB are known Rob - regulated genes ( 5 , 21 ) . RI 10850996 212 Our analysis revealed that inaA , marRAB , aslB , ybaO , mdlA , yfhD , and ybiS were transcriptionally activated by constitutive levels of Rob , while galT expression was repressed . RI 10850996 213 Rob contributes significantly to the expression of the marRAB operon , which regulates the intrinsic resistance of E . coli to various antibiotics , bactericidal agents , and organic solvents ( 5 , 10 , 13 ) . RI 10850996 217 Among functions that could contribute to cellular resistance to environmental agents , Rob enhances expression of mdlA , which encodes a multiple - drug - resistance - like ATP - binding component of a transport system ( 3 ) , and strongly enhances expression of micF , the antisense RNA that downregulates the outer membrane porin OmpF . RI 10850996 223 Several Rob - regulated genes , such as marA , noted above , seem to encode other regulatory proteins . RI 10850996 231 In this study , galT was the only gene repressed by Rob . RI 10850996 246 Additional effects on the expression of many genes may occur through Robmediated activation of other regulatory proteins ( MarA , AslB , and YbaO ) . RI 20023096 134 On the other hand , activation of the P araB promoter was delayed when glucose was present , consistent with the regulation of this promoter also by CRP ( 6 ) . RI 20023096 256 Given the proximity of this binding site to the start site for transcription , these results suggest a model where AraC sterically inhibits the binding of XylR and RNA polymerase to the P xylA promoter , consistent with our experimental results . RI 20023096 741 Organization and regulation of the D - xylose operons in Escherichia coli K - 12 : XylR acts as a transcriptional activator . RI 8980677 18 The most intensely examined operon is araBAD , where AraC is required for repression and activation of BAD transcription ( reviewed by Schleif , 1992 ) . RI 1560456 13 This loop helps prevent AraC protein from entering its inducing state and helps hold the uninduced , or basal level , of expression of the araBAD genes at a low level . RI 1560456 138 Epst , ream repredsion and CRP stimulation of the Escherichia coli r , - arabinose operon . RI 1560456 176 In vitro activation of the transcription of araBAD operon by AraC activator . RI 3041410 169 One of the ara loops , that involving AraC bound at araO2 and araI , was previously shown to generate repression of araBAD ( 5 - 7 ) . RI 1447222 342 The repression of the araBAD operon occurs by cooperative interaction between two distal AraC binding sites araOz and urd ( 19 , 39,40 ) . RI 6262769 16 The expression of araC is repressed by its own product and stimulated by the CAP system ( 5 ) . RI 6262769 109 We believe that the low - affinity CAP site located between - 130 and - 140 probably does not constitute the site for CAP - stimulation of - the araC promoter , not only because its position ( in relation to the polymerase binding site and transcriptional start point ) is at variance with other CAP sites ( 16 , 17 ) , but also be - A A FIG . 5 . RI 6262769 129 Based on these four sites , the . known start point for araBAD transcription at + 1 ( 6 ) , and an estimated start point for araC transcription near - 170 , Ogden et aL proposed ( i ) that araBAD and araC are stimulated by the . binding of CAP to distinct regions on the ' DNA - CRPBAD and CRPC , respectively , and ( ii ) that the araO , when bound to araC protein , prevents the binding of CAP to either CRPc or CRPBAD , and this in turn de - B756 Biochemistry : Lee et aL Proc . RI 6262769 152 Schematic representation of regulation of araBAD and araC promoters by CAP , P1 , and P2 . negative control of Pc by P1 and P2 . RI 19906180 1 Molecular Microbiology ( 2009 ) 74 ( 6 ) , 1513 – 1526 doi : 10.1111 / j . 1365 - 2958.2009.06950 . x First published online 19 November 2009 Competitive activation of the Escherichia coli argO gene coding for an arginine exporter by the transcriptional regulators Lrp and ArgPmmi _ 6950 1513 .. 1526 Eveline Peeters , Phu Nguyen Le Minh , Maria Foulquié - Moreno and Daniel Charlier * Erfelijkheidsleer en Microbiologie , Vrije Universiteit Brussel ( VUB ) , Pleinlaan 2 , B - 1050 Brussels , Belgium . RI 19906180 2 Summary In vivo and in vitro analyses indicate that transcription of the argO gene coding for an arginine exporter is regulated by the global transcriptional regulator Lrp , an effect that went by unnoticed in previous genome - scale screenings of the Lrp regulatory network in Escherichia coli . RI 19906180 4 Activation by Lrp interferes with the previously demonstrated activation of the argO promoter by ArgP . RI 19906180 10 Combined , the effector modulated activation of argO transcription by ArgP and Lrp must ensure an adapted and fine - tuned synthesis of the transporter in response to environmental conditions . RI 19906180 49 Expression of the E . coli argO gene is activated by ArgP , also known as IciA , which is a transcriptional activator of the LysR type transcriptional regulator family ( Nandineni and Gowrishankar , 2004 ; Laishram and Gowrishankar , 2007 ) . RI 19906180 53 More recently , ArgP was shown to bind to an approximately 60 - bp - long sequence partially overlapping the argO promoter ( Fig . 1 ; Laishram and Gowrishankar , © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd , Molecular Microbiology , 74 , 1513 – 1526Lrp and ArgP activation of argO transcription 1515 2007 ) . RI 19906180 84 Autoradiographs of EMSA analyses of Lrp binding to the argO control region without and with 7 mM L - leucine in the binding buffer respectively . RI 19906180 101 Furthermore , we have demonstrated that Lrp - mediated activation of P argO is also operative , and even more efficient , in an argP deletion mutant ( see below ) . RI 19906180 105 The L - leucine concentration was set at 7 mM , corresponding to the concentration of total leucine in cells grown in © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd , Molecular Microbiology , 74 , 1513 – 1526Lrp and ArgP activation of argO transcription 1517 leucine - supplemented medium ( Quay et al . , 1977 ) . RI 19906180 108 This complex binding pattern and its Lrp concentration - dependent profile strongly suggest the cooperative binding of Lrp dimers to multiple binding sites in the argO control region and the formation of complexes with a different stoichiometry . RI 19906180 114 In silico analyses and experimental verification of potential binding sites for Lrp in the argO control region The formation of multiple Lrp – argO operator complexes with a different apparent stoichiometry suggests the existence of several binding sites for Lrp in the argO control region . RI 19906180 116 Three potential Lrp binding sites can be found in the argO control region ( Pot - box 1 , 2 and 3 ; Fig . 1 ) . RI 19906180 124 For example , binding of Lrp to the isolated strong binding site 2 of the ilvIH control region occurred in the nM range ( Cui et al . , 1996 ) but , in contrast , binding to site 2 of the pap operon could not be detected by EMSA , although this site was successfully used for the growth of co - crystals ( de los Rios and Perona , 2007 ) . RI 19906180 166 Since both Lrp and ArgP stimulate argO expression and bind to overlapping targets in the argO control region , we were interested in analysing potential interferences in the action of these transcriptional activators on argO expression , in response to environmental conditions . RI 19906180 168 The major conclusions are that : ( i ) both Lrp and ArgP stimulate P argO in an effector - dependent manner , but to a different extent , ( ii ) the two regulators behave as competitive activators , and ( iii ) ArgP is intrinsically a more potent activator for P argO as compared with Lrp . RI 19906180 200 The cooperative binding of Lrp to the argO control region and the footprinting data ( Figs 3 and 4 ) suggest the binding of at least three Lrp dimers to regularly spaced binding sites ( Fig . 1 ) and wrapping of the operator DNA . RI 19906180 210 The artPIQM operon is , besides being downregulated by Lrp ( Hung et al . , 2002 ) , also repressed by ArgR ( Caldara et al . , 2007 ) . RI 19906180 352 ( 2007 ) ArgR - dependent repression of arginine and histidine transport genes in Escherichia coli K - 12 . RI 19906180 454 ( 1997 ) The binding of two dimers of IciA protein to the dnaA promoter 1P element enhances the binding of RNA polymerase at to the dnaA promoter 1P . RI 19906180 501 ( 2005 ) Lrp and H - NS – cooperative partners for transcription regulation at Escherichia coli rRNA promoters . RI 19906180 540 ( 1999 ) Lrp binds to two regions in the dadAX promoter region of Escherichia coli to repress and activate transcription directly . RI 17504942 39 Results ArgP - mediated regulation of E . coli argO Reconstitution of ArgP - mediated regulation of argO transcription in vitro Toward reconstitution of argO transcriptional regulation in vitro , the in vivo start site of argO transcription was first mapped ( in an argP d strain where such transcription is high and constitutive ) by primer extension analysis to an A residue situated 28 bases upstream of the predicted translation start site of the structural gene ( see Supplementary Fig . S1 ) . RI 17504942 46 ArgP - mediated regulation of argO transcription was then tested in a defined in vitro run - off transcription system using 70 - bearing RNAP holoenzyme and a linear DNA template that encompasses the argO region from 293 to + 109 ( Fig . 1C ) . RI 17504942 62 Although a 26 - bp near - perfect palindromic region from 102 to 77 exists upstream of argO ( see Fig . 1A ) , neither its deletion ( designated 1 ) nor that of an overlapping 1260 GENES & DEVELOPMENT upstream region from 115 to 90 ( designated 2 ) led to any significant loss of either ArgP binding to argO in vitro ( Fig . 2B ) or to Arg - and Lys - modulated transcriptional regulation in vivo ( Fig . 1B , top panel ) . RI 17504942 65 Since the results above suggested that the argO regulatory region upstream of 89 is not required for ArgP binding , we tested fragments with increasing length of downstream sequence in the EMSA experiments . RI 17504942 73 As earlier , with ArgP alone added , all radioactivity was shifted to a sharp band of retarded mobility , indicative of formation of a binary complex that ArgP - mediated regulation of E . coli argO Figure 2 . RI 17504942 183 Likewise , GalR - mediated repression of the gal operon P1 promoter ( in the absence of galactose ) is postulated to occur by a mechanism involving trapped RNAP in a complex that is an intermediate between the closed and the open complexes ( Choy et al . 1995 ) . RI 17504942 430 The yeaS ( leuE ) gene of Escherichia coli encodes an exporter of leucine , and the Lrp protein regulates its expression . RI 17504942 447 Transcriptional activation of the dnaA gene encoding the initiator for oriC replication by IciA protein , an inhibitor of in vitro oriC replication in Escherichia coli . RI 17504942 453 The binding of two dimers of IciA protein to the dnaA promoter 1P element enhances the binding of RNA polymerase to the dnaA promoter 1P . RI 18502871 10 Purified His 6 - tagged ArgP protein binds with an apparent K d of 35 nM to the dapB promoter in a gel retardation assay , provided that sequences up to 103 are present . RI 18502871 12 These results fit with a model in which ArgP contributes to enhanced transcription of dapB when lysine becomes limiting . * Corresponding author . RI 18502871 102 Upon transformation into strain JCP75 , this plasmid led to red colonies on MacConkey lactose agar plates supplemented with lysine , confirming that ArgP is able to enhance dapB transcription . RI 18502871 112 As shown in Fig . 2 , induction of ArgP synthesis was soon followed by a sharp stimulation of dapBlacZ transcription . RI 18502871 113 Altogether , these data indicate that ArgP is able to activate transcription from the dapB promoter , provided that some sequences upstream of 81 are present , and is necessary for lysine to repress dapB expression . RI 18502871 115 To further investigate ArgP - mediated activation of dapB transcription , we purified His - tagged ArgP protein ( 17 ) and used it to perform EMSE . RI 18502871 125 ArgP binds a site upstream of the dapB promoter . RI 18502871 132 Samples were loaded on a polyacrylamide gel , run , and analyzed by autoradiography . gave a single retarded band of mobility similar to that observed with purified His 6 - ArgP protein , whereas crude extracts devoid of ArgP gave no retarded band ( Fig . 3B ; see also Fig . 5A ) , indicating that no protein other than ArgP is able to bind significantly to the dapB promoter region . RI 18502871 206 Transient repressor effect of Fis on the growth phase - regulated osmE promoter of Escherichia coli K12 . RI 15150242 29 Based on data from in vitro studies , Celis suggested that wild - type ArgP both represses its own transcription and activates that of argK ; he also sought to explain the Can r phenotype associated with the argP d mutation on the assumption that it is a loss - of - function ( that is , dominant - negative ) allele whose product is unable to efficiently activate the genes involved in Arg uptake , including argK ( 12 ) . RI 15150242 35 We show in this study that strains with null mutations in either argP or yggA exhibit abnormally increased sensitivities to CAN ( Can ss ) and that ArgP is a transcriptional regulator of yggA that mediates the latter ’ s induction by Arg . RI 15150242 46 Our results indicate that yggA encodes an ArgP - regulated Arg exporter in E . coli . RI 15150242 194 Therefore , a straightforward interpretation of our results , as further discussed below , is that ( i ) ArgP is a transcriptional activator of yggA , ( ii ) ArgP ’ s activator function is enhanced by Arg and inhibited by Lys , and ( iii ) yggA encodes an Arg ( and CAN ) exporter in E . coli . RI 15150242 197 The model proposed by Celis ( 11 , 12 ) to explain the Can r phenotype of an argP d mutant assumed ( i ) that the mutation represents a loss - of - function ( that is , dominant - negative ) allele of argP and ( ii ) that wild - type ArgP is a transcriptional activator of argK , whose product in turn acts to enhance Arg uptake through two different transport systems . RI 15150242 199 Furthermore , even the finding of Celis ( 12 ) that argK is a target for transcriptional activation by ArgP may need to be reexamined , given that ( i ) there is a discrepancy between the expected argK runoff transcript size ( approximately 310 bases ) determined from the promoter mapping data published by Celis et al . in an earlier paper ( 13 ) and that reported by him ( 215 bases ) for ArgP activation experiments ( 12 ) and ( ii ) the promoter ( for argK ) mapped by his group ( 13 ) is within the argK coding region per the genome sequence published by Blattner et al . ( 5 ) ( GenBank accession number AE000375 , wherein argK is annotated ygfD ) . RI 15150242 200 Coeffectors of ArgP in yggA regulation . RI 15150242 202 The ArgP - mediated induction of yggA expression is also observed upon the addition of the Arg biosynthetic precursor citrulline ( Fig . 3 ) or ornithine ( data not shown ) , most likely by virtue of its conversion to Arg within the cells . RI 21441513 55 Lrp was also found to be involved in the regulation of cadBA expression . RI 21441513 139 Analysis of the lysP promoter revealed a conserved T - N 11 - A motif , a typical binding site of LysR - type transcriptional regu - VOL . RI 21441513 152 To evaluate whether ArgP was involved in the transcriptional regulation of lysP , a nonfunctional argP allele ( argP : : Cam r ) was transduced into strain MG - LR carrying the chromosomal P lysP : : lacZ fusion . RI 21441513 155 To confirm the role of ArgP in the regulation of lysP transcription , the argP gene was cloned into plasmid pBAD24 under the control of the arabinose - inducible promoter ( 18 ) . RI 21441513 165 These results indicated that ArgP is responsible for the transcriptional activation of lysP in the absence of lysine . RI 21441513 188 To determine whether ArgP directly regulates lysP transcription , we tested binding of ArgP to the lysP promoter / control region . RI 21441513 197 As already mentioned , a potential ArgP - binding site , ATG AAGGTGTCTTAT , is centered at position 59 in the lysP promoter / control region ( Fig . 2 ) . RI 21441513 201 To study the ArgP - binding site in the lysP promoter / control region in more detail , DNase I footprinting analysis was performed in the absence and presence of lysine ( Fig . 5 ) . RI 21441513 220 So far , our results demonstrate that ArgP directly binds to the lysP promoter / control region and that the T - N 11 - A sequence located close to the RNA polymerase binding site is important for binding and crucial for lysP transcription . RI 21441513 231 This scenario is plausible , because ArgP and Lrp competitively activate argO ( 35 ) . RI 21441513 255 Lrp stimulates transcription of lysP by direct binding to its control region . RI 21441513 270 Peeters et al . have recently demonstrated that argP expression was not regulated by Lrp ( 35 ) . RI 21441513 288 However , under inducing conditions , the P cadBA activity in the lrp : : Km r mutant strain was 2 - fold lower than that in the parent strain , suggesting that Lrp stimulates expression of cadBA . RI 21441513 310 Together , these results indicate that Lrp upregulates expression of cadBA . RI 21441513 323 It is important to note that the molecular mechanisms of lysinedependent regulation by ArgP differ between argO and dapB ( 2 , 24 ) . RI 21441513 345 The ArgP - binding site for argO extends up to nucleotide 20 and thereby overlaps the 35 promoter motif , whereas the ArgP - binding site for lysP extends up to position 47 , which is upstream of the 35 promoter site ( Fig . 2 ) . RI 21441513 352 Lrp - dependent regulation of lysP and cadBA . RI 21441513 372 Recently , the implication of Lrp in the regulation of the gene encoding the arginine exporter ( argO ) was reported . RI 21441513 375 Here , we found that Lrp binds to several sites at the lysP control region and is able to potentiate the transcriptional activation mediated by ArgP when lysine becomes limiting . RI 21441513 376 Considering the in vivo and in vitro data presented in this work , the mechanism by which ArgP regulates argO transcription ( 24 ) , and the known capacity of Lrp to alter the shape of the DNA by inducing bending and wrapping ( 57 ) , a model for lysP regulation in which binding of Lrp to the lysP control region may favor and / or stabilize the ArgP - RNA polymerase - DNA complexes or introduce DNA conformational changes is conceivable . RI 21441513 379 According to the results described here , Lrp participates in the activation of cadBA under inducing conditions . RI 21441513 625 CadC - mediated activation of the cadBA promoter in Escherichia coli . RI 21441513 650 The H - NS protein modulates the activation of the ilvIH operon of Escherichia coli K12 by Lrp , the leucine regulatory protein . RI 21441513 747 Competitive activation of the Escherichia coli argO gene coding for an arginine exporter by the transcriptional regulators Lrp and ArgP . RI 15066032 4 Pellegrini Molecular Microbiology ( 2004 ) 52 ( 2 ) , 437 – 449 doi : 10.1111 / j . 1365 - 2958.2004.03986 . x Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors : NagC , ChbR and CAP Jacqueline Plumbridge * and Olivier Pellegrini Institut de Biologie Physico - Chimique ( CNRS UPR9073 ) , 13 , rue Pierre et Marie Curie , 75005 Paris , France . RI 15066032 7 Expression of the chb operon is repressed by NagC , which regulates genes involved in amino sugar metabolism . RI 15066032 15 The need for two specific inducing signals , one for ChbR to activate the expression of the operon and a second for NagC to relieve its repression , ensure that the chb operon is only induced when there is sufficient flux through the combined chb - nag metabolic pathway to activate expression of both the chb and nag operons . RI 15066032 110 This shows that whereas both NagC and ChbR appear to act as repressors for the chbB operon in the absence of the inducing sugar chitobiose , ChbR is necessary for induction and so behaves as a dual - function repressor – activator . RI 15066032 128 We can deduce that cellobiose - 6P is capable of generating an inducing signal for ChbR but not an inducing signal for NagC and that induction of both regulators is necessary for derepressed expression of the chb operon . RI 15066032 133 Thus the role of CAP is to act in concert with ChbR to activate expression of the chb operon . RI 15066032 172 Repression by NagC and ChbR in vivo A plasmid overexpressing ChbR produces very strong repression of the chbB – lacZ fusion in a wild - type strain ( 37 - fold ) , whereas a plasmid overexpressing NagC produces less than twofold repression ( Table 5 ) . RI 15066032 173 In a nagC strain , the threefold increase in chbB – lacZ expression compared with the wild type is , as expected , eliminated by the NagC plasmid . RI 15066032 177 So there is no strong repression of chbB – lacZ by plasmid - encoded NagC . RI 15066032 179 Effect of excess ChbR and NagC on repression of the chbB – lacZ fusion . wild type nagC chbR pTZ ( NagC ) 48 ± 6 47 ± 3 83 ± 4 pTZ ( Mlc ) 42 ± 4 62 ± 3 56 ± 4 pTZ ( ChbR ) 2.3 ± 0.2 32 ± 2 2.8 ± 0.5 pTZ control . RI 15066032 183 Results are the mean of two independent cultures . © 2004 Blackwell Publishing Ltd , Molecular Microbiology , 52 , 437 – 449Regulation of the chbB promoter 443 represses weakly by itself and strongly in the presence of nagC + , implying that part of the repression by ChbR involves enhancing the binding of NagC to its promoter proximal site . RI 15066032 225 Although these results are consistent with the requirement for CAP and ChbR for chbB activation there is a significant difference between the in vivo and in vitro data . RI 15066032 232 Discussion The results described here show that three transcription factors , ChbR , NagC and cAMP / CAP , cooperate in the © 2004 Blackwell Publishing Ltd , Molecular Microbiology , 52 , 437 – 449Regulation of the chbB promoter 445 repression and induction of the chbBCARFG operon in E . coli . RI 15066032 276 Compared with other operons regulated by NagC , nagE - BACD and glmUS , it is very likely that the repression complex involves an interaction between NagC bound to its two sites , NagC1 and NagC2 , so that a DNA loop is formed . RI 15066032 281 NagC is required for full repression of the chbB operon and it serves to limit induction when the supply of chitobiose is low . RI 15066032 289 For example , the galETK operon is regulated by three factors GalR , CAP and the nucleoid - associated protein HU . RI 15066032 291 Another example is the NarL / NarP - and FNR - stimulated expression of nirB and nrf genes , which are repressed by Fis and IHF ( Browning et al . , 2002 ) . RI 15066032 305 The NagC - regulated nagB – lacZ fusion has been described previously ( Plumbridge and Kolb , 1998 ) . RI 15066032 477 ( 2001 ) DNA binding sites for the Mlc and NagC proteins : regulation of nagE , encoding the N - acetylglucosamine specific transporter in Escherichia coli . RI 18028317 9 Introduction of chbR from two Cel + mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose , in reporter strains carrying disruptions of the chromosomal chbR and nagC . RI 18028317 28 It has been shown that the three proteins ChbR , CRP and NagC regulate the expression of the chb operon . RI 18028317 70 Many Cel + strains carry mutations in nagC The NagC repressor that regulates the nag operon involved in N - acetylglucosamine metabolism was also shown to regulate the chb operon ( Plumbridge and Pellegrini , 2004 ) . RI 18028317 247 The derepression of the chb operon due to the loss of NagC regulation is still not sufficient to confer a Cel + phenotype . RI 18028317 411 ( 2004 ) Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors : NagC , ChbR and CAP . RI 10601201 14 We show that the mutant CRP constitutively activates transcription from both the IS5 - disrupted and the wild - type fucPIK promoters , and we identify the CRP - binding site that is required for this activity . RI 10601201 15 Our results show that the fucPIK promoter , a complex promoter which ordinarily depends on both CRP and the fucose - specific regulator FucR for its activation , can be activated in the absence of FucR by a mutant CRP that uses three , rather than two , activating regions to contact RNA polymerase . RI 10601201 184 These findings indicate that , whereas the binding of wild - type CRP to site 1 is not sufficient to activate transcription from the fucPIK promoter , binding of CRP K52N is sufficient , at least for the IS5 - disrupted promoter . RI 10601201 204 This suggests that at the complex fucPIK promoter , any interactions between CRP and the - CTD do not contribute significantly to promoter activation . RI 10601201 439 Transcriptional co - activation at the ansB promoters : involvement of the activating regions of CRP and FNR when bound in tandem . RI 15659677 23 P1 drives cotranscription of edd and eda and is induced by growth on gluconate ( 7 ) ; P1 appears to be controlled by GntR ( 20 , 21 , 24 ) , although binding of GntR to the P1 regulatory region was not established prior to this work . RI 15659677 217 These data indicate that eda P2 has a high basal level of transcription , is induced by glucuronate , and is repressed by KdgR . RI 15659677 225 PhoB - dependent regulation of P4 . RI 15659677 269 CsrA appears not to affect eda transcript stability but appears to activate eda , since eda transcription was 1.6 - to 1.8 - fold lower in a csrA background . RI 15659677 273 CsrA appears to activate eda expression but does so by an unknown mechanism . RI 15659677 524 Positive and negative transcriptional regulation of the Escherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP ( cAMP ) - cAMP receptor protein complex . RI 9657988 125 Nystrom showed that induction of the RelA protein results in high expression of eda ( 47 ) . RI 9657988 150 Since gluconate is an intermediate of the L - idonate pathway , there must be cross talk with the GntR regulon which leads to induction of the ED pathway . RI 9657988 171 The dgo operon , encoding the enzymes of galactonate metabolism , is negatively regulated by the dgoR product and specifically induced by galactonate ( 19 ) . RI 14593252 1 Research Article J Mol Microbiol Biotechnol 2003 ; 6 : 41 – 56 DOI : 10.1159 / 000073407 Dual Control by Regulators , GntH and GntR , of the GntII Genes for Gluconate Metabolism in Escherichia coli Ryouichi Tsunedomi Hanae Izu Takuya Kawai Mamoru Yamada Department of Biological Chemistry , Faculty of Agriculture , Yamaguchi University , Yamaguchi , Japan Key Words Gluconate metabolism W Gntll W gntR W gntH W Divergent promoter W Expressional regulation Abstract Escherichia coli possesses two systems , GntI and GntII , for gluconate uptake and catabolism , whose genes are regulated by GntR as a repressor and GntH as an activator , respectively . RI 14593252 11 These genes for the GntI system have been extensively characterized and shown to be regulated negatively by GntR and positively by the cyclic AMP ( cAMP ) - cAMP receptor protein ( CRP ) complex [ Tong et al . , 1996 ; Izu et al . , 1997a , b ; Porco et al . , 1997 ; Peekhaus and Conway , 1998 ] . RI 14593252 20 Recently , we have uncovered a unique regulation that the activator of GntII genes , GntH , exerts negative control of GntI genes [ Tsunedomi et al . , 2003 ] . RI 14593252 30 In the present study , we have shown not only the positive regulation by GntH , but also a negative and positive regulation by GntR , a repressor for the GntI system , of the GntII genes . RI 14593252 35 In the first point , such a case has been reported in the galETK ( M ) operon concerning galactose utilization , which is regulated by GalR and GalS [ Weickert and Adhya , 1993a b ] , but its molecular mechanism is clearly different from that of the GntII gene regulation . RI 14593252 36 In the second point , there is also an example that AraC positively and negatively regulates the araBAD operon , the mechanism of which might be similar to some extent to that of the GntII regulation . RI 14593252 101 This stimulates us to examine the possibility that GntR is also involved in the expressional regulation of the GntII genes . RI 14593252 105 These results suggest that GntR negatively regulates the expression of the GntII genes . RI 14593252 194 Discussion In the previous study , we found the cross - regulation between GntI and GntII systems through GntH as a positive regulator for the GntII genes , which negatively controls the expression of GntI genes [ Tsunedomi et al . , 2003 ] . RI 14593252 195 The negative regulation by GntH may allow the GntI gene expression to cease after the maximum expression level and its positive regulation causes induction of the GntII genes . RI 14593252 219 This and the finding that expression of gntV - lacZ fusion was relatively high even in the absence of cAMP ( table 4 ) may suggest that binding of GntR to all 3 sites slightly activates gntV expression . RI 14593252 234 General de Bibliotecas 198.143.33.17 - 1 / 11 / 2016 5 : 09 : 44 PMand then the GntII genes become induced by GntR bound to HRII followed by GntH as described above . RI 14593252 235 GntH expressed as a result represses the expression of the GntI genes as proposed [ Tsunedomi et al . , 2003 ] . RI 14593252 237 The data obtained thus revealed the results after induction by GntH of the GntII genes . RI 14593252 400 Positive and negative transcriptional regulation of the Escherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP ( cAMP ) - cAMP receptor protein complex . RI 14593252 447 The activator of GntII genes for gluconate metabolism , GntH , exerts negative control of GntR - regulated GntI genes in Escherichia coli . RI 18346968 2 36 , No . 8 2667 – 2676 doi : 10.1093 / nar / gkn119 Autoregulation of the Escherichia coli melR promoter : repression involves four molecules of MelR Shivanthi Samarasinghe 1 , Mohamed Samir El - Robh 1 , David C . RI 18346968 6 Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites . RI 18346968 16 MelR is a member of the AraC - XylS family of bacterial gene regulatory proteins ( 6 ) and our previous studies have shown that MelR , together with the cyclic AMP receptor protein , CRP , regulates expression of the melAB operon that encodes products essential for melibiose metabolism ( 7 ) . RI 18346968 27 The melR promoter and its repression by MelR . RI 18346968 38 Hence , in this work , we used in vivo and in vitro methods to investigate the determinants needed for MelR - dependent repression of the melR promoter . RI 18346968 76 Thus , the melR promoter is not efficiently repressed by MelR , and MelR is over - expressed . RI 18346968 106 To measure MelR - dependent repression of melR promoter activity , the pRW50 derivatives carrying the TB20 , TB22 , TB23 or TB10 fragments were transformed into WAM1321 melR lac cells containing either pJW15 , encoding melR , or empty control vector , pJWmelR and b - galactosidase activities were measured . RI 18346968 109 In contrast , and in full agreement with the previous results of Wade et al . ( 17 ) , the longer deletion in the TB23 fragment , that removes MelR binding site 2 , results in a sharp reduction in the repression of the melR promoter by MelR . RI 18346968 111 Next , we attempted to reproduce MelR - dependent repression of the melR promoter in vitro . RI 18346968 117 The experiment illustrated in Figure 2 shows that MelR has little or no effect on the synthesis of RNA - 1 but represses synthesis of the transcript from the melR promoter . RI 18346968 119 Orientation of MelR bound at site R Wade et al . ( 17 ) showed that MelR - binding site R is essential for MelR - dependent repression of the melR A MelR RNA - 1 B TB23 TB23 TB22 TB22 MelR + + % Relative Intensity 150 100 50 0 TB23 MelR + MelR TB22 Figure 2 . RI 18346968 130 MelR - dependent repression of the melR promoter carried by each derivative was then measured , as above . RI 18346968 156 Results in Figure 3C show that Val273 MelR gives a small enhancement in repression of the melR promoter carrying the 13T substitution in site R , B . RI 18346968 177 Results illustrated in Figure 5B show that MelR - dependent repression of the melR promoter is unaffected when CRP binding at this target is prevented . RI 18346968 180 This change confers partial CRP independence on the melR promoter ( data not shown ) and also causes a small reduction in MelR - dependent repression . RI 18346968 182 Results illustrated in Figure 5B show that the MelR - dependent repression of the melR promoter in the TB210 fragment is unchanged in the TB211 and TB222 derivatives . RI 18346968 197 Thus , TB201 is a derivative of TB20 in which the DNA site for CRP located between MelR site 1 Other determinants for MelR - dependent repression of the melR promoter To investigate the effects of altering the spacing between MelR - binding site R and the upstream sites , derivatives of the TB22 fragment with 5 - or 10 - bp deletions , or with 5 - , 41 - or 91 - bp insertions were constructed ( see Figure 6A and Materials and methods section ) . RI 18346968 198 MelR - dependent repression of the melR promoter in each of the new constructs was measured as above and the results are illustrated in Figure 6B . RI 18346968 201 In our previous study ( 17 ) , we reported that repression of the melR promoter by MelR was unaffected by point mutations in either site 1 or site 1 0 . RI 18346968 204 Results illustrated in Figure 6B show that MelR - dependent repression of the melR promoter in the TB28 fragment is reduced . RI 18346968 206 In vitro studies of MelR binding at the melR promoter Our results show that MelR - dependent repression of the melR promoter is contingent on MelR binding to site R and is somehow modulated by MelR binding to upstream sites 2 , 1 and 1 0 . RI 18346968 256 In the images shown in Figure 9 , the MelR focus is 15 33 nm across the shortest and longest axes of the oval in the complex with the shortened arm ( right hand panel ) , compared to 9 12 nm in the other complex R R DISCUSSION At the E . coli arabinose operon regulatory region , transcription is repressed by the binding of two AraC molecules at two targets that are in the same orientation and separated by 210 bp , and a repression loop forms ( 4,5 ) . RI 18346968 257 Our aim in this work was to ascertain if such a simple model applied to MelR - dependent repression of the melR promoter . RI 18346968 265 We previously suggested that a MelR dimer simultaneously occupies site R and site 2 , creating a repression loop and the effects of different insertions and deletions on MelR - dependent repression of the melR promoter ( Figure 6 ) are consistent with this ( 17 ) . RI 18346968 326 ( 2000 ) Transcription activation at the Escherichia coli melAB promoter : the role of MelR and the cyclic AMP receptor protein . RI 18346968 366 ( 2000 ) Repression of the Escherichia coli melR promoter by MelR : evidence that efficient repression requires the formation of a repression loop . RI 18346968 380 ( 2004 ) Transcription activation at the Escherichia coli melAB promoter : interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase s subunit . RI 18346968 452 ( 2004 ) Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors : NagC , ChbR and CAP . RI 10747919 8 MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions 120.5 , 100.5 , 62.5 , and 42.5 upstream of the transcript start point . RI 10747919 27 Recent studies ( 11 ) have shown that the occupation of site 2 by MelR is the most crucial for the activation of the melAB promoter , but that , of the four target sites , site 2 binds MelR most weakly . RI 10747919 29 This suggested that the increased melAB expression in the W3133 - 2 mutant could be due to better binding of MelR to site 2 . RI 10747919 146 The resulting recombinant plasmids were used to monitor MelR - dependent activation at the melAB promoter ; i . e . transcripts initiating at the melAB promoter run to the oop terminator and give discrete transcripts that are easy to detect by gel electrophoresis . RI 10747919 170 We suppose that this improvement in MelR binding is responsible for the increased melAB promoter activity in the W3133 - 2 mutant , although we cannot prove that it is solely responsible . RI 8010957 7 A melAB promoter fragment that is very weakly activated by the meiR gene product has been made by creating multiple symmetrical changes in both MelR - binding sites . RI 8010957 18 Here we report that most of the changes we studied had but small effects on MelR - dependent activation of pmelAB . RI 8010957 123 Cells carrying KK43 score as Lac ' on Maconkey indicator plates , due to MelR - dependent activation of pmelAB . RI 8010957 124 With KK433 , cells score as Lac - because MelR binding and pmelAB activation are reduced . RI 8010957 127 After screening 10000 colonies , we purified six independent pJW15 derivatives carrying simple substitutions in MelR that resulted in detectable activation of mutant pmelAB . RI 8010957 145 This shows that we had failed to identify MelR mutants with improved specificity for the changed KK433 MelR - binding sequences ; mutants with specificity for the KK433 sequence would have given lower levels of activation with the wild - type melAB promoter . RI 16621812 13 MelR carrying each of the single substitutions is less able to repress the melR promoter , while MelR carrying some combinations of substitutions is completely unable to repress the melR promoter . RI 16621812 14 These results argue that different conformational states of MelR are responsible for activation of the melAB promoter and repression of the melR promoter . RI 16621812 37 Thus , melibiose toggles MelR between a state where it represses pmelR and is unable to activate pmelAB to a state where pmelR is derepressed and pmelAB is activated . RI 16621812 86 Activities were used to measure MelR - dependent activation of pmelAB and MelR - dependent repression of pmelR . RI 16621812 94 We previously showed that MelR - dependent activation of pmelAB could be readily monitored using the low - copy - number , broadhost - range lac fusion plasmid pRW50 , carrying the KK43 pmelAB promoter fragment ( KK43 - pRW50 ) ( 2 ) . RI 16621812 100 Plasmid pJW15 was used to supply MelR to activate expression of the pmelAB : : lac fusion carried by KK43 - pRW50 . RI 16621812 145 With one exception , each of the different single MelR substitutions results in a small reduction in the MelR - dependent repression of pmelR . RI 16621812 169 However , this second screening step identified 11 pJW15 derivatives encoding MelR that were still able to repress pmelR , despite being defective in the activation of pmelAB . RI 16621812 182 We reasoned that the difference between the form of MelR unable to activate pmelAB but competent for repression of pmelR and the alternative form that can activate pmelAB but is unable to repress pmelR might be , in part , due to subunitsubunit interactions . RI 16621812 206 This shows that MelR carrying these substitutions has a reduced requirement for CRP to activate pmelAB . RI 16621812 216 AraC - dependent transcription regulation has been most studied at the araBAD and araC genes , which are expressed from divergent promoters , paraBAD and paraC , whose transcription start sites are separated by 166 bp . RI 16621812 249 Since it is likely that MelR - MelR interactions are important for both activation of pmelAB and repression of pmelR , we suppose that that differences recorded in Table 5 are due to differences in these interactions . RI 16621812 290 Transcription activation at the Escherichia coli melAB promoter : the role of MelR and the cyclic AMP receptor protein . RI 16621812 379 Transcription activation at the Escherichia coli melAB promoter : interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase sigma subunit . RI 16621812 584 Repression of the Escherichia coli melR promoter by MelR : evidence that efficient repression requires the formation of a repression loop . RI 10760178 1 Molecular Microbiology ( 2000 ) 36 ( 1 ) , 211 ± 222 Transcription activation at the Escherichia coli melAB promoter : the role of MelR and the cyclic AMP receptor protein Tamara A . RI 10760178 13 These studies also show that the cyclic AMP receptor protein ( CRP ) interacts with the melAB promoter and increases MelR - dependent transcription activation . RI 10760178 25 Optimal expression of the melAB promoter requires Sites 1 0 , Site 1 , Site 2 and Site 2 0 ; CRP acts as a ` bridge ' between MelR bound at Sites 1 0 and 1 and at Sites 2 and 2 0 , increasing expression from the melAB promoter . RI 10760178 33 Interestingly , AraC - dependent transcription initiation at the araBAD promoter is increased by the cyclic AMP receptor protein ( CRP ) , a well - characterized global regulator , controlled by cyclic AMP ( cAMP ) , that activates the expression of a number of genes in response to a variety of stresses , including glucose starvation ( reviewed by Kolb et al . , 1993 ) . RI 10760178 43 In order to advance our understanding of the AraC family , we have focussed on the E . coli melibiose operon , which is regulated by MelR , a close homologue of AraC . RI 10760178 49 In subsequent work ( Williams et al . , 1994 ) , we were able to prove that MelR binding to both these sites ( called Site 1 and Site 2 ; see Fig . 1A ) is necessary for melibiose - dependent activation of the melAB promoter in vivo . RI 10760178 62 The horizontal shaded boxes denote 22 bp DNA sites for CRP : one site centred at position 2195.5 is responsible for the activation of the melR promoter , while the other , centred at position 281.5 , is discussed in this paper . RI 10760178 66 Q 2000 Blackwell Science Ltd , Molecular Microbiology , 36 , 211 ± 222Activation at the E . coli melAB promoter 213 in vitro and that this activation is dependent on MelR and melibiose . RI 10760178 71 Further studies showed that the melR promoter is a typical Class II CRPdependent promoter , activated by a single dimer of CRP , that binds to a 22 bp DNA site centred at position 241.5 with respect to the transcription start point ( reviewed by Busby and Ebright , 1997 ) . RI 10760178 98 In a second set of experiments , we used the in vitro transcription assay to investigate whether CRP affected activation at the melAB promoter . RI 10760178 167 The role of different MelR sites in activation of the melAB promoter The above footprinting experiments suggest that five MelR molecules can bind upstream of the melAB transcript start Fig . 6 . RI 10760178 186 Strikingly , all induction is lost with the JK14 fragment , showing that MelR binding to Site 1 and Site 1 0 is essential for activation of the melAB promoter . RI 10760178 202 Role of CRP in activation of the melAB promoter The results from the in vitro studies reported above suggest that CRP interacts directly at the melAB promoter . RI 10760178 216 Conclusions In this paper , we have reported an in vitro system for the study of transcription activation of the E . coli melAB promoter by MelR . RI 10760178 222 Note that the magnitude of the effect of CRP on activation of the melAB promoter in the in vitro transcription experiment ( Fig . 2B ) is less than might have been expected from the footprinting and in vivo experiments ( Fig . 4 and Table 1 ) . RI 10760178 235 Q 2000 Blackwell Science Ltd , Molecular Microbiology , 36 , 211 ± 222Activation at the E . coli melAB promoter 219 which transcription activation at the melAB promoter is absolutely dependent on the occupation of Site 2 0 by MelR . RI 10760178 264 Model for transcription activation at the melAB promoter . In the absence of melibiose , MelR subunits ( M ) occupy Site 1 0 , Site 1 and Site 2 and the CRP dimer is recruited to the DNA between Site 1 and Site 2 . RI 12675795 16 In order to simplify our analysis , we exploited an engineered derivative of the melAB promoter in which MelR binding to site 2 and site 2 ¢ , in the absence of CRP , is sufficient for transcription activation . RI 12675795 42 MelRdependent activation of the melAB promoter requires the binding of MelR subunits to four 18 bp sites and the binding of a single CRP dimer to a low - affinity target sequence ( Belyaeva et al . , 2000 ; Wade et al . , 2001 ; see Fig . 1A ) . RI 12675795 179 Measurements of b - galactosidase activities in the resulting transformants indicated that none of the single base changes in MelR binding site 2 significantly affected MelR - dependent activation of the JK22 melAB promoter ( data not shown ) . RI 12675795 247 For example , with AraC at the araBAD promoter , a combination of genetics and biochemistry has shown that the binding orientation is such that the C - terminally located HTH motif is adjacent to the promoter - 35 element ( Niland et al . , 1996 ) , and that , during activation , AraC binds to two adjacent sites as a direct repeat ( Reeder and Schleif , 1993 ) . RI 12675795 262 Figure 10 illustrates the likely organization of MelR subunits during transcription activation at the melAB pro - © 2003 Blackwell Publishing Ltd , Molecular Microbiology , 48 , 335 – 348MelR binding at the E . coli melAB promoter 345 Fig . 10 . RI 12675795 263 Model for MelR and CRP binding during activation at the melAB promoter . RI 12675795 337 ( 2000 ) Transcription activation at the Escherichia coli melAB promoter : the role of MelR and the cyclic AMP receptor protein . RI 10760179 1 Molecular Microbiology ( 2000 ) 36 ( 1 ) , 223 ± 229 Repression of the Escherichia coli melR promoter by MelR : evidence that efficient repression requires the formation of a repression loop Joseph T . RI 10760179 29 This results in the formation of a repression loop and the repression of the araC promoter by AraC ( reviewed by Schleif , 1996 ) . RI 10760179 31 MelR is essential for induction of the melAB operon that is responsible for melibiose metabolism . RI 10760179 51 We show that the melR promoter is repressed by MelR and that this autoregulation requires MelR binding to Site R . RI 10760179 70 The shaded boxes denote 22 bp DNA sites for CRP : one site centered at position 241.5 is responsible for the activation of the melR promoter , while the other , centered at position 2155.5 is involved in activation of the melAB promoter . RI 10760179 83 However , cells that also contain pJW15 , encoding melR , score clearly as Lac minus , due to the MelR - dependent repression of the melR promoter ( i . e . they give pale pink ± white colonies ) . RI 10760179 87 We reasoned that MelR - dependent repression of the melR promoter must be less efficient in these mutants . RI 10760179 92 From this , we conclude that the MelR - binding Site R plays an essential role in MelR - dependent repression of the melR promoter . RI 10760179 112 The figure also shows the measured amount of MelR - dependent repression of the melR promoter on the KK81 fragment carrying the different Site R substitutions . RI 10760179 119 Optimal MelR - dependent repression of the melR promoter depends on upstream sequences Transcription initiation of the melR promoter is dependent on the binding of CRP to a 22 bp DNA site centered at position 241.5 , but sequences further upstream are not essential ( Webster et al . , 1988 ) . RI 10760179 122 The results shown in Fig . 4 indicate that , with the shorter KK101 fragment , repression of the melR promoter by MelR differs from the repression found with the longer KK81 fragment . RI 10760179 126 We reasoned that some part of the melAB promoter might be responsible for the high level of MelRdependent repression of the melR promoter seen with the KK81 fragment . RI 10760179 130 Results in Fig . 5 show that MelR - dependent repression of the melR promoter is little altered by the changes in Site 1 ( KK81S3 ) or Site 1 0 ( KK81S4 ) . RI 10760179 133 Thus , when MelR - binding Site 2 is altered in the KK81 fragment , the pattern of MelRdependent repression of the melR promoter is similar to that seen with the shorter KK101 fragment ( Fig . 4 ) . RI 10760179 136 Repression of the melR promoter carried on different KK81 fragments . The figure shows the degree of MelR - dependent repression of the melR promoter carried on the starting KK81 fragment and the mutant derivatives , S2 ± S5 . RI 10760179 144 However , the situation is not so simple , and , in the absence of melibiose , efficient repression requires DNA sequences upstream of the melR promoter ; in particular , MelRbinding Site 2 is required . RI 10760179 147 Together with the present results , this suggests a simple model ( illustrated in Fig . 6 ) to explain how MelR - dependent repression of the melR promoter is modulated by the elements at the upstream melAB promoter . RI 10760179 171 Model for MelR - dependent repression of the melR promoter . In the absence of melibiose , MelR subunits occupy Site R and Site 2 forming a repression loop . RI 10760179 183 Thus , the melR promoter is not efficiently repressed by MelR and MelR is overexpressed . RI 10760179 187 References Belyaeva , T . , Wade , J . , Webster , C . , Howard , V . , Thomas , M . , Hyde , E . , et al . ( 2000 ) Transcription activation at the Escherichia coli melAB promoter : the role of MelR and the cyclic AMP receptor protein . RI 11742992 8 We have identi ® ed residues within activating region 1 ( AR1 ) of CRP that are important in transcription activation of the melAB promoter . RI 11742992 21 CRP binding upstream of the E . coli malK promoter repositions three molecules of MalT on the DNA such that they are able to activate transcription ( Richet et al . , 1991 ) . RI 11742992 23 FNR can only activate transcription at the E . coli nirB promoter when the FIS and IHF repressors are removed by the binding of NarL or NarP ( Browning et al . , 2000 ) . RI 11742992 81 Therefore , we compared transcription activation by wild - type CRP and RH180 CRP at the wild - type melAB promoter and at the promoter containing the substitution at position ± 75 . RI 11742992 82 As expected , the RH180 substitution had little effect on CRPdependent activation of the wild - type melAB promoter . RI 11742992 110 Substitutions within CRP that affect transcription activation at the melAB promoter To identify the residues of CRP that are important in transcription activation at the melAB promoter , a library of randomly mutated crp genes was created using error - prone PCR . RI 11742992 136 Taken together , our results argue that residues Thr158 , Pro160 , Gln164 and Lys166 of CRP participate in transcription activation at the melAB promoter : these residues identify a surface of CRP that overlaps with AR1 ( Figure 4 ) . RI 11742992 140 Thus , we have investigated the involvement of aCTD in CRP - dependent activation at the melAB promoter by using a preparation of E . coli RNAP , reconstituted with truncated a subunits that lack the C - terminal 73 residues . RI 11742992 146 However , CRP is able to activate transcription from the melAB promoter with RNAP containing both fulllength and truncated a subunits . RI 11742992 147 The fact that the same level of CRP - dependent transcription activation is seen in vitro with both RNAP preparations argues that CRP activates transcription at the melAB promoter independently of aCTD . RI 11742992 149 In order to investigate further the role of residues in and around AR1 in transcription activation at the melAB promoter , we puri ® ed CRP carrying the TI158 substitution that improves CRP - dependent activation of the melAB promoter in vivo . RI 11742992 162 MelR and CRP interact at the melAB promoter to activate transcription , but this interaction can be exploited to repress transcription ( Figure 3 ) . RI 11742992 166 Similarly , at the melAB promoter , activation by CRP appears to be dependent on direct protein ± protein interactions with MelR . RI 11742992 172 In conclusion , MelR and CRP bind to the melAB promoter co - operatively to adjacent sites in order to activate transcription in a co - dependent manner . RI 11742992 225 ( 2000 ) Transcription activation at the Escherichia coli melAB promoter : the role of MelR and the cyclic AMP receptor protein . RI 11742992 326 ( 1995 ) Transcriptional co - activation at the ansB promoters : involvement of the activating regions of CRP and FNR when bound in tandem . RI 8729576 43 This investigation reveals that the similarity sufficient to guarantee homology is hardly found between the regulatory protein and the regulated protein , except for the relation between the repressor RbsR and the regulated operon rbsDACBK , where RbsR has been indicated to show considerable similarity to the ribose binding protein RbsB ( Mauzy & Hermodson , 1992 ) , and for some of the regulatory proteins that are known to be self - regulated at the level of transcription . RI 8729576 199 In spite of such high similarity , Fnr is associated not with the transport system but with the switching of respiration styles ; i . e . under the anaerobic condition , Fnr activates the transcription of anaerobic enzyme genes , repressing the transcription of oxidase gene ( Spiro & Guest , 1991 ) , together with ArcA , one of the response regulators in Group 1 , that represses the transcription of aerobic respiration enzyme genes ( Iuchi et al . , 1990 ; Iuchi & Lin , 1993 ) . RI 8729576 201 ( iii ) Most of the sugar transport operons , whose transcription is activated by CRP , are also under the control of repression by other transcriptional regulatory proteins . RI 8729576 206 Some operons such as malEFG are under the transcriptional activation by the response regulators in Group 1 as well as by CRP , suggesting that they can be transcribed in response to the sensing of respective sugars regardless of the absence or presence of cAMP - CRP . RI 8729576 217 In practice , considerable similarity between the regulatory protein and the regulated protein is only seen in the regulatory protein RbsR and a periplasmic component RbsB of HAT , which is encoded by the operon rbsDABCK under the repression by RbsR . RI 8729576 819 Characterization of OmpR binding sequences in the upstream region of the ompF promoter essential for transcriptional activation . RI 9484892 9 Plasmids overproducing either the NagC protein or the Mlc protein repress the expression of manX , but the effect of the Mlc protein is stronger . RI 9484892 158 A plasmid carrying the mlc gene expressed from its own promoter ( pMlc2 , a gift from S . Ichihara ) produced a 10 - fold repression of the manX – lacZ fusion , while a plasmid in which Mlc was under the control of the lac promoter , pTZ ( Mlc ) , reduced expression of the fusion to less than measurable values ( greater than 100 - fold ; Table 5 ) . RI 9484892 217 The repression of the manXYZ operon by a plasmid expressing NagC is in agreement with the original observations of Vogler and Lengeler ( 1989 ) . RI 9484892 242 Similarly , the homologous NarL and NarP proteins of the nitrate regulon bind to the same sites but regulate differentially ( Darwin et al . , 1997 ) . RI 15743943 9 nanC expression is also activated by the regulators cyclic AMP - catabolite activator protein , OmpR , and CpxR . RI 15743943 47 We show in this work that yjhA is indeed regulated by NanR and several other regulators and that it encodes a Neu5Ac - specific channel . RI 15743943 111 nanC expression was predicted to be regulated by the two - component regulatory system CpxR - CpxA by transcriptome analysis ( 16 ) . RI 15743943 220 However , it is clear that they allow coordination of nanC and fimB expression , since NanR and NagC repress one and activate the other of these divergently arranged genes . RI 15743943 222 Two other regulators , OmpR and CpxR , activate nanC expression , although a direct control has not been proven . RI 9469834 53 This is particularly true in the case of nagB , but activation of nagE requires in addition protein – protein interactions between CAP and the RNA polymerase machinery . RI 9469834 78 The mutation CAP + 6 puts the CAP site at a non - functional distance for nagE ( – 67.5 ) , while the CAP – 6 mutation puts the CAP site at – 65.5 for nagB , also expected to be non - functional for cAMP / CAP activation ( 37,38 ) . RI 9469834 92 The greater expression of nagB in the CAP + 6 context than in the CAP – 6 case correlates with the fact that activation by CAP is still possible with the CAP + 6 mutation ( the CAP site is still at – 71.5 ) but not at CAP – 6 , where the CAP site is now located at – 66.5 , i . e . on the other side of the DNA helix compared with RNA polymerase ( 5,18 ) . RI 9469834 93 There is no obvious effect of the CAP + 6 mutation on nagE expression but this is presumably due to two opposing effects which are of similar magnitude : an 10 - to 15 - fold increase in nagE expression due to loss of repressor binding ( equivalent to that seen for the nagB fusion ) and a 10 - to 15 - fold decrease in nagE expression due to a lack of cAMP / CAP activation ( 5 ; see also below ) . RI 9469834 162 This idea seemed a real possibility as similar contacts have been detected in the CytR – cAMP / CAP repression complex at deoP1 ( 44 ; reviewed in 17 ) . RI 9469834 201 Despite the fact that the residual activity of the CAP pc proteins at the two promoters is different in the induced and uninduced conditions , there is a clear qualitative difference in the response of nagE and nagB to the mutant CAPs : 50 – 100 % activation at nagB and maximally 20 % activation at nagE . RI 9469834 203 Since the same level of activation is seen with the truncated single operator site nagE – lacZ fusion it implies that no DNA important for CAP stimulation of nagE – lacZ expression is situated beyond – 100 . RI 9469834 210 This was indeed suggested by Détiollaz et al . ( 49 ) on the basis that IHF sites replacing CAP sites allowed activation of the malT promoter , albeit less than with CAP . RI 9469834 227 Although the role of CAP in repression of nagE – B is likely to be purely architectural , it is clear that a major part of the CAP activation of nagE is due to protein – protein contacts between CAP and RNA polymerase . RI 24593230 13 NagC repression is critical in maintaining chiP mRNA levels low enough , relative to ChiX , to allow full silencing by this sRNA . RI 24593230 44 In addition , the repression of chiP translation by ChiX induces premature Rho - dependent transcription termination within the chiP gene , preventing expression of the chiQ gene ( encoding a putative lipoprotein of unknown function ) located immediately downstream of chiP and normally cotranscribed with the latter ( Bossi et al . , 2012 ) . RI 24593230 47 As the chb transcript accumulates , ChiX pairing with the intercistronic chbBC mRNA elicits the degradation of the sRNA resulting in the progressive relief of chiP repression ( Figueroa - Bossi et al . , 2009 ; Overgaard et al . , 2009 ) . RI 24593230 152 Translation of the chiP mRNA is repressed by base - pairing with the ChiX sRNA . RI 24593230 161 The latter step is critical for the initiation of the entire induction cascade , as the chbBCARFG operon itself is repressed by NagC ( Plumbridge and Pellegrini , 2004 ) . RI 24593230 197 First , expression of the inner membrane transporter chbCBA is activated directly by the ChbR protein , which allows entry of Chb - 6P and generation of the specific ChbR - inducing signal ( by action of ChbG enzyme ; Verma and Mahadevan , 2012 ) . RI 24593230 198 Activation of chbBCA expression by ChbR is essential to relieve ChiX repression of chiP . RI 1629153 3 174 , No . 15 Localization of Upstream Sequence Elements Required for Nitrate and Anaerobic Induction of fdn ( Formate Dehydrogenase - N ) Operon Expression in Escherichia coli K - 12 JING LI ' AND VALLEY STEWART ' 2 * Sections of Microbiology ' and Genetics & Development , 2 Cornell University , Ithaca , New York 14853 - 8101 Received 25 March 1992 / Accepted 19 May 1992 Two transcriptional activators , the FNR and NARL proteins , are required for induction of the fdnGHI operon , encoding Escherichia coli formate dehydrogenase - N . RI 11929525 8 Summary Expression from the Escherichia coli nrfA promoter ( pnrfA ) is activated by both the FNR protein ( an anaerobically triggered transcription activator ) and the NarL or NarP proteins ( transcription activators triggered by nitrite and nitrate ) . RI 11929525 15 Both Fis and IHF repress in vivo expression from pacsP1 , but have smaller repressive effects on expression from pnrfA . RI 11929525 35 At the nirB promoter ( pnirB ) , it has been shown that IHF and Fis drive the promoter DNA into an inhibitory architecture , which represses FNR - dependent transcription . RI 11929525 48 Thus , at pnirB , NarL and NarP activate transcription by an indirect mechanism , rather than by interacting directly with the transcription machinery ( Browning et al . , 2000 ) . RI 11929525 116 Similarly , incubation of the pnrf97 EcoRI – HindIII fragment with Fis binds to the nrfA – acs intergenic region and represses transcription At the E . coli nirB promoter , transcription initiation is repressed by the association of the nucleoid - associated factors , Fis and IHF ( Wu et al . , 1998 ; Browning et al . , 2000 ) . RI 11929525 299 For these reasons , we believe that NarL and NarP activate expression at pnrfA by a similar mechanism to that at pnirB , i . e . by counteracting the effects of repressive factors . RI 11929525 303 In relation to pacsP1 , the DNA site for Fis is centred at position – 61 , and its distal nature suggests that pacsP1 repression operates via a different mechanism . RI 11929525 414 ( 2000 ) Suppression of FNR - dependent transcription activation at the Escherichia coli nir promoter by Fis , IHF and H - NS : modulation of transcription by a complex nucleo - protein assembly . RI 11929525 466 ( 2000 ) FNR dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR - activating regions 1 and 3 . RI 9696769 16 This suggests that Fnr and NarP may act synergistically to activate napF operon expression . RI 9696769 54 The inability of NarL to activate napF operon expression led us to hypothesize that NarL is deficient in the mechanism by which NarP activates transcription from the 44.5 binding site ( 9 ) . RI 9696769 105 This Fnr ( D154A ) protein was able to activate ( napF - lacZ ) expression in vivo ( data not shown ) . RI 9696769 111 By analogy with Crp - dependent promoters ( 3 ) and synthetic Fnr - dependent promoters ( 29 , 30 ) , we hypothesized that the mechanism by which napF expression is activated by Fnr is distinct from the mechanism involved in the activation of most4194 DARWIN ET AL . J . RI 9696769 160 In a control experiment , the activity of the Fnr ( D154A ) protein was confirmed by its ability to activate transcription of the Fnr - dependent dmsA promoter in vitro ( data not shown ) . RI 9696769 170 It is possible that NarP and Fnr may activate napF transcription synergistically whereas Fnr and NarL cannot , as discussed below . RI 9696769 171 Note that in a control experiment , the activity of the MBP - NarL protein was confirmed by its ability to activate transcription of the NarL - dependent fdnG promoter in vitro ( data not shown ) . RI 9696769 184 However , the napF control region has a very different architecture , with an Fnr - binding site centered at position 64.5 and activation by NarP being mediated by a binding site downstream , at position 44.5 ( Fig . 1 ) . RI 9696769 185 In this study we confirmed the location of the Fnr - binding site at position 64.5 and supported the idea that the mechanism of Fnr - dependent activation of napF expression is distinct from that of promoters with the Fnr - binding site at position 41.5 . RI 9696769 207 The fact that most Fnrdependent promoters have the latter architecture may be due to the fact that many of them are also activated by the NarL protein ( e . g . , the narG , fdnG , nrfA , and nirB promoters ) . RI 9696769 209 The napF operon provides the only known example of a promoter activated by NarP but not NarL . RI 9696769 385 Upstream sequence elements required for NarL - mediated activation of transcription from the narGHJI promoter of Escherichia coli . RI 17720788 31 For the nirBDC and nrfABCDEFG operons , Fnr protein , bound near position 41.5 , activates transcription maximally only when phospho - NarL or - NarP protein is bound further upstream to block inhibition by other proteins ( 2 , 8 , 59 ) . RI 18227264 15 Thus , although the region of the ydhY – T promoter containing the ” 16 and + 15 heptamers was recognized by NarL in vitro , mutation of these heptamers did not affect NarL - mediated repression in vivo . RI 18227264 238 Thus , it appears that NarL and NarP adopt overlapping mechanisms to inhibit ydhY – T expression . RI 18227264 242 NarL - mediated repression of the frdA promoter is achieved by NarL binding over a large region centred near the transcription start site and including the FNR site ( Li et al . , 1994 ) . RI 18227264 243 Similarly , at the FNR - activated NarL - repressed dmsA promoter NarL protects a large region that includes the sites for both FNR and RNA polymerase binding ( Bearson et al . , 2002 ) . RI 18227264 244 Thus , NarL - mediated repression of the ydhY promoter follows the general pattern established by the frdA and dmsA promoters . RI 18227264 250 Nevertheless , in contrast to the ydhY – T promoter , where NarP - mediated repression requires a 7 - 2 - 7 site centred at + 10.5 , occupation of which will probably inhibit RNA polymerase binding , the fdnGHI promoter possesses a NarP - binding 7 - 2 - 7 site centred at 2104.5 that is thought to antagonize activation by NarL ( Wang & Gunsalus , 2003 ) . RI 18227264 252 The relative degrees of repression of the ydhY – lacZ fusion in the presence of nitrite ( approximately twofold ) or nitrate ( ~ 13 - fold ) suggest that NarL - mediated inhibition of FNR recruitment is a key component of the regulation of the ydhY – T promoter , and that NarP - mediated inhibition of RNA polymerase binding is partially overcome by the ability of FNR to recruit RNA polymerase . RI 22101843 163 Therefore , the induction of dcuB depends on functional DcuS / DcuR . RI 22101843 175 However , the inactivation of DcuS ( IMW553 ) led to a decrease of citC - lacZ expression by a factor of 1.5 , which could be due to interaction between CitA and DcuS . RI 22101843 176 A 3.4 - fold decrease of citC - lacZ induction was observed when DcuR was inactivated ( IMW554 ) . RI 22101843 177 The impact of DcuR on citC expression might be due to a transcriptional activation of citAB by DcuR , as discussed below . RI 11073923 8 To address the mechanisms of activation by CRP and the RNA polymerase - subunit C - terminal domain ( - CTD ) at rhaSR , we tested the effects of alanine substitutions in CRP activating regions 1 and 2 , overexpression of a truncated version of ( - 235 ) , and alanine substitutions throughout - CTD . RI 11073923 32 RhaR regulates transcription of rhaSR by binding promoter DNA spanning 32 to 82 relative to the rhaSR transcription start site . RI 11073923 35 Subsequent to rhaSR expression , RhaS binds DNA upstream of rhaBAD at 32 to 81 relative to the transcription start site to increase rhaBAD expression by approximately 1,000 - fold ( to 10 Miller units in single copy ) . RI 11073923 36 An additional 50 - fold activation of rhaBAD expression occurs when CRP occupies its binding site centered at 92.5 , which places CRP adjacent to RhaS ( 9 ) . RI 11073923 39 To explore the origin of the indirect effect , we tested whether CRP was involved in regulation of rhaSR expression . RI 11073923 131 RESULTS CRP both directly and indirectly activates rhaBAD . RI 11073923 132 During the course of our studies on CRP activation of the rhaBAD operon , we compared expression from various rhaBAD promoter fusions ( Fig . 1 ) in crp and crp strain backgrounds . RI 11073923 148 We hypothesize that CRP binding to some or all of these sites may influence transcription activation at rhaSR . RI 11073923 161 These results suggest that the DNA region between 90 and 128 is important for activation at rhaSR and that putative CRP sites 2 , 3 , and / or 4 may be functional CRP sites . RI 11073923 162 Deletion of crp ( Table 5 ) resulted in a level of expression from each fusion that was similar to the expression from ( rhaS - lacZ ) 90 in the crp strain background , supporting our hypothesis that full rhaSR activation requires CRP . RI 11073923 164 We assayed the plasmid - borne lacZ fusions carrying these mutations to determine whether either of these two CRP sites was required for rhaSR activation . RI 11073923 170 This suggests that CRP site 3 is responsible for at least part of the CRP activation of rhaSR expression . RI 11073923 190 Taken together , our results suggest that CRP site 3 is the major site required for CRP activation of rhaSR expression . RI 11073923 192 Since we now had evidence that CRP was a direct activator of rhaSR expression , we wished to determine whether AR1 and / or AR2 of CRP were necessary for this activation . RI 11073923 197 Interestingly , these same three CRP mutants had similar small activation defects at the divergent rhaBAD promoter ( 14 ) . RI 11073923 200 To more directly test for a role of - CTD in rhaSR activation , we assayed the effect of expressing a derivative of with the entire C - terminal domain deleted , - 235 ( 14 ) , on expression from several rhaSR promoter fusions . RI 11073923 218 To identify specific residues in - CTD that are involved in rhaSR activation , we assayed an - CTD plasmid library with independent alanine substitutions at each residue in - CTD ( 10 , 30 ) at the ( rhaS - lacZ ) 216 promoter fusion ( Fig . 3 ) . RI 11073923 227 Effects of - CTD alanine substitution mutants on rhaSR activation . RI 11073923 244 DISCUSSION CRP activates rhaSR from at least one newly identified binding site . RI 11073923 246 Although there are many moderate to weak matches to the consensus CRP - binding site sequence in the rhaSR - rhaBAD intergenic region , three of these , sites 2 through 4 , seemed most likely to directly influence rhaSR activation . RI 11073923 250 It is unlikely that CRP binding to site 4 contributes directly to an increase in rhaSR expression , since transcription activation by CRP requires that its binding site be on the same face of the DNA as the promoter ( 6 ) . RI 11073923 285 Mechanism of activation by CRP at rhaSR . RI 11073923 286 Clearly there is a component ( 13 - to 20 - fold ) to the activation by CRP at rhaSR that is independent of both - CTD and RhaR and hence does not function by the same mechanism used at simple class I CRP - dependent promoters , nor does it function through cooperative binding with RhaR . RI 11073923 288 This component of CRP activation at rhaSR could account for the majority of the CRP activation in a wild - type context and may involve a mechanism , such as DNA bending , that can act from a distance . RI 11073923 300 Role of - CTD in rhaSR activation . RI 11073923 307 Alternatively , - CTD residues 321 , 322 , and 323 were defective at a rhaBAD promoter fusion that was activated only by the RhaS protein . RI 11073923 442 Role of multiple CytR binding sites on cooperativity , competition , and induction at the Escherichia coli udp promoter . RI 11073923 558 Separate contributions of UhpA and CAP to activation of transcription of the uhpT promoter of Escherichia coli . RI 17513476 18 Given that cyclic AMP receptor protein ( CRP ) , the second activator required for full rhaBAD expression , cannot activate rhaBAD expression in a rhaS strain , it was of interest to test whether CRP could activate transcription in combination with RhaS - CTD . RI 17513476 24 RhaR activates transcription of the operon that encodes the two activator proteins , rhaSR ( 37 , 38 ) . RI 17513476 55 This domain alone could activate transcription of araBAD up to 15 % as well as full - length AraC when alone or to the same level as fulllength AraC when fused to an unrelated dimerization domain ( 7 , 36 ) . RI 17513476 188 DNA sequence inspection and previous results indicating that RhaS was required for activation of rhaT expression ( 40 ) suggested that RhaS binds to DNA at the rhaT promoter , but direct evidence of RhaS protein binding to rhaT promoter DNA had not been obtained . RI 17513476 202 Given that the activation by full - length , chromosomally expressed RhaS at rhaBAD is approximately 33 - fold higher than that of chromosomally expressed RhaR at rhaSR , comparable efficiencies of activation by the CTDs to their full - length counterparts would have resulted in His 6 - RhaR - CTD activating rhaSR by approximately 30 - fold . RI 17513476 222 Two gels were prepared , with identical culture samples on each gel , and each blot was probed with the primary antibody corresponding to the purified protein samples loaded , as indicated to the right . - S , anti - RhaS antibody ; - R , anti - RhaR antibody . allow CRP to contribute to rhaBAD activation . RI 17513476 233 Finally , at the ( rhaB - lacZ ) 110 fusion , which contains the CRP site required for full rhaBAD activation as well as the full RhaS binding site , there was a twofold contribution to activation by CRP when in combination with RhaS - CTD and a fivefold contribution to activation4990 WICKSTRUM ET AL . J . RI 17513476 264 ( B ) Transcription activation by RhaS expressed from the chromosome in SME1048 ( wild - type rhaSR ) . RI 17513476 279 We found that CRP alone was not capable of activating rhaBAD expression , similar to previous findings in vivo , and that His 6 - RhaS - CTD alone substantially activated rhaBAD expression ( Fig . 6 ) . RI 17513476 281 The threefold contribution to activation by CRP in this experiment is very similar to the twofold contribution found in the above in vivo experiment ( Table 3 ) , confirming that RhaS - CTD is sufficient to allow at least partial CRP activation of rhaBAD expression . RI 17513476 755 Cyclic AMP receptor protein and RhaR synergistically activate transcription from the L - rhamnose - responsive rhaSR promoter in Escherichia coli . RI 9393706 109 Thus , activation by Fis is dependent on the presence of MarA or Rob ( or induced levels of SoxS ; see below ) . RI 9393706 123 In a strain with a wild - type chromosomal marRAB operon , Rob activates the promoter , and therefore the rob + mar + strain produces more MarR repressor than the rob mar + strain . RI 9393706 125 For the marbox transversion mutants ( no . 7 and 8 ) which are insensitive to stimulation by Rob , the presence of Rob increases the transcription of the chromosomal marRAB promoter and therefore only increases the repression of the mutant fusions . RI 9393706 130 In a similar experiment with a rob + mar strain , excess MarA stimulated mar transcription of a wild - type mar : : lacZ fusion by only 1.6 - fold ( 27 ) . RI 9393706 136 This result again demonstrates that SoxS activation of the mar promoter is dependent on a functional marbox ( 27 ) . RI 9393706 140 We previously demonstrated that in vitro transcriptional activation by MarA at the zwf promoter requires interaction with the carboxy - terminal domain of the α subunit of RNA polymerase ( 21 ) and inferred that MarA activates the mar promoter by enhancing the binding of RNA polymerase ( 27 ) . RI 9393706 154 Whatever the mechanism for this apparent reduction in mar transcription by Fis , it cannot be the result of the specific repression of the mar operon , since the same reduction is observed whether or not the promoter is capable of binding Fis . RI 9393706 171 We have considered three mechanisms for the activation by Fis of the mar promoter . RI 9393706 196 Previously , overexpression of Rob was shown to induce multiple antibiotic and superoxide resistance phenotypes ( 3 ) , to activate expression of fumC , inaA , and sodA in vivo ( 3 ) , and to activate the transcription of six mar / soxRS regulon promoters in vitro by mechanisms that are strikingly similar to that of MarA and SoxS ( 22 ) . RI 9393706 218 The mar promoter is regulated by two independent mechanisms , transcriptional activation , depending on Fis and the MarA / Rob / SoxS activators , and repression due to MarR . RI 12791142 17 Thus , it is likely that E . coli is protecting itself by the Rob - mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract . RI 12791142 73 Presumably , uninduced levels of MarA and / or SoxS mediate a low - level induction of the inaA gene ; the lower basal level of inaA expression in Dmar strains was noted earlier ( Rosner and Slonczewski , 1994 ) . RI 12791142 121 The transcription of the marA gene is regulated by the repressor MarR ( Alekshun and Levy , 1997 ) . RI 10715008 6 JOHNSON AND ROBERT F . SCHLEIF * Department of Biology , Johns Hopkins University , Baltimore , Maryland 21218 Received 20 September 1999 / Accepted 12 January 2000 Full activation of transcription of the araFGH promoter , p FGH , requires both the catabolite activator protein ( CAP ) and AraC protein . RI 10715008 79 Only the CAP - proximal AraC site , araFGH1 , is required for normal activation of p FGH , and all upstream DNA can be deleted without significant effect . RI 10715008 182 Why , then , is CAP sufficient to activate the gal operon but not p FGH ? RI 10715008 407 Transcriptional co - activation at the ansB promoters : involvement of the activating regions of CRP and FNR when bound in tandem . RI 9358057 3 Primer extension and lacZ - operon fusion analyses revealed that gntT has one strong and two weak promoters , all of which are regulated positively by cAMP - CRP and negatively by GntR . RI 9358057 11 The gntR gene is constitutively expressed , gluconate permease , and a thermoresistant glu - whereas the gntKU genes are positively regulated by the cAMP – CRP complex and negatively by GntR , and gntU expression is further repressed by attenuation , * Corresponding author . RI 9358057 94 GntR thus seems to function as Inhibition of gluconate uptake by alternative sugars in Hfr G6MD2 a negative regulator of gntT by binding to the putative harboring pGNTTK sequence ( see below ) . RI 9358057 117 gntR gene ( Fig . 4A ) , suggesting that the promoters are However , no such reduction of bGal activity by GntR negatively regulated by GntR . RI 2182324 1 The EMBO Journal vol . 9 no . 4 pp . 973 - 979 , 1990 The three operators of the lac operon cooperate in repression Stefan Oehler , Elisabeth R . Eismann , Helmut Kramer1 and Benno Muller - Hill Institut fur Genetik der Universitiit zu Koin , Weyertal 121 , D 5000 Koin 41 , FRG ' Present address : Department of Biological Chemistry , UCLA , School of Medicine , Los Angeles , CA , USA Communicated by B . Muller - Hill We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor . RI 2182324 21 However , the hybrid mac promoters , containing only part of the lac promoter and the first lac operator , are just - 5 - fold repressed by Lac repressor ( Vidal - Ingigliari and Raibaud , 1985 ) . RI 2182324 28 We show that cooperativity between the three lac operators , mediated through tetrameric Lac repressor , is crucial for repression of the lac operon . ( Oxford University Press 973S . Oehler et al . Results We constructed a set of eight plasmids , each encompassing the lacZ gene under control of the natural lac promoter and the three lac operators ( 01 , 02 and 03 ) either active or inactivated by site directed mutagenesis ( Figure 1 ) in all possible combinations . RI 2182324 95 Discussion Full repression of the lac operon requires both 02 and 03 Kania and Muller - Hill ( 1977 ) concluded from studies with a Lac repressor - fl - galactosidase chimera that two repressor subunits are sufficient for operator binding and that therefore tetrameric Lac repressor must be able to bind two lac operator sequences at the same time . RI 2182324 148 To calculate the percentages of the various structures , we used the following information : ( i ) the wild - type lac operon is 1300 - fold repressed ; ( ii ) a lac operon in which 02 and 03 are destroyed is 18 - fold repressed , ( iii ) a lac operon in which O1 is destroyed is 2 - fold repressed , ( iv ) 02 and 03 have a 20 - and 100 - fold lower affinity for Lac repressor than O1 in vitro and ( v ) lac transcription is 50 - fold activated by CAP . RI 8626315 10 We found that Rob ( i ) activates the transcription of zwf , fpr , fumC , micF , nfo , and sodA , ( ii ) requires a 21 - bp soxbox - marbox - robbox sequence to activate zwf transcription , ( iii ) protects the soxbox / marbox / robbox from attack by DNase I , ( iv ) is ambidextrous , i . e . , requires the C - terminal domain of the α subunit of RNA polymerase for activation of zwf but not fumC or micF , ( v ) bends zwf and fumC DNA , and ( vi ) binds zwf and fumC DNA as a monomer . RI 8626315 24 Moreover , they found that Rob overexpression activates expression of fumC , sodA , and inaA ( unknown function ) , but not zwf , and that purified Rob forms DNA - protein complexes with DNA fragments containing the micF , sodA , nfo , and zwf promoters ( 3 ) . RI 8626315 79 Sufficiency of Rob for in vitro transcriptional activation of zwf , fpr , fumC , micF , nfo , and sodA , promoters known to be activated by MalE - SoxS and MarA . RI 8626315 81 Since we have shown previously that MalE - SoxS and MarA are sufficient for the in vitro transcriptional activation of the zwf , fpr , fumC , micF , nfo , and sodA promoters ( i . e . , additional factors are not required ) ( 18 , 19 ) , we conducted similar studies with purified Rob protein . RI 8626315 88 Coincidence of the sites for SoxS - and Rob - mediated activation of zwf transcription in vitro . RI 8626315 92 Recently , we demonstrated that activation of in vitro zwf transcription by MalE - SoxS ( 18 ) , and by MarA as well ( 19 ) , requires this same DNA binding element and that this soxbox / marbox can bring in vitro gnd transcription under control of these two activators ( 18 , 19 ) . RI 8626315 94 As with MalE - SoxS and MarA ( 18 , 19 ) , Rob stimulated in vitro transcription about threefold from the zwf promoter of templates Z1 , Z2 , Z3 , Z4 , and Z5 ( 9 ) , whose 5 ´ deletion end points reside at positions - 140 , - 86 , - 76 , - 67 , and - 62 , respectively ( Fig . 3A ) but failed to enhance transcription from construct Z6 , whose 5 ´ deletion end point lies at position - 57 ( 9 ) . RI 8626315 434 Auto - activation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli . RI 8522515 12 Like MalE - SoxS , MarA ( i ) activated the transcription of zwf , fpr , fumC , micF , nfo , and sodA ; ( ii ) required a 21 - bp " soxbox " sequence to activate zwf transcription ; and ( iii ) was " ambidextrous , " i . e . , required the C - terminal domain of the α subunit of RNA polymerase for activation of zwf but not fumC or micF . RI 8522515 75 To determine whether the purified MarA protein affected gene expression , we tested its ability to activate the in vitro transcription of zwf , sodA , micF , and fumC , genes known to be under mar control in vivo , and of fpr and nfo , genes not known to be regulated by mar in vivo but activated by SoxS in vivo and by MalE - SoxS in vitro . RI 8522515 110 Miller et al . ( 28 ) have found that SoxS activates mar transcription in vivo , and we have recently found a marbox sequence in the promoter region upstream of marRAB which binds MarA and enables either purified MalE - SoxS or MarA to activate transcription of marRAB in vitro ( 18 , 26 ) . RI 23010927 30 Expression of acrZ is coregulated with acrAB and tolC by the MarA , Rob , and SoxS transcription factors . RI 23010927 212 acrZ is regulated by MarA , Rob , and SoxS . RI 21097628 27 This is likely due to the greater extent of activation by SoxS of promoters involved in superoxide defense , e . g . , acnA , fpr , zwf , fumC , and sodA . RI 21097628 35 For example , MarA binds tightly to the marRAB promoter in vitro yet stimulates transcription by only ~ 3 - fold ; no significant binding to inaA can be demonstrated by gel shift experiments , yet transcription is stimulated > 5 - fold . RI 21097628 93 In every case , MarA ( E89A ) bound as well as or more tightly ( i . e . , had a lower K D [ equilibrium dissociation constant ) than WT MarA . For several promoter marboxes this change was dramatic : the K D dropped from > > 150 to 50 for inaA , from 200 to 75 for nfsB , and from > > 150 nM to ~ 100 nM for both ybjC and yhbW . In spite of this , MarA ( E89A ) activation was greater than that of WT MarA for inaA , sodA , and ybjC ; it was comparable to that of WT MarA for micF and less than that of WT MarA for fumC , mdaB , nfsB , and ybhW ( Table 3 and Fig . 1 ) . We conclude that the WT glutamic acid of MarA at position 89 is an inhibitor of MarA binding to many marboxes . In an effort to understand why E89 is inhibitory , we examined the sequences of the 14 marboxes for which we have data ( Table 3 ) . We noticed that five haveaTatposition 12 and that the binding to none of these sequences is increased by the E89A substitution ( less than 1.7 - fold ) . In contrast , of the remaining nine sequences that do not have aTatthis position , the binding of E89A was increased for six of them by > 2.5 - fold and for the seventh by 1.7 - fold ; only two do not show increased binding . Although the cocrystal structure of MarA with the marRAB marbox DNA ( with aTatposition 12 ) indicates no interaction between the two molecules at this position ( 36 ) , we considered the possibility that steric hindrance between the marbox DNA and MarA could limit activation by MarA when position 12 is not a T residue ( see the Discussion for a fuller treatment ) . To examine one facet of this possibility , namely , that the glutamic acid side chain of E89 sterically inhibits interaction with marbox DNAs lacking aTatposition 12 , we tested the effects of several amino acid substitutions at residue 89 on the fpr : : lacZ fusion and the mdtG : : lacZ fusion , the two fusions that showed the greatest effects of E89A on activation ( see above ) . Plasmids carrying WT MarA , the MarA variant MarA ( E89A ) ( with a nonpolar single methyl group side chain ) , MarA ( E89G ) ( with no side chain ) , MarA ( E89D ) ( with a side chain one methylene group shorter than glutamic acid ) , MarA ( E89V ) ( with the amino acid present at the corresponding position of SoxS and having a dimethyl methylene side chain , ) , WT SoxS , and SoxS ( V83E ) were introduced into these fusions and assayed for β - galactosidase . Again ( Table 2 ) , activation of these promoters by SoxS was much greater than that by MarA ( Fig . 3 ) . Similarly , MarA ( E89A ) was considerably more active than WT MarA . In contrast , the E89D variant was less active than WT MarA for fpr and completely inactive ( indistinguishable from the control plasmid ) for mdtG . E89G was approximately twice as active as WT MarA for fpr and 4 - fold more active for mdtG although for both promoters it was considerably less active than E89A . The E89V variant was marginally more active than the WT for both promoters but substantially less so than E89A . SoxS ( V83E ) reduced the activation of these promoters relative to WT SoxS , but SoxS ( V83E ) was still more active than WT MarA . ( The activations shown here are greater than those apparent in Table 2 because the expression of the plasmids carrying MarA , SoxS , and their mutants is not entirely shut off by lacI q so that the increases expressed in Table 2 appear smaller . ) As outlined in the Discussion , these results , namely , ( i ) that variant E89D is virtually inactive , ( ii ) that E89G is very active although to a lesser extent than E89A , ( iii ) that E89V is only marginally more active than the WT , and ( iv ) that SoxS ( V83E ) has reduced activation although not to the low levels expressed by WT MarA , are consistent with the possibility that that the side chain of E89 sterically inhibits interaction with the DNA for marbox sequences lacking aTat position 12 . If , as outlined above and presented in greater detail in the Discussion , steric interference with the phosphate between positions 12 and 13 and the glutamic acid side chain at position 89 is responsible for the very poor activation of promoters such as fpr , then it would be predicted that binding of WT MarA to the fpr marbox would be enhanced if that phosphate were absent . We therefore compared the binding affinity of WT MarA to either a 36 - bp double - stranded DNA containing the marbox sequence of fpr or with dsDNA of the same sequence and length but prepared so that the phosphate linkage between positions 12 and 13 of the marbox was eliminated ( see Materials and Methods ) . Again ( Fig . 2 and Table 3 ) , MarA bound very poorly to the fpr marbox ( Fig . 4 ) . However , binding increased significantly when the fpr DNA lacked the phosphate group between positions 12 and 13 ( Fig . 4A and B ) . In contrast , SoxS bound more tightly to dsDNA than to the discontinuous DNA ( Fig . 4B ) . When the phosphate located 3 nt farther upstream ( between positions 9 and 10 ) was absent , no significant alteration in binding was observed ( Fig . 4 ) although a small increase was observed when the phosphate between positions 15 and 16 was absent . We conclude that the phosphate group between nt 12 and 13 of the consensus sequence inhibits the ability of MarA to bind . Activation and marbox binding by MarA ( Q91A ) . The only other MarA mutation found here to differ significantly from WT MarA in the activation of class I promoters was Q91A ( Table 2 ) . Of the seven class I promoters examined , MarA ( Q91A ) activated the acrAB , marRAB , and zwf promoters to similar extents as WT MarA but activated acnA , fpr , mdtG , and poxB to only 60 % or less of MarA WT levels ( Table 2 ; see also below ) . For the nine class II promoters , MarA ( Q91A ) significantly reduced the activation of fumC , inaA , micF , pqiA , and ybjC but had no significant effect on nfsB , mdaB , sodA , oryhbW . MarA - ( Q91 ) has been identified as forming van der Waals interactions with the methyl groups of the two thymidines that are complementary to the adenines at positions 17 and 18 of the consensus sequence ( 36 ) . Thus , it would be expected that the Q91A substitution might reduce activation of the seven class II promoters that have at least one A at position 17 or 18 but not the two promoters , nfsB and sodA , that have no A residues at these positions . Indeed , nfsB and sodA are among the promoters that Q91A activated to the same extent as WT MarA . Since Q91A reduced the expression of four of the seven class I promoters and four of the nine class II promoters , our results are inconsistent with the proposition that Q91 is principally required for interactions at class II promoters , as has been proposed for the analogous site ( Q85 ) in SoxS ( 16 ) . For the majority of these promoters , the gel mobility assay for binding of MarA to marboxes was insufficiently precise to determine whether there is a correlation between binding and activation by MarA ( Q91A ) relative to MarA ( Table 3 ) . Among the class I promoters that exhibited measurable binding to MarA , MarA ( Q91A ) showed no greater binding or activation for acrAB , a modest reduction in activation and binding for mdtG and zwf , and a reduction in binding but not in activation for marRAB . A modest reduction in binding with a small increase in activation was seen for the class II mdaB promoter . Although not observed in these experiments , a more detailed analysis of the binding of MarA ( Q91A ) to the micF marbox ( using protein without the His 6 tag ) showed a very small reduction in binding concomitant with the reduced ability of MarA ( Q91A ) to activate the class II micF promoter ( 13 ) . Thus , relative to MarA , there may be a correlation between activation and binding for Q91A at class I promoters , but none is obvious with regard to the class II promoters . Activation of superoxide resistance by MarA ( E89A ) . If the glutamic acid at position 89 of MarA is a major determinant in vivo of the reduced activity of MarA at many promoters where SoxS is more active , we would expect cells carrying MarA ( E89A ) to be more resistant to superoxides than cells carrying WT MarA . This was tested with gradient plate assays of sensitivity to two superoxide - generating compounds , phenazine methosulfate ( PMS ) and menadione ( Table 4 ) . Cells constitutively expressing MarA ( E89A ) were more resistant than WT MarA to PMS ( 1.7 - fold ) and , to a lesser extent , to menadione ( 1.3 - fold ) . Comparable MICs with SoxS for PMS were 1.6 - fold and for menadione 2.0 - fold . Clearly , MarA ( E89A ) activates superoxide resistance to a greater extent than WT MarA . Curiously , the Q91A substitution had no effect on resistance to the superoxide generator PMS ( MIC of 35 μM for both the Q91A mutant and the WT MarA ) but lowered resistance to the superoxide generator menadione ( MIC of 0.9 mM for the Q91A mutant and 1.8 mM for the WT ) . DISCUSSION . The MarA - SoxS - Rob regulon of E . coli consists of a variety of genes that enable cells to adapt to multiple stresses . It contains genes that render the cell multidrug and organicsolvent resistant ( acrAB , tolC , and micF ) and that defend against superoxide stress ( e . g . , acnA , fpr , fumC , nfsA , sodA , and zwf ) ( 1 , 2 , 5 , 26 , 31 , 33 ) . Not surprisingly , there are quantitative differences in the extents of activation of particular promoters by the paralogous activators so that the phenotypic outcomes depend on which activator is upregulated ( 2 , 26 , 34 , 43 ) . E89 inhibition . We have shown here that the MarA glutamic acid residue E89 is responsible for decreasing the binding of WT MarA relative to SoxS for the class I marbox promoters , acnA , mdtG , fpr , and zwf , thereby decreasing the relative activation of these promoters and hence the resistance engendered to superoxides by MarA . We think the following may explain these results . A detail of the MarA structure ( Fig . 5 ) shows that E89 is oriented in the cocrystal with its acidic groups exposed to the solvent away from the DNA backbone . In so doing , methyl hydrogens on the β - and - carbons are positioned restrictively close to an oxygen of the phosphate group between bases T12 and G13 of the marRAB marbox . Even this orientation of E89 is only possible as the result of a small “ clash ” ( as predicted by the MolProbity program [ http : / / molprobity . biochem . duke . edu / ] ) between E89 and S90 that permits the rotation of E89 into the MarA core and away from the DNA backbone . RI 21097628 295 Transcription activation at the Escherichia coli melAB promoter : interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase sigma subunit . RI 18673442 7 Transcriptional activation by MarA , SoxS and Rob of two tolC promoters using one binding site : a complex promoter configuration for tolC in Escherichia coli . RI 18673442 77 Concomitant with upregulation of tolC , EvgA upregulated several multidrug resistance genes ( acrAB , emrKY , mdfA and yhiUV ) and showed increased antibiotic resistance that was fully dependent on TolC but only partially dependent on AcrA . RI 16478729 9 As for the hdeA and purA promoters , which are repressed by MarA , the rob marbox is also in the “ backward ” orientation . RI 16478729 21 Transposon mutagenesis experiments aimed at defining the Rob regulon revealed eight Rob - regulated targets , namely inaA , marRAB , aslB , ybaO , mdlA , yfhD , ybiS , and galT ( 10 ) , some of which are also known members of the mar and sox regulons ( 11 ) . RI 16478729 26 It has been shown previously that all three transcriptional factors ( MarA , SoxS , and Rob ) regulate the expression of themselves ( 16 , 17 ) and each other ( 15 – 18 ) . RI 16478729 28 Thus far , only the MarA / SoxS / Rob - binding site within the mar promoter has been identified and shown to be responsible for activation by all three transcription factors ( 17 ) . RI 16478729 29 In contrast , the repression of both soxS and rob by SoxS , Rob , or MarA occurs via a yet to be identified binding site ( s ) within the sox and rob promoter regions ( 15 , 16 ) . RI 16478729 106 Because previous studies have shown that the MarA - binding sites of two other repressed promoters ( hdeA and purA ) lie in a backward orientation ( 20 ) and either partially or completely overlap the - 35 hexamer , we examined whether the putative rob marbox might be in a similar configuration and position . RI 16478729 159 However , in the presence of MarA the rob transcript levels decreased over time , but as expected the levels of the gnd transcript ( the control ) remained unaffected ( Fig . 6C ) . RI 16478729 160 Thus MarA is able to repress the rob promoter after the preformed open complex clears the promoter . RI 16478729 169 This study demonstrates that MarA can repress the expression of the rob gene both in vivo and in vitro via a marbox sequence located within its promoter . RI 16478729 183 When RNAP was added to the rob promoter at 0 ° C prior to MarA addition and then transfer to 37 ° C , MarA inhibited rob transcription ( see Fig . 6B ) . RI 16478729 206 In contrast , in all three known examples of MarA - repressed genes ( hdeA , purA , and rob ) , the marbox is in the backward orientation and is proximal to or overlapping the RNAP - binding site ( see Fig . 8 ) . RI 11395452 29 The active ( oxidized or nitrosylated ) form of SoxR activates transcription of the soxS gene up to 100 - fold . RI 11395452 32 The known SoxS - activated genes are sodA ( encoding Mnsuperoxide dismutase ) , fpr ( NADPH - ferredoxin oxidoreductase ) , micF ( antisense RNA , repressor of OmpF translation ) , ribA ( cyclic GMP hydrolase ) , inaA ( unknown function ) , fldA and fldB ( flavodoxins A and B ) , nfo ( endonuclease IV ) , marRAB ( multiple - antibiotic - resistance operon ) , nfsA ( also called mdaA , a nitroreductase ) , zwf ( glucose - 6 - phosphate dehydrogenase ) , fur ( an iron - binding repressor of iron uptake ) , fumC ( fumarase C ) , acnA ( aconitase ) , tolC ( outer membrane protein ) , acrAB ( drug efflux pump ) , and rimK ( a modifier of ribosomal protein S6 ) . RI 11395452 44 While the soxS gene is under the redox - regulated , positive control of SoxR , marA is under negative control by MarR , a repressor whose DNA - binding activity is regulated by the binding of small molecules with toxic effects ( 1 , 31 , 39 ) . RI 11395452 136 Of the remaining seven SoxS - activated genes , five ( nfo , rimK , tolC , fldB , and mdaA ) were activated with values below threshold while one ( ribA ) appeared slightly repressed . RI 11395452 200 In addition , the PQ - or SoxSresponsive genes gatABD , gltA , nfnB , and ybjC were recently shown to be activated by constitutive expression of MarA ( 5 ) . RI 11395452 201 The ybjC gene was also activated by expression of SoxS in our experiments ( Table 1 ) . RI 11395452 246 Recently , a direct regulatory connection between oxidative stress and iron metabolism was shown by Zheng et al . , who demonstrated the transcriptional activation of fur by SoxS and OxyR ( 49 ) . RI 11395452 248 In addition to the increase in fur expression , superoxide stress resulted in the down - regulation of sodB , a Fur - activated gene ( 13 ) . RI 11395452 454 marA locus causes decreased expression of OmpF porin in multiple - antibiotic - resistant ( Mar ) mutants of Escherichia coli . RI 11395452 699 Auto - activation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli . RI 11321589 31 Expression of the EmrAB multidrug transport system of E . coli ( Lomovskaya and Lewis , 1992 ) is regulated by repressor EmrR ( Lomovskaya and Lewis , 1995 ) , encoded by the upstream gene and induced by various uncouplers of oxidative phosphorylation , salicylic acid and carbonyl cyanide m - chlorophenylhydrazone ( CCCP ) . RI 11321589 34 In addition to AcrAB , MarA also positively regulates expression of the outer membrane porine TolC which is required for functioning of the AcrAB efflux pump ( Aono et al . , 1998 ; Fralick et al . , 1997 ) . RI 11321589 38 Besides , MarA activates fumC , fpr , nfo and other genes possibly involved in response to the oxidative stress . RI 11321589 83 The first gene of the emrRAB operon encodes transcription factor EmrR that represses transcription of this operon . RI 11321589 89 This resembles the known co - regulation of acrAB and tolC by the transcriptional factor MarA . RI 10802742 103 Recent genetic data show that Rob represses transcription of the galT gene ( M . H . J . B . and B . D . , unpublished results ) . RI 10802742 125 In vitro binding of Rob to the micF promoter 6,22 , and transcriptional activation of micF in vitro 9 have been demonstrated . RI 10564485 64 The results demonstrate that stimulation of the inaA promoter by MarA and SoxS in vivo requires only sequences from - 51 to + 3 ( data not shown ) . RI 9473050 7 The TolC level was regulated positively by MarA , Rob , or SoxS . RI 9473050 156 Production of OmpF and LamB was regulated negatively by MarA also in MC4100 . RI 9473050 207 OmpX synthesis is likely to be regulated positively by MarA , Rob , or SoxS ( Fig . 2 ) . RI 9473050 227 We have found that the amount of AcrA expressed from the chromosomal acrAB operon was elevated in OST3408 , OST3410 , OST3408T , JA300R , and JA300 , each carrying marA , robA , and soxS ( Fig . 3 ) . RI 7777530 95 To determine whether site II is essential for repression by MarR , marO133 , a mutant deleted for the 20 bp immediately preceding marR ( pos . 1425 - 1444 ) but wild - type for marRAB ( unpublished data ) , was studied . RI 11751765 214 Fis , an accessorial factor for transcriptional activation of the mar ( multiple antibiotic resistance ) promoter of Escherichia coli in the presence of the activator MarA , SoxS , or Rob . RI 15225313 30 Additional activation of the marRAB operon is achieved by the binding of Fis to a site just upstream of the marbox at bp - 92 to - 73 ( Martin and Rosner , 1997 ) . RI 15225313 36 To evaluate the role of the two MarR binding sites in the repression of marRAB operon transcription ( Fig . 1A ; Cohen et al . , 1993a ; Martin and Rosner , 1995 ) , fusions of the marRAB regulatory region to lacZ were constructed and integrated in the chromosome as single - copy λRS45 lysogens ( Simons et al . , 1987 ) . RI 12060687 19 MelR activates expression of the melA and melB genes , encoding an α - galactosidase and a melibiose permease , respectively . RI 12060687 39 AraC - dependent transcription initiation at the araBAD promoter is increased by CRP , which binds to a single DNA site upstream of I1 and I2 ( 11 ) . RI 12060687 260 The current studies support our previous suggestion that transcription activation at pmelAB requires the binding of MelR to site 2 , which overlaps the – 35 sequence of the promoter ( 20 ) . RI 12060687 270 30 , No . 12 The fact that a poor binding site at – 35 enhances transcription activation by MelR emphasises the importance of the cooperative interactions between MelR proteins at adjacent sites in pmelAB . RI 12060687 369 ( 2000 ) Repression of the Escherichia coli melR promoter by MelR : evidence that full repression requires the formation of a repression loop . RI 12060687 374 ( 2000 ) Transcription activation at the Escherichia coli melAB promoter : the role of MelR and the cyclic AMP receptor protein . RI 12060687 430 ( 2001 ) Transcriptional regulation in the Escherichia coli melibiose operon : interations of MelR and CRP . RI 16515535 142 The metR gene , which activates metE and metH , was only slightly derepressed . RI 16515535 176 Gene LU106 - pFM45 / LU106 - pFM20 ( average + S . D . ) metA 1.6 + 0.1 metB 1.5 + 0.2 metC 1.3 + 0.04 metE 5.7 + 0.3 metF 1.5 + 0.1 metG 1.2 + 0.2 metH 1.6 + 0.1 metJ 7.0 + 0.1 metK 1.6 + 0.1 metL 1.3 + 0.1 metR 1.3 + 0.1 Figure 3 RT – PCR analysis of met transcripts M indicates marker lanes containing a 100 bp ladder ; c : lanes where aliquots have been loaded with control assays in which RT was omitted ; R : a sample from LU106 ( pFM20 ) cells in which the met genes are repressed ; D : a sample from LU106 ( pFM26 ) cells in which there is no functional metJ gene and consequently expression of met genes is de - repressed . RI 16515535 212 First , when we replaced the knockout with a wild - type metJ gene expressed from a plasmid , thus increasing gene copy number , transcript and presumably repressor protein concentrations , there was a remarkable increase in the ability of MetJ to repress the metE locus . RI 12218041 108 The three metD genes are regulated by the MetJ repressor . RI 8898382 172 The mefA operator had the highest apparent affinity in these assays , where the repression complex would be expected to contain at least four MetJ dimers , compared with two in the minimal complexes formed on the synthetic sites . RI 21070640 157 First , repression of nanC and fimB divergent transcripts by NagC has been observed both in vivo and in vitro [ 32 ] . RI 19007420 31 An isorepressor , GalS , has also been identified as © 2008 The Authors Journal compilation © 2008 Blackwell Publishing LtdNagC , GalR and GalS repression at galP 147 Fig . 1 . RI 19007420 88 The expression of both fusions was increased by growth on galactose and to a lesser extent by growth on GlcNAc , supporting the hypothesis that NagC is involved in repression of galP ( Table 2 ) . RI 19007420 94 Note that 0.5 mM GlcNAc or glucose , when present alone , is depleted by A 650 = 0.12 . © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd , Molecular Microbiology , 71 , 146 – 157NagC , GalR and GalS repression at galP 149 corresponds to the strength of the galactose - derepressed galP promoter and implies that the long fusion is better repressed than the short fusion . RI 19007420 147 The mutation in nagO1 behaved as expected : expression was higher in glycerol , giving a © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd , Molecular Microbiology , 71 , 146 – 157NagC , GalR and GalS repression at galP 151 value comparable to the nagC mutation and expression was strongly increased by growth on galactose . RI 19007420 179 Although a similar arrangement of sites around the transcription initiation regions of other genes of the gal regulon might have suggested that they all are regulated by a similar mechanism to galE ( Weickert and Adhya , 1993a , b ; Semsey et al . , 2007 ) , the results presented in this paper demonstrate that repression of the galP gene , is mediated by a distinctly different nucleoprotein complex involving NagC as well as GalR and GalS and including at least 280 bp of DNA . RI 19007420 200 Indeed , the GalS isorepressor was discovered because , in the absence of GalR , there was some residual repression of the galE gene . RI 19007420 207 Only one NagC operator has been identified as important for repression at galP . RI 19007420 217 It is interesting to note that the three central operators , galOE2 , OE3 and OE4 , which have higher affinity for GalS than GalR , all have A / T at positions 8 of the operators ( Fig . 1B ) which is also the case for the mglB and galS operators ( Weickert and Adhya , 1993a , b ) which are preferentially regulated by GalS . RI 19007420 220 Indeed , the effect of the mutation in OE5 was similar to © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd , Molecular Microbiology , 71 , 146 – 157NagC , GalR and GalS repression at galP 153 that in the weakly binding OE2 and OE3 sites ( a mutation in OE4 has not yet been tested ) . RI 1474901 4 Summary Expression of the narK gene of Escherichia coli , like the narGHJI operon , is positively regulated by two frans - acting factors : Fnr , which is activated by anaerobic conditions , and NarL , which is activated by the presence of nitrate . RI 1474901 17 Introduction The expression of respiratory nitrate reductase in Escherichia cotiis regulated at the level of transcription by two pleiotropic rrans - acting factors , Fnr and NarL ( Stewart , 1982 ) . RI 1474901 19 NarL , activated in the presence of nitrate , modulates the pattern of anaerobic gene expression by inducing further the Fnr - dependent expression of formate dehydrogenase N ( Berg and Stewart , 1990 ) and nitrate reductase ( Stewart , 1982 ; Stewart and Parales , 1988 ) and by repressing the expression of the gene which encodes the fermentative enzyme alcohol dehydrogenase { Kalman and Gunsalus , 1988 ) as well as reductases for alternative electron acceptors , such as dimethylsulphoxide and fumarate ( luchi and Lin , 1987 ; Cotter and Gunsalus , 1989 ) , whose reductions are characterized by tower oxidation - reduction potentials . RI 1474901 28 To explore the possible mechanisms involved in the integrated transcriptional activation by Fnr and NarL we have extended our studies to the c / s - acting regions required for expression of the narKgene , which encodes another component involved in nitrate respiration , a nitrate - nitrite antiporter ( Noji et ai , 1989 ; DeMoss and Hsu , 1991 ) . RI 10601216 13 In addition , it was discovered that RpoS activates adhE transcription at both start sites . RI 10601216 135 Thus , it seems that Fnr can regulate adhE expression from the downstream transcriptional start site only in the physical absence of the upstream region . RI 11357510 233 Melville and Gunsalus ( 1996 ) reported that the hemA gene , which is also repressed by FNR , contains only one half - site of FNR consensus sequence . RI 15225315 17 In previous work , we found that NarL or NarP activates the nir promoter by binding to a site centred at position - 69.5 and counteracting the repressive effects of IHF and Fis . RI 15225315 19 We suggest that NarL interferes with IHF binding at the nir promoter by distorting the minor groove at its target site , and we argue that the resulting activation by NarL results from remodelling of the local nucleoprotein structure to facilitate FNR - dependent transcription . RI 15225315 33 Recent studies have shown that transcription initiation at pnir is repressed by the binding of H - NS ( histone - like nucleoid structuring factor ) , IHF ( integration host factor ) and Fis ( factor for inversion stimulation ) ( Wu et al . , 1998 ; Browning et al . , 2000 ) , DNA - binding proteins that play a role in shaping the folded bacterial chromosome ( Azam and Ishihama , 1999 ) . RI 15225315 47 The binding of Fis and IHF inhibits FNRdependent transcription at the nir promoter ( – ve ) . RI 15225315 52 The binding of phospho - NarL / NarP displaces IHF from the IHF I site and remodels the architecture of the nir promoter , enabling FNRdependent transcription activation . RI 15225315 75 Thus , although Fis and IHF repress FNR - dependent activation of pnir , they do not prevent FNR binding . RI 15225315 77 IHF bound to the IHF II site activates FNR - dependent transcription Previously , we demonstrated that IHF binding to the IHF I site represses FNR - dependent transcription at pnir ( Wu et al . , 1998 ; Browning et al . , 2000 ) . RI 15225315 129 This suggests that occupation of the IHF II site increases FNR - dependent activation of pnir . RI 15225315 133 As expected , the introduction of the p99G and p146A substitutions derepressed expression from the wild - type promoter , confirming that IHF and Fis binding to the IHF I and Fis I sites represses pnir . RI 15225315 154 NarL , FNR and Fis can bind to pnir together , but NarL interferes with the binding of IHF To investigate the mechanism by which NarL counteracts IHF - and Fis - mediated repression at pnir , we used gel retardation assays and DNase I footprinting to examine the binding of NarL , FNR , Fis and IHF . RI 15225315 223 As the NarL / NarP binding site at pnir overlaps with the IHF I site ( Fig . 3 ) , NarL / NarP might compete with IHF , and this would provide a simple mechanism by which NarL / NarP could counteract IHFmediated repression . RI 15225315 333 ( 2000 ) Suppression of FNR - dependent transcription activation at the Escherichia coli nir promoter by Fis , IHF and H - NS : modulation of transcription by a complex nucleo - protein assembly . RI 23651393 11 Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis . RI 23651393 27 The rbsDACBK operon is the only target of regulation so far identified to be under the control of RbsR ( Lopilato et al . , 1984 ; Mauzy & Hermodson , 1992a ; Laikova et al . , 2001 ) . RI 23651393 131 Since RbsR influences transcription of the udk and dcd operons , RsbR may also be involved in the regulation of salvage pathway of pyrimidine nucleotides . RI 3316663 11 S1 mapping located transcriptional start points and showed that RhaR , and possibly RhaS , positively regulate transcription from thcb structural gene promoters as well as transcription from their own promoter . RI 3316663 219 We conclude that RhaR positively regulates transcription from the promoters pl , p2 and p3 , and that RhaS may regulate p1 and p3 weakly . RI 3316663 287 It is conceivable that the primary function of the RhaS protein is to positively activate transcription from a gene ( s ) encoding a rhamnose transport protein ( s ) . RI 7896685 13 Both intact Rob and the N - terminal fragment activated expression of stress genes ( inaA , fumC , sodA ) but with a pattern distinct from that found for SoxS and MarA . RI 7896685 30 Oxidative stress genes that are transcriptionally activated by both the marRAB and soxRS systems include sodA ( encoding a superoxide dismutase ) , zwf ( encoding glucose - 6 - phosphate dehydrogenase [ G6PD ] ) , fumC ( encoding a heat - stable fumarase ) , soi - 17 / - 19 , and the genes encoding two other oxidative stress proteins of unknown function ( 4 , 17 , 18 , 26 , 28 ) . RI 7896685 132 Despite overlapping regulation by SoxS , MarA , and Rob , the three systems exhibit differential control in terms of both the strength of their effects on individual genes and activation of some promoters uniquely ( e . g . , zwf by SoxS or MarA but not by Rob ) . RI 7896685 308 marA locus causes decreased expression of OmpF porin in multiple - antibiotic - resistant ( Mar ) mutants of Escherichia coli . RI 22465792 69 Instead , only R603A ( 73 % ) of 70 reduced Rob - dependent activation of the micF promoter . RI 22465792 73 With a tri - alanine scanning library of amino acid residues of 70 from positions 531 – 590 ( residues 531 – 540 lie in the distal portion of 70 R3 . 2 , residues 541 – 570 lie in 70 R4 . 1 , and residues 571 – 590 lie in the N - terminal portion of 70 R4 . 2 ) , 1,46,47 we recently determined that none of the amino acid residues within this region are required for SoxS - dependent activation of transcription from class II promoters fumC and micF , including some substitutions that replace amino acids that are known to interact with nucleotides of the 35 hexamer . RI 22465792 79 Again , with the use of the criterion for a significant effect of a substitution as one that reduces transcription to < 80 % of that produced by wild - type 70 , none of the library ’ s substitutions significantly reduced Robdependent transcription activation of the three class II promoters ( i . e . , fumC , micF , and inaA ) ( Fig . 2a – c ) . RI 22465792 80 In our previous work with the tri - alanine scanning library , we also determined that several members of the library reduced SoxS - dependent activation of transcription from the class I fpr promoter , as expected , because the SoxS binding site of class I promoters lies upstream of the 35 hexamer , which is therefore exposed and available for binding by 70 R4 . RI 22465792 82 The experiments in Fig . 2a – c suggest that the binding of Rob to the robboxes of the fumC , micF , and inaA promoters occludes the binding of 70 R4 to the 35 hexamer of these class II promoters , as is also the case during SoxS - dependent activation of the fumC and micF promoters . RI 22465792 94 20 - 22,39 Indeed , changing C5 to any of the other three nucleotides severely reduces SoxS - dependent transcription activation of the fpr and zwf promoters . RI 22465792 180 Table 1 shows that two amino acid residues of 70 R4 are required for Rob - dependent activation of fumC and that only one is required for activation of micF . RI 22465792 474 Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein . RI 10559180 389 Ribose catabolism of Escherichia coli : characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene , which is involved in regulation of rpiB expression . RI 8576032 303 The inducer for ribose phosphate isomerase B and the mechanism by which RpiR regulates rpiB gene expression remain to be elucidated . RI 9401019 176 The transcriptional fusions ( Fig . 2 ) in the als operon were Lac in their original background but became repressed when AlsR was supplied from a plasmid , implying that the AlsR is a negative regulator for the als operon ( data not shown ) . RI 9401019 229 Therefore , it was deduced that AlsR regulates the als operon , which contains genes for the transport and metabolism of D - allose . RI 9401019 556 Ribose catabolism of Escherichia coli : characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene , which is involved in regulation of rpiB expression . RI 2643114 7 476 - 480 , January 1989 Biochemistry In vivo DNA loops in araCBAD : Size limits and helical repeat ( supercoiling / twist / periodicity / linking number deficit / looping energetics ) DONG - HEE LEE AND ROBERT F . SCHLEIF Biochemistry Department , Brandeis University , Waltham , MA 02254 Communicated by Alexander Rich , October 14 , 1988 ABSTRACT Formation of a DNA loop by AraC proteins bound at the aral and araO2 sites , whose center - to - center distance is 211 base pairs , is necessary for repression of the araBAD promoter , PBAD , of Escherichia coli . RI 2643114 12 Regulation of the repressed basal level as well as the induced level of expression of the arabinose operon in Escherichia coli involves DNA looping mediated by the AraC protein , AraC proteins bound to two distinct DNA sites and bound to each other to form a DNA loop ( 1 - 4 ) . RI 1848302 178 Additionally . the activation of t , he lac promoter and arnC ’ B . 4 I ) 1 ) ~ CRP is similar in that CRT ’ shows the S ~ IIIP orientation tlependcnce in both operons : as in nrtc ( ‘ H ~ ~ lO , CRT ’ can partially activate the lnc promoter when its distance from the promoter is increased by 11 base - pairs , but not by five base - pairs , both in uiuo ( Mandecki $ Carut ’ hers , 1984 ) and in r * itro ( St . raney et n / .. 1989 ) . RI 7768852 16 Studies of the araBAD promoter , p BAD , show that to activate transcription the AraC protein binding site must overlap the 35 region of the promoter by 4 bp ( 25 ) ( Fig . 1 ) . RI 7768852 23 This work is a first step in studying the mechanism ( s ) by which AraC regulates transcription at the araE promoter , p E , and at p FGH . RI 9600836 6 Charles Street Baltimore , MD 21218 , USA * Corresponding author In the absence of arabinose and interactions with other proteins , AraC , the activator - repressor that regulates the araBAD operon in Escherichia coli , was found to prefer participating in DNA looping interactions between the two well - separated DNA half - sites , araI 1 and araO 2 at their normal separation of 211 base - pairs rather than binding to these same two half - sites when they are adjacent to one another . RI 9600836 281 The DNA loop model for ara repression : AraC protein occupies the proposed loop sites in vivo and repression - negative mutations lie in these same sites . RI 2962192 159 Therefore , arafl is essential for both repression and activation and acts as a switch to allow both the repressor and the activator forms of the AraC protein to control the araBAD promoter . RI 2962192 175 The extent of AraC stimulation of araBAD is at least 25 - fold larger . RI 2231717 38 Studies of araE transcription in vitro confirm the results in vivo and show that promoter activity is directly stimulated by AraC and CRP ( Stoner & Schleif , 1983 ) . RI 2231717 170 Dominant - negative AraC mutants that fail to activate transcription of araBAD were selected , and their interactions with specific bases of the binding site were probed with chemically modified DNA . RI 9254708 2 25 , No . 17 © 1997 Oxford University Press The binding of two dimers of IciA protein to the dnaA promoter 1P element enhances the binding of RNA polymerase to the dnaA promoter 1P Yong Lee 1,2 , Ho Lee 1,2 , Jeongbin Yim 1,2 and Deog Hwang 1 , * 1 The Institute for Molecular Biology and Genetics , 2 Department of Microbiology , Seoul National University , Seoul 151 - 742 , Korea Received May 6 , 1997 ; Revised and Accepted July 11 , 1997 ABSTRACT Transcription of the dnaA gene from the promoter 1P has been shown to be activated in vitro and in vivo by the binding of IciA protein to two sites on the dnaA promoter region [ Lee , Y . S . , Kim , H . , and Hwang , D . S . ( 1996 ) Mol . Microbiol . 19 , 389 – 396 ; Lee , Y . S . , and Hwang , D . S . ( 1997 ) J . Biol . Chem . 272 , 83 – 88 ] . RI 9254708 12 The extent of oligomerization of DnaA protein appeared to be proportional to the inhibition of RNA polymerase binding to the promoters and the inhibition of transcription from the promoters . RI 9254708 18 Among the two dnaA promoters , transcription from the promoter 1P was specifically enhanced by the in vivo overproduction of IciA protein or using in vitro transcription assays of the dnaA gene . RI 9254708 20 In this report , we determined the cis - element responsible for the IciA - dependent activation of the dnaA promoter 1P and the binding mode of IciA protein to the IciA I site . RI 9254708 68 As shown previously ( 16 ) , IciA protein activated transcription from the dnaA promoter 1P in the 488 bp EcoRI / HindIII fragment ( + IciA I , + IciA II ) of plasmid pHJ1 without affecting transcription from the promoter 2P ( Fig . 2A and B ) . RI 9254708 72 From these results , we conclude that the IciA I site is essential and alone sufficient for the activation of transcription from the dnaA promoter 1P by IciA protein . RI 9254708 91 According to previous results ( 16 ) , IciA protein was able to activate in vitro transcription from dnaA promoter 1P in the presence of limiting amounts of RNA polymerase . RI 9254708 98 This result implies that the binding of two dimers of IciA protein to the IciA I site is required for the activation of RNA polymerase binding to the dnaA promoter 1P . RI 9254708 99 The enhanced binding of RNA polymerase to the dnaA promoter 1P by IciA protein was confirmed using a DNase I protection assay ( Fig . 4C ) . RI 9254708 103 DISCUSSION Physically and functionally heterogeneous complexes containing IciA protein bound to the dnaA promoter were isolated and analyzed in order to address the binding mode of IciA protein to DNA containing the IciA I site and how this binding activates transcription from the dnaA promoter 1P . RI 9254708 104 We demonstrate in this paper that the mechanism of stimulation of transcription by IciA protein may include : ( i ) the binding of two dimers of IciA protein to the IciA I site , which is located – 50 to – 63 from the transcription start site of dnaA promoter 1P ; and ( ii ) the stimulation of RNA polymerase binding to the promoter by two IciA protein dimers on the IciA I site . RI 8825783 1 Molecular Microbiology ( 1996 ) 19 ( 2 ) , 389 – 396 Transcriptional activation of the dnaA gene encoding the initiator for oriC replication by IciA protein , an inhibitor of in vitro oriC replication in Escherichia coli Yong Sun Lee , 1 , 2 Hakjung Kim 1 and Deog Su Hwang 1 * 1 The Institute for Molecular Biology and Genetics , and 2 Department of Microbiology , Seoul National University , Seoul 151 - 742 , Korea . RI 8825783 8 These findings suggest that IciA protein may regulate the initiation of chromosomal DNA replication at oriC by controlling expression of the dnaA gene , as well as by inhibiting the initiation of chromosomal DNA replication at oriC . RI 8825783 48 One site , designated the IciA I site , was located upstream IciA protein binds two sites in the dnaA promoter region DNase I - protection experiments were performed to identify binding sites of the IciA protein in the dnaA promoter region ( Fig . 3 ) . RI 8825783 51 The 32 P - end - labelled fragments were incubated with the indicated amounts of IciA protein and subjected to gel - shift analysis as described in the Experimental procedures . # 1996 Blackwell Science Ltd , Molecular Microbiology , 19 , 389 – 396Transcriptional activation of the dnaA gene by IciA protein 391 Fig . 3 . RI 8825783 52 IciA protein binds to two sites in the dnaA promoter region . RI 8825783 66 These indicate that DnaA and IciA proteins independently bind to their respective binding sites on the dnaA # 1996 Blackwell Science Ltd , Molecular Microbiology , 19 , 389 – 396392 Y . RI 8825783 72 DnaA and IciA proteins also independently bound to their binding sites in oriC ( Hwang and Kornberg , 1992b ) . RI 8825783 73 Overproduction of IciA protein enhances in vivo transcription from dnaA promoter 1P The recombinant plasmid pIC1 contains the iciA coding region under control of the inducible araB promoter of vector pING1 ( Hwang et al . , 1992 ; Johnston et al . , 1985 ) . RI 8825783 93 Thus , overproduction of IciA protein specifically increased transcription from the dnaA promoter 1P . RI 8825783 97 As shown previously ( Wang and Kaguni , 1987 ) , purified DnaA protein repressed transcription from the dnaA promoters , 1P and 2P ( Fig . 5A ) . RI 8825783 98 Although DnaA protein , at concentrations of 75 ng and greater , reduced transcription from rpmH promoter 1P , repression was preferential for the dnaA promoters . RI 8825783 101 Whereas # 1996 Blackwell Science Ltd , Molecular Microbiology , 19 , 389 – 396Transcriptional activation of the dnaA gene by IciA protein 393 Fig . 6 . RI 8825783 112 In vivo S1 nuclease - protection assays ( Fig . 4 ) and in vitro run - off transcription assays ( Figs 5 and 6 ) indicate that IciA protein specifically activates transcription from dnaA promoter 1P . RI 8825783 119 The expression of dnaA gene decreased after initiation of chromosomal replication ( Theisen et al . , 1993 ) . RI 8825783 133 Hwang we have shown that IciA protein activates transcription from the promoter 1P of the dnaA gene . RI 8825783 309 ( 1987 ) Transcriptional repression of the dnaA gene of Escherichia coli by DnaA protein . RI 8825783 312 ( 1989 ) DnaA protein regulates transcription of the rpoH gene of Escherichia coli . RI 8995231 4 Occlusion of RNA Polymerase by Oligomerization of DnaA Protein over the dnaA Promoter of Escherichia coli * ( Received for publication , June 25 , 1996 , and in revised form , September 13 , 1996 ) Yong Sun Lee § ¶ and Deog Su Hwang From the Institute for Molecular Biology and Genetics and the § Department of Microbiology , Seoul National University , Seoul 151 - 742 , Korea DnaA protein , the initiator protein for initiation of Escherichia coli chromosomal replication , has been shown to repress its own expression from two dnaA promoters , 1P and 2P . RI 8995231 7 This results in the repression of transcription , suggesting that the extent of oligomerization of DnaA proteins over two dnaA promoters contributes to the autoregulation of expression of the dnaA gene . RI 8995231 30 In vitro transcription of the dnaA gene was inhibited by DnaA protein ( 15 ) . RI 8995231 32 Also , DnaA protein functions as a transcriptional repressor for the expression of other genes including rpoH ( 18 ) , mioC ( 19 ) , the guaBA operon ( 20 ) , and uvrB ( 21 ) , while the expression of the nrd gene appeared to be enhanced by DnaA protein ( 22 ) . RI 8995231 36 Also , IciA protein binds to two sites , IciA I and IciA II , in the dnaA promoter region ( 25 ) . RI 8995231 84 RESULTS Oligomerization of DnaA Protein to the dnaA Promoter — Sequence - specific DNA binding of DnaA protein to the dnaA promoter region ( Fig . 1 ) was studied in detail using a combined gel - shift and chemical footprinting assay to determine the extent of DNA binding to the region flanking the consensus DNA - binding site , the DnaA box . RI 8995231 109 These observations indicate that the inhibition of binding of RNA polymerase to the dnaA promoters is dependent upon the oligomerization of DnaA protein to regions flanking the DnaA box and that the binding of one or two DnaA monomers to the DnaA box is not sufficient for occlusion of RNA polymerase from the dnaA promoters . RI 8995231 127 The binding of two dimers of IciA protein to the IciA I site is responsible for the activation of dnaA promoter 1P . RI 8995231 128 2 When the two dnaA promoters were repressed by DnaA protein , IciA protein was able to restore transcription from the 1P promoter with little effect on transcription from the 2P promoter ( 25 ) . RI 8995231 129 The mechanism of IciA protein stimulation of transcription from dnaA promoter 1P in the presence of inhibitory amounts of DnaA protein was examined . RI 8995231 141 RNA polymerase binding to the region containing dnaA promoter 1P ( lane 3 ) was inhibited by DnaA protein ( lane 4 ) . RI 8995231 144 DISCUSSION Physically and functionally heterogeneous complexes containing DnaA protein bound to the dnaA promoter were isolated and analyzed in order to address the mode of binding of DnaA protein to DNA containing a single DnaA box and how this binding inhibits transcription by RNA polymerase . RI 8995231 149 IciA protein stimulates RNA polymerase binding to dnaA promoter 1P in the presence of inhibitory amounts of DnaA protein . RI 8995231 159 We investigated whether DnaA protein and RNA polymerase can coexist at the dnaA promoter by gel - shift and footprinting analyses and whether a direct protein - protein interaction between DnaA protein and RNA polymerase is the mechanism of inhibition of transcription . RI 8995231 160 Our results indicate that the repression of transcription from the two dnaA promoters ( 1P and 2P ) by DnaA protein is promoted by the oligomerization of DnaA protein over dnaA promoters 1P and 2P and that this binding directly prevents RNA polymerase binding to the two promoters . RI 8995231 163 The binding of two dimers of IciA protein to the IciA I site , which is located 50 to 63 nucleotides from the transcription start site of dnaA promoter 1P , is required to stimulate RNA polymerase binding to the 1P promoter . RI 8995231 167 Our previous experiments have shown that when both dnaA promoters 1P and 2P were repressed by DnaA protein , IciA protein specifically activated transcription from dnaA promoter 1P , while dnaA promoter 2P was still repressed ( 25 ) . RI 10600368 39 Here , it is shown that the product of the argP gene , the ArgP protein , is a modulator molecule that regulates the expression of the arginine transport system . RI 10600368 80 The ArgP protein enhances the expression of the argK gene To test the biological activity of the ArgP protein on the arginine transport system , the amounts of mRNA synthesized by a 502 bp KpnI - EcoRV DNA fragment containing the N terminus and the control region of the argK gene was investigated . RI 10600368 87 The ArgP protein represses its own synthesis Sequences consistent with the consensus for E . coli promoters ( 35 and 10 ) have not been found upstream of the start codon for the iciA ( argP ) gene ( ThoÈny et al . , 1991 ) . RI 10600368 454 Transcriptional repression of the dnaA gene of Escherichia coli by DnaA protein . RI 9135111 6 CAT fusion analysis revealed that the operon has a promoter for gntR and another for gntKU , and that the gntR gene is constitutively expressed , while that of gntKU is regulated positively by the cAMP - CRP complex and negatively by GntR . RI 9135111 7 Read - through transcription from the gntR promoter into gntK was decreased in the presence of GntR , although GntR did not repress its own promoter . RI 11115104 89 ExuR regulates expression of the uxaCA , uxaB and exuT operons involved in galacturonate catabolism . RI 11115104 93 The transport of glucuronides and their subsequent interconversion to the glucuronate in E . coli is mediated by the genes of the uidAB operon , which is under negative regulation by UidR and UxuR ( Blanco , 1987 ) . RI 11115104 191 Moreover , almost all genes from the KdgR regulon are regulated by CRP in E . chrysanthemi ( Nasser et al . , 1997 ) . RI 3062173 21 The physiological function of the GutM protein is shown to be activation of gut operon transcription , while that it involves participation of both a glucitol - Gxpressional regulation of the operon is unusual in that it invokes participation of both a glucitolspecific repressor and a glucitol - specific activator in addition to the general activator , the cAMP - CRP complex . RI 3062173 184 Experiments using the cat gene cartridge revealed that induction of the gut operon enhances expression of the gutR gene as well as the gutABD and M genes . RI 11094278 30 The marR gene encodes a repressor protein that binds to marO and represses transcription of marRAB . RI 2419307 10 The metBL and metF genes each had a single transcript which was repressed by metj , while the metJ gene had three transcripts , of which the first was strongly repressed by metj , the second was less strongly repressed , and the third was not repressed . RI 2419307 16 The metJ gene product exists as a dimer with an Mr of 24,000 and has been shown to bind specifically , in vitro , to the region of DNA between metJ and metBL and to the DNA near the metF promoter , causing repression of all four genes ( 24 , 26 ) . RI 2419307 103 Thus , the single chromosomal copy of metJ in JJ100 produces enough product to cause about a 35 - fold repression of the multiple , plasmid - borne metB promoters . RI 2419307 112 Deletion of all of the metJ coding region and half of the intercistronic region ( pKmJAHN ) lowered the galK expression , but the remaining activity was ' still subject to repression by chromosomal metJ . RI 2419307 148 165 , 1986 REGULATION OF IN VIVO TRANSCRIPTION OF metJBLF 675 very effectively represses transcription from the first metJ promoter . RI 2419307 164 In the repressed strains , JJ100 and its pRCG161 transformant , no metF message was detectable , showing that transcription of both the chromosomal and plasmid - borne metF gene is blocked by the metJ - metK regulatory system . RI 2419307 196 It appears that binding of the metJ gene product may prevent binding of RNA polymerase to the metB and Jl promoters and , under strongly repressing conditions , may also decrease the binding to the J2 promoter . RI 2419307 209 Our results confirm that metJ is autoregulated and demonstrate that much of the regulation is indeed at the level of transcription ( i . e . , repression of the Jl and J2 promoters ) . RI 7545108 62 There are two NagC operator sites upstream of gimU The perfect inverse correlation between the induction of nag and repression of glm genes posed the question as to whether the regulation of glmU expression involved the NagC protein . RI 7545108 151 An alternative explanation is that repression occurs by the ' roadblock ' mechanism described for PurR repression of the purB promoter ( He and Zalkin , 1992 ) . RI 7545108 154 It is interesting to compare the characteristics of NagCmediated repression of nag degradative genes with the NagC - mediated activation of the glm genes . RI 7545108 171 Expression of the FNR - dependent nitrate regulon is further activated by NarL binding to an upstream site , situated 70 - 200 bp upstream of the transcription start site , depending upon the promoter ( reviewed in Stewart , 1993 ) . RI 7545108 173 Although the proximal site , BoxGl , is located at a position typical of prokaryotic activators ( Collado - Vides et al . , 1991 ; Gralla , 1991 ) , it is not sufficient to allow NagC to to activate glmU by itself ; this could be because NagC occupation of this lower affinity site is inadequate in the absence of the higher affinity upstream site . RI 7545108 179 The AraC protein activates and represses the araBAD genes , depending upon the presence of the inducer and cAMP / CAP which change the occupancy of the different sites ( Reeder and Schleif , 1993 and references therein ) . RI 17238917 9 In contrast , IHF does not enhance the ability of NanR to activate fimB expression in the wild - type background . RI 17238917 11 However , placing the NanR , IHF and NagC binding sites closer to the fimB promoter enhances the ability of the regulators to activate fimB expression . RI 17238917 86 Taken together , these results support a model in which O NC2 enhances the binding of NagC to O NC1 , with the two sites functioning jointly to control fimB expression . RI 17238917 87 Scanning - replacement mutagenesis of the region separating O NC1 from O NC2 and the effect of IHF on fimB expression Although the results described above show that O NC1 and O NC2 function together , the distance between the centre of the two operators ( 212 bp ) is longer than for any other NagC - regulated operon . RI 17238917 122 To investigate how IHF activates fimB expression further , the IHF binding site mutation described above was combined with those in O NC1 , O NC2 and O NR alone , and O NC1 O NR in combination , and the expression of the FimB – LacZ chimera was measured ( Fig . 6 ) . RI 17238917 130 The wild - type ( W + ) and mutant strains indicated were analysed as in the legend to Fig . 2A . © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd , Molecular Microbiology , 63 , 1223 – 1236Regulation of fimB by Neu 5Ac and GlcNAc 1229 lower level of fimB expression , as would be expected if it prevents activation by both NagC and NanR . RI 17238917 134 This suggests that IHF binding to ibs has a small inhibitory effect on the ability of NanR to activate fimB expression . RI 17238917 138 Taking these results together with those presented above , it is concluded that the cooperative interaction of NagC bound to O NC1 and O NC2 , with IHF bound to the site identified in this work , forms a nucleoprotein complex that activates fimB expression . RI 17238917 143 Thus , NanR and NagC appear to activate fimB expression independently . RI 17238917 144 Accordingly we reasoned that a loss of fimB expression as a result of decreased NagC activity in the D3 background might be compensated for by enhanced activation by NanR for some reason . RI 17238917 158 It thus seems likely that the ability of IHF alone to activate fimB expression is enhanced in the D3 mutant background . RI 17238917 161 Thus the D3 region is not apparently required for NanR to activate fimB expression in the wildtype background , and it could contain specific sequences that inhibit this . RI 17238917 174 While the basis for this effect is uncertain , it is nevertheless evident that neither NanR nor NagC requires the wild - type segment of D7 DNA in order to be able to activate fimB expression . RI 17238917 176 To test this hypothesis further , and to examine the effect of operator - to - promoter distance on NagC activation , the effect of deleting the 274 bp region ( D7 ) described above on fimB expression was also analysed ( Fig . 8 ) . RI 17238917 177 The results of this analysis confirm this assertion , and show additionally that NagC is also better able to We previously presented evidence suggesting that the D2 region , and hence NanR and NagC , could activate fimB expression by blocking the inhibitory effect of additional cis - acting element ( s ) positioned closer to nanC ( El - Labany et al . , 2003 ) . RI 17238917 183 Discussion We showed in earlier work that fimB expression , and hence the OFF - to - ON phase switching of type 1 fimbriation , is activated by NanR and NagC and hence repressed Fig . 9 . RI 17238917 187 NanR and NagC activate fimB expression by binding to operator sequences ( O NR and O NC1 respectively ; Fig . 1 ) situated further upstream of the regulated promoter than is usual in bacteria . RI 17238917 189 Furthermore , as NagC binding to O NC1 prevents CRP - dependent activation of nanC ( Condemine et al . , 2005 ) , this study should enhance our understanding of the regulation of this gene as well . RI 17238917 196 Considered together , these data show that IHF promotes an interaction between NagC bound to O NC1 and O NC2 , forming a stable nucleoprotein complex that activates fimB expression . RI 17238917 199 Thus , not only does IHF stimulate both FimB and FimE recombination , as well as activate fimbrial expression in phase ON cells , but it is now clear that it enhances fimB expression too . RI 17238917 212 Thus , activation of fimB expression by NanR , as judged by the effect of mutations in nanR , orin its operator sequence ( O NR ) , is enhanced considerably in the D3 deletion mutant . RI 17238917 213 This observation not only allows us to understand how a modest mutation in O NC2 can affect fimB expression whereas the D3 mutation does not , but it also supports our prior assertion that NanR activates fimB expression without NagC ( Sohanpal et al . , 2004 ) . RI 17238917 214 The results described above also suggest that when O NR is placed closer to the fimB promoter , NanR is better able to activate fimB expression , and further work supports this idea . RI 17238917 216 By replacing the 274 bp of wild - type DNA from - 467 to - 194 with heterologous sequences it was shown that neither NanR nor NagC requires this segment of DNA in order to activate fimB expression . RI 17238917 221 NagC is best characterized as a repressor ( Plumbridge , 2001 ; Plumbridge and Pellegrini , 2004 ) , although the regulator does activate one of the glmUS operon promoters albeit by binding at the more conventional position of - 47 relative to the transcriptional start site ( Plumbridge , 1995 ) . RI 17238917 222 At the moment we do not know how NagC activates the fimB promoter , but two general scenarios seem most plausible . RI 17238917 234 We note that such an effect is not unprecedented for a member of the GntR / FadR family of transcriptional regulators like NanR ( Rigali et al . , 2002 ; Kalivoda et al . , 2003 ) ; activation of the glcDEFGB operon by GlcC and IHF is thought to occur in this way , with IHF bound to a site between the promoter and a distant ( > - 170 bp ) upstream activating site for GlcC ( Pellicer et al . , 1999 ) . RI 17238917 236 Thus , if the model proposed above is correct , it seems probable that NanR must make contact with RNA polymerase , or another regulatory factor bound closer to the promoter , in order to activate fimB expression in the wild - type background as well . RI 17238917 454 ( 2004 ) Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors : NagC , ChbR and CAP . RI 15534208 140 The identification of NagC as an activator of fimB expression indicated that the expression of the recombinase would be inhibited by GlcNAc . RI 15534208 165 101 no . 46 16325yjhA encodes an outer membrane channel that can facilitate Neu 5 Ac uptake ( G . Condemine , personal communication ) , and this gene is also repressed strongly by NanR , an effect which is modulated by NagC binding to the NagC1 site . RI 15534208 178 fimE is regulated by the fim invertible element , and hence , like the Pap adhesin regulators PapB and PapI , both of the fim regulatory proteins are probably controlled by phase variation as well ( 48 , 49 ) . RI 1848637 276 Expression of both nagE and nagB was shown to be stimulated by CAP in viva , although nagE was much more dependent than nagB ( Plumbridge , 1990 ) . RI 1848637 326 In experiments using native nag DNA , both sites are of DNA looping was reinforced by the strict phasing requirement of the AraC binding sites for repression of the araBAD operon ( Dunn et al . , 1984 ; Lee & Schleif , 1989 ) . RI 1848637 359 The simultaneous binding of repressor and activator type proteins to nag DNA implies two corollaries : ( 1 ) NagC does not repress by competition with CAP and ( 2 ) CAP does not activate by displacing NagC . RI 1848637 861 DNA - protein recognition : demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor . RI 1848637 866 CRP / cAMP and CytR regulated promoters in Escherichia coli K12 : the cdd promoter . RI 3035558 7 3901 - 3905 , June 1987 Microbiology The narL gene product activates the nitrate reductase operon and represses the fumarate reductase and trimethylamine N - oxide reductase operons in Escherichia coli ( frd gene / tor gene / regulation ) S . RI 3035558 120 ( ii ) Relief of the repression by the narL mutation indicated that the gene product , which activated the narC operon ( 36 ) , was also required for repression of thefrd operon . RI 3035558 159 After the emergence of nitrate respiration , the narL product probably first evolved as a specific activator protein for the narC operon and subsequently broadened its role to include repression . RI 19251855 8 In this study , we show that the global transcription factor FNR is a class II activator at the ynfEFGHI promoter and that NarL represses activation by binding to a single target that overlaps the promoter 10 element . RI 19251855 41 Figure 4 shows autoradiographs from electromobility shift assays designed to investigate how NarL represses FNR - dependent activation of the ynfE promoter . RI 19251855 85 Thus , the dmsA promoter is repressed by NarL binding to multiple sites spread across 80 bp covering the entire promoter ( 1 , 5 ) . RI 19251855 88 Our results suggest that repression by NarL at the ynfE promoter may be unusual and might be due to locking of the FNR - RNAP - promoterVOL . RI 19251855 146 Integration of three signals at the Escherichia coli nrf promoter : a role for Fis protein in catabolite repression . RI 1304369 405 MalI , a novel protein involved in regulation of the maltose system of Escherichia coli , is highly homologous to the repressor proteins GalR , CytR , and LacI . RI 2405166 251 This may mean that RhaR stimulates transcription from the psr promoter by interacting directly with RNA polymerase . RI 15342598 8 RhaR activates transcription of rhaSR , and RhaS activates transcription of the operon that encodes the L - rhamnose catabolic enzymes , rhaBAD , as well as the operon that encodes the L - rhamnose transport protein , rhaT . RI 15342598 12 In the present study , we first identified two positively charged amino acids in 70 , K593 and R599 , and three negatively charged amino acids in RhaR , D276 , E284 , and D285 , that were important for RhaR - mediated transcription activation of the rhaSR operon . RI 15342598 34 The Ada protein has two activation domains , one of which is an AraC / XylS family domain which is required to activate transcription of the alkA operon ( 33 ) . RI 15342598 37 RhaS activates transcription of the rhaBAD and rhaT operons by binding as a dimer to sites that overlap the 35 hexamers of the promoters by 4 bp and extend upstream to 81 and 82 , respectively ( see Fig . 1 for the rhaBAD promoter ) ( 10 , 45 ) . RI 15342598 38 Similarly , RhaR activates transcription of the rhaSR operon by binding as a dimer to a site that overlaps the RNAP binding site by 4 bp and extends upstream to 82 ( Fig . 1 ) ( 43 ) . RI 15342598 50 The CRP site required for full activation at rhaSR is located upstream but not adjacent to the RhaR binding site and is centered at 111.5 ( Fig . 1 ) ( 17 ) . RI 15342598 57 We identified amino acid residues in the C - terminal domain of 70 and in RhaR that are important for RhaR - mediated transcription activation at the rhaSR promoter . RI 15342598 59 We also extended the previous studies by Bhende and Egan ( 4 ) of RhaS - mediated transcription activation at rhaBAD . RI 15342598 127 We assayed this library of alanine substitutions in 70 at two RhaR - activated single - copy translational fusions , ( rhaS - lacZ ) 216 and ( rhaS - lacZ ) 92 . RI 15342598 147 RhaR residues D276 , E284 , and D285 are important for rhaSR transcription activation . RI 15342598 222 Previous results reported by Bhende and Egan ( 4 ) identified two amino acid residues in 70 , K593 and R599 , that were important for RhaSmediated transcription activation at rhaBAD and rhaT . RI 15342598 224 BACTERIOL . present study , we identified four amino acid residues in 70 , K593 , L595 , R599 , and R608 , which were important for RhaRmediated transcription activation of rhaSR ( Fig . 2B ) . RI 15342598 234 Previous results showing an interaction between RhaS D241 and 70 R599 at rhaBAD ( 4 ) led us to investigate whether an interaction between RhaR D276 and 70 R599 might be required for RhaR activation at rhaSR . RI 15342598 436 Transcription activation at the Escherichia coli melAB promoter : interactions of MelR with the C - terminal domain of the RNA polymerase alpha subunit . RI 15342598 450 Transcription activation at the Escherichia coli melAB promoter : interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase sigma subunit . RI 15342598 459 Interdependence of activation at rhaSR by cyclic AMP receptor protein , the RNA polymerase alpha subunit C - terminal domain and RhaR . RI 15342598 534 The Escherichia coli Ada protein can interact with two distinct determinants in the 70 subunit of RNA polymerase according to promoter architecture : identification of the target of Ada activation at the alkA promoter . RI 7608078 205 The treB treC operon is negatively regulated by TreR , whose gene treR is located upstream of treB but is not part of the operon . RI 11750821 52 Two regions of enhanced XylR binding in the presence of xylose were revealed in the xylAB / xylFGH intergenic space by DMS footprinting [ 4 ] . RI 9371449 26 The expression of xylF is negatively regulated by Fis and RpoS at the transcriptional level ( 50 ) , and a putative regulator , xylR , was found downstream of xylFGH . RI 9371449 35 The arabinose operon , structurally similar to the xyl operon , receives both positive and negative modes of regulation involving AraC protein ( 29 ) . RI 9371449 259 These genes , together with xylAB , are positively regulated by the same protein ( XylR [ Fig . 4 ] ) , which appears to recognize the consensus sequences located in their promoterVOL . RI 9371449 741 Identification of the DNAbinding domain of the OmpR protein required for transcriptional activation of the ompF and ompC genes of Escherichia coli by in vivo DNA footprinting . RI 9371449 785 Fis activates the RpoS - dependent stationary - phase expression of proP in Escherichia coli . RI