cAMP Receptor Protein Binding Research Articles

Visualization of two architectures in class-II CAP-dependent transcription activation.

Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters via a recruitment and isomerization mechanism. However, whether and how it modifies RNA polymerase (RNAP) to initiate transcription remains unclear. Here, we report cryo–electron microscopy (cryo-EM) structures of an intact Escherichia coli class-II CAP-dependent transcription activation complex (CAP-TAC) with and without de novo RNA transcript. The structures reveal two distinct architectures of TAC and raise the possibility that CAP binding may induce substantial conformational changes in all the subunits of RNAP and transiently widen the main cleft of RNAP to facilitate DNA promoter entering and formation of the initiation open complex. These structural changes vanish during further RNA transcript synthesis. The observations in this study may reveal a possible on-pathway intermediate and suggest a possibility that CAP activates transcription by inducing intermediate state, in addition to the previously proposed stabilization mechanism.

Serotype-shifting gene rfbT is a direct transcriptional target of cAMP receptor protein (CRP) in V. cholerae O1

Ogawa and Inaba are two main serotypes of O1 V. cholerae and alternate among cholera epidemics. The rfbT gene encodes a methyltransferase and is required for Ogawa serotype. The Inaba serotype is the consequence of genetic alterations in rfbT gene which results in loss-of-function enzyme product. However, the expression and regulation of rfbT has not been understood yet. Here we demonstrated that a global regulator, cAMP receptor protein (CRP), positively regulates rfbT transcription through a non-canonical CRP binding site (CBS) in its promoter region. This finding is supported by the analyses of rfbT mRNA abundance, rfbT–lacZ fusions and electrophoretic mobility shift assay (EMSA). The analyses of rfbT mRNA level in wild type (WT), Δcrp, and lower or higher level of cAMP backgrounds revealed that CRP is required for rfbT expression in response to intracellular cAMP level. Subsequent rfbT–lacZ fusions and EMSA collectively displayed that cAMP-CRP complex transcriptionally activates rfbT through directly binding to CBS in rfbT promoter region. Consistently, serological microagglutination test showed that crp deletion resulted in at least 4-fold decrease in titer of Ogawa serum compared to its WT. These results expanded our knowledge of understanding the genetic determinants and probable regulatory mechanism of V. cholerae O1 serotype shift between Ogawa and Inaba.

Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917.

Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription. (1)H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.

SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross-talk and sheds light on regulation by effector molecules.

Surface plasmon resonance monitoring of the binding of transcription factors cAMP receptor protein (CRP) and nitrogen control factor of cyanobacteria (NtcA) from Synechocystis sp. PCC6803 to promoter fragments of glnA, glnN (NtcA regulon) and cccS (CRP regulon), revealed exclusive CRP binding to cccS, whereas NtcA was bound to all three promoters with different affinities, which were strongly increased by the NtcA activator 2-oxoglutarate. Effective NtcA affinity for 2-oxoglutarate varied with the promoter. High-affinity promoters and the NtcA-coactivating protein PII-interacting protein X (PipX) increased NtcA affinity towards 2-oxoglutarate, suggesting PipX-stabilization of the 2-oxoglutarate-bound NtcA conformation. PipX binding to NtcA required 2-oxoglutarate and was much tighter (Kd≈85 nM) than to the PipX-sequestering PII protein. NtcA appears to require more strongly PipX and 2-oxoglutarate (2OG) for estimulating gene expression at promoters having "imperfect" NtcA binding sites.

CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication

The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.

ThetibAdherence Locus of EnterotoxigenicEscherichia coliIs Regulated by Cyclic AMP Receptor Protein

Enterotoxigenic Escherichia coli (ETEC) is a Gram-negative enteric pathogen that causes profuse watery diarrhea through the elaboration of heat-labile and/or heat-stable toxins. Virulence is also dependent upon the expression of adhesive pili and afimbrial adhesins that allow the pathogen to adhere to the intestinal epithelium or mucosa. Both types of enterotoxins are regulated at the level of transcription by cyclic AMP (cAMP) receptor protein (CRP). To further our understanding of virulence gene regulation, an in silico approach was used to identify putative CRP binding sites in the genome of H10407 (O78:H11), an ETEC strain that was originally isolated from the stool of a Bangledeshi patient with cholera-like symptoms circa 1971. One of the predicted binding sites was located within an intergenic region upstream of tibDBCA. TibA is an autotransporter and afimbrial adhesin that is glycosylated by TibC. Expression of the TibA glycoprotein was abolished in an H10407 crp mutant and restored when crp was provided in trans. TibA-dependent aggregation was also abolished in a cyaA::kan strain and restored by addition of exogenous cAMP to the growth medium. DNase I footprinting confirmed that the predicted site upstream of tibDBCA is bound by CRP. Point mutations within the CRP binding site were found to abolish or significantly impair CRP-dependent activation of the tibDB promoter. Thus, these studies demonstrate that CRP positively regulates the expression of the glycosylated afimbrial adhesin TibA through occupancy of a binding site within tibDBp.

Computational prediction of cAMP receptor protein (CRP) binding sites in cyanobacterial genomes

BackgroundCyclic AMP receptor protein (CRP), also known as catabolite gene activator protein (CAP), is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed.ResultsWe have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution.ConclusionThe CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The loss of CRPs in these species leads to the rapid loss of their binding sites in the genomes.

CRP Binding and Transcription Activation at CRP-S Sites

In Haemophilus influenzae, as in Escherichia coli, the cAMP receptor protein (CRP) activates transcription from hundreds of promoters by binding symmetrical DNA sites with the consensus half-site 5′-A 1A 2A 3T 4G 5T 6G 7A 8T 9C 10T 11. We have previously identified 13 H. influenzae CRP sites that differ from canonical (CRP-N) sites in the following features: (1) Both half-sites of these noncanonical (CRP-S) sites have C 6 instead of T 6, although they otherwise have an unusually high level of identity with the binding site consensus. (2) Only promoters with CRP-S sites require both the CRP and Sxy proteins for transcription activation. To study the functional significance of CRP-S site sequences, we purified H. influenzae ( Hi)CRP and compared its DNA binding properties to those of the well-characterized E. coli ( Ec)CRP. All EcCRP residues that contact DNA are conserved in HiCRP, and both proteins demonstrated a similar high affinity for the CRP-N consensus sequence. However, whereas EcCRP bound specifically to CRP-S sites in vitro, HiCRP did not. By systematically substituting base pairs in native promoters and in the CRP-N consensus sequence, we confirmed that HiCRP is highly specific for the perfect core sequence T 4G 5T 6G 7A 8 and is more selective than EcCRP at other positions in CRP sites. Even though converting C 6 → T 6 greatly enhanced HiCRP binding to a CRP-S site, this had the unexpected effect of nearly abolishing promoter activity. A + T-rich sequences upstream of CRP-S sites were also found to be required for promoter activation, raising the possibility that Sxy binds these A + T sequences to simultaneously enable CRP–DNA binding and assist in RNA polymerase recruitment.

A Cyclic AMP Receptor Protein-Regulated Cell-Cell Communication System Mediates Expression of a FecA Homologue in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a DSF, and seven structural derivatives, which require rpfF and rpfB for synthesis. Acquisition of iron from the environment is important for bacterial growth as well as the expression of virulence genes. We identified a gene homologous to fecA, which encodes a ferric citrate receptor that transports exogenous siderophore ferric citrate from the environment into the bacterial periplasm. Western blot analysis with anti-FecA-His(6) antibody showed that the FecA homologue was induced in the iron-depleted medium supplemented with a low concentration of ferric citrate. Deletion of rpfF or rpfB resulted in reduced FecA expression compared to the wild type. Synthetic DSF restored FecA expression by the DeltarpfF mutant to the wild-type level. Reverse transcription-PCR showed that the fecA transcript was decreased in the DeltarpfF mutant compared to the wild type. These data suggest that DSF affected the level of fecA mRNA. Transposon inactivation of crp, which encodes cyclic AMP (cAMP) receptor protein (CRP) resulted in reduced FecA expression and rpfF transcript level. Putative CRP binding sites were located upstream of the rpfF promoter, indicating that the effect of CRP on FecA is through the rpf/DSF pathway and by directly controlling rpfF. We propose that CRP may serve as a checkpoint for iron uptake, protease activity, and hemolysis in response to environmental changes such as changes in concentrations of glucose, cAMP, iron, or DSF.

Coactivation of Vibrio vulnificus putAP operon by cAMP receptor protein and PutR through cooperative binding to overlapping sites

The cAMP receptor protein (CRP) positively regulates the expression of Vibrio vulnificus putAP genes encoding a proline dehydrogenase and a proline permease. In the present study, an open reading frame encoding PutR was identified downstream of the putAP genes and a mutational analysis revealed that the PutR protein was also involved in regulating the putAP transcription by activating Pput promoter. Although CRP acts as a primary activator and the influence of PutR on Pput is mediated by CRP, the level of Pput activity observed when PutR and CRP functioned together was greater than the sum of Pput activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of PutR and CRP were not significantly affected by each other, indicating that PutR and CRP coactivate Pput rather than function sequentially in a regulatory cascade. Two adjacent binding sites for PutR mapped by in vitro DNase I protection assays were found to overlap the CRP binding sites and were centred -91.5 (PCBI) and -133.5 bp (PCBII) upstream of the transcription start site of Pput respectively. PutR and CRP bind to the sites cooperatively and a dissection of the role of the binding sites revealed that CRP at PCBI plays the most crucial role in the activation of Pput. Accordingly, the present results revealed that PutR and CRP coactivate the expression of Pput and exert their effect by cooperatively binding to the promoter.

Dps expression in Escherichia coli O157:H7 requires an extended − 10 region and is affected by the cAMP receptor protein

The DNA binding protein from starved cells (Dps) is a general stress protein that provides Escherichia coli protection from osmotic, oxidative, and acid stresses. While Dps production and accumulation is primarily associated with stationary phase, during log phase, this protein protects against oxidative stress in an OxyR-dependent manner. In this study, evidence is provided that expands the role of Dps in acid tolerance to both log- and stationary-phase E. coli O157:H7. The transcription of dps occurred in log-phase cells without OxyR or stress and was upregulated during entry into stationary phase. The expression in log and stationary phase involved σ 70 and σ s, respectively, with both sigma factors recognizing the same promoter region. Site-directed mutagenesis identified an extended − 10 region that was essential to both σ 70 and σ s transcription of dps. cAMP receptor protein (CRP) was found to repress dps expression as a crp mutant had a significant increase in the dps mRNA level. However, a CRP binding site was not found in the dps promoter and upregulation of dps in the crp mutant was absent in a crp rpoS double mutant. The findings from this study demonstrated that dps was expressed at a basal level during growth, both σ 70- and σ s-driven transcription required an extended − 10, and CRP repression is mediated through the alternative sigma factor σ s ( rpoS).

Characterization of Mycobacterium tuberculosis Rv3676 (CRPMt), a cyclic AMP receptor protein-like DNA binding protein.

Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRP(Mt), is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP(Mt)'s interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP(Mt) DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for Escherichia coli. CRP(Mt) binding sites were identified in a total of 73 promoter regions regulating 114 genes in the M. tuberculosis genome, which are being explored as a regulon. Specific CRP(Mt) binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP(Mt). cAMP enhanced CRP(Mt)'s ability to bind DNA and caused allosteric alterations in CRP(Mt) conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in M. tuberculosis, suggesting a role for cAMP signal transduction in M. tuberculosis and implicating CRP(Mt) as a cAMP-responsive global regulator.

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l -Rhamnose-Responsive rhaSR Promoter in Escherichia coli

The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position -111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the alpha-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

Isotope‐edited UV Raman spectroscopy of protein–DNA interactions: binding modes of cyclic AMP receptor protein to a natural DNA recognition site

AbstractIsotope‐edited UV Raman spectroscopy was applied to the study of the binding mode of cyclic AMP (cAMP) receptor protein (CRP) to a 22‐mer oligonucleotide (LacDNA) representing the primary CRP binding site of the E. coli lac promoter. LacDNA contains four guanine residues in the consensus pentamer regions (G5 and G7 on the sense strand and G5′ and G7′ on the anti‐sense strand) and they were individually labeled with deuterium at C(8) on the guanine ring. In the UV (251 nm) Raman difference spectrum between unlabeled and C(8)‐D‐labeled LacDNA, a sharp positive/negative peak pair appeared at ∼1490/1465 cm−1, which was assigned to a guanine ring vibration (ν6/ν6′) sensitive to the hydrogen bonding state at C(6)O. CRP is a dimeric protein and forms two complexes with the cofactor cAMP, i.e. half‐filled CRP–(cAMP)1 and fully liganded CRP–(cAMP)2. The ν6/ν6′ wavenumbers measured in the presence of CRP showed that CRP–(cAMP)1 binds to G5 and G5′ in a symmetric manner, whereas CRP–(cAMP)2 exhibits an additional binding to G7. Since the base sequence of LacDNA and the structure of CRP–(cAMP)1 are both asymmetric, the symmetric LacDNA–CRP–(cAMP)1 interaction suggests that CRP–(cAMP)1, which is considered to be dominant under physiological conditions, has a conformational flexibility to conform to structural asymmetry of natural DNA sequences. In contrast, the symmetric complex CRP–(cAMP)2 binds to LacDNA asymmetrically, suggesting a decreased flexibility of CRP in the fully liganded form. Isotope‐edited UV Raman spectroscopy provides unique information on the DNA recognition by CRP. Copyright © 2005 John Wiley & Sons, Ltd.

A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor.

Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

Because the phosphoenolpyruvate:sugar phosphotransferase system plays multiple regulatory roles in addition to the phosphorylation-coupled transport of many sugars in bacteria, synthesis of its protein components is regulated in a highly sophisticated way. Thus far, the cAMP receptor protein (CRP) complex and Mlc are known to be the major regulators of ptsHIcrr and ptsG expression in response to the availability of carbon sources. In this report, we performed ligand fishing experiments by using the promoters of ptsHIcrr and ptsG as bait to find out new factors involved in the transcriptional regulation of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, and we found that the anaerobic regulator ArcA specifically binds to the promoters. Deletion of the arcA gene caused about a 2-fold increase in the ptsG expression, and overexpression of ArcA significantly decreased glucose consumption. In vitro transcription assays showed that phospho-ArcA (ArcA-P) represses ptsG P1 transcription. DNase I footprinting experiments revealed that ArcA-P binds to three sites upstream of the ptsG P1 promoter, two of which overlap the CRP-binding sites, and the ArcA-P binding decreases the CRP binding that is essential for the ptsG P1 transcription. These results suggest that the response regulator ArcA regulates expression of enzyme IICB(Glc) mediating the first step of glucose metabolism in response to the redox conditions of growth in E. coli.

Calorimetric Analysis of Mutagenic Effects on Protein–Ligand Interactions

Analysis of the effects of indirect T127→L mutagenesis on the thermodynamic changes in cooperativity of cAMP binding to cAMP receptor protein (CRP) has relied heavily on analysis of the structural changes induced in CRP by this mutation. This analysis results in the development of a structural model that best describes how the conformational changes induced by the T127→L mutation result in the observed thermodynamic changes in the cyclic nucleoside monophosphate (cNMP)–CRP binding interaction. The structural methods employed in this analysis of indirect mutagenesis effects included X-ray crystallography, small-angle neutron scattering (SANS), and circular dichroism (CD) measurements. Furthermore, it should be emphasized that this structural model offers an explanation based on the presently available structural and thermodynamic data about this system and could, thus, be changed as more experimental data about this intriguing system become available. Analysis of the effect of indirect mutagenesis on the thermodynamics of protein–ligand interactions involves development of a structural model to explain how mutations away from the binding site change the observed thermodynamics of the binding reaction: this is not as straight-forward as analysis of the effect of direct mutagenesis on the thermodynamics of protein–ligand interactions in terms of changes in the solvent-accessible surface areas at the binding site.

The cyclic AMP-cyclic AMP receptor protein complex regulates activity of the traJ promoter of the Escherichia coli conjugative plasmid pRK100.

The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system. A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100. The defect in the cya mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region. DNase I footprint experiments mapped the CRP binding site around position -67.5 upstream of the putative traJ promoter. Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100.

Promoter Analysis and Regulatory Characteristics of vvhBAEncoding Cytolytic Hemolysin of Vibrio vulnificus

Cytolytic hemolysin, a gene product of vvhA, is a putative virulence factor of the pathogenic bacterium Vibrio vulnificus. We have previously shown that hemolysin production is repressed by adding glucose to culture media and that production can be restored by adding cAMP. In this study, hemolysin activity and the level of vvh transcript were determined to reach a maximum in late exponential phase and were repressed when cells entered stationary phase. Northern blot and primer extension analyses revealed that vvhA is cotranscribed with a second gene, vvhB, located upstream of vvhA. Transcription of the vvhBA operon begins at a single site and is under the direction of a single promoter, P(vvh). A crp null mutation decreased hemolysin production and the level of vvhBA transcript by reducing the activity of P(vvh), indicating that the P(vvh) activity is under the positive control of cAMP receptor protein (CRP). A direct interaction between CRP and the regulatory region of the vvhBA operon was demonstrated by gel-mobility shift assays. The CRP binding site, centered at 59.5 bp upstream of the transcription start site, was mapped by deletion analysis of the vvhBA promoter region and confirmed by DNase I protection assays. These results demonstrate that the vvhBA expression is activated by CRP in a growth-dependent manner and that CRP exerts its effects by directly binding to DNA upstream of P(vvh).

ToxR interferes with CRP-dependent transcriptional activation of ompT in Vibrio cholerae.

In pathogenic Vibrio cholerae, the transmembrane DNA-binding protein ToxR co-ordinates the expression of over 20 genes, including those encoding important virulence factors such as cholera toxin and the toxin-co-regulated pilus. The outer membrane protein OmpT is the only member of the ToxR regulon known to be repressed by ToxR. In this study, we examined the environmental conditions that regulate OmpT expression and demonstrated that ompT transcription is upregulated 14-fold when the bacteria enter late log phase from early log phase. Deletion of the crp gene completely abolishes OmpT expression. Comparison of ompT transcription levels in the isogenic crp-, toxR- and crp-toxR- mutants revealed that (i) in the absence of ToxR, constitutive high-level ompT transcription is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP-dependent ompT activation, but also abolishes the CRP-independent, basal level ompT transcription; thus, the mechanism by which ToxR represses ompT transcription involves both antiactivation and direct repression; (iii) both CRP and ToxR are required for the regulation of OmpT expression by growth phase. To provide further insights into the molecular mecha-nism of CRP-dependent activation of ompT transcription, we demonstrated that CRP-dependent activation requires a CRP binding site centred at -310 of the ompT promoter, without which the interaction of CRP with other CRP binding site(s) more proximal to the promoter results in repression. Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase (RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from -310 of the ompT promoter via DNA looping.