Quorum-sensing Regulator HapR Research Articles

Regulation mechanism of the quorum sensing regulator AphA on the type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis

Objective: To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods: Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), ΔaphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter-lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp (tssD2) and vgrG (tssI2), and the quorum sensing regulator HapR in WT and ΔaphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results: The mRNA expression levels of tssB2, hcp(tssD2), vgrG (tssI2), and hapR as well as the protein expression and secretion levels of Hcp in ΔaphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in ΔaphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions: AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).

Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR

ObjectiveThis study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR. MethodsThe roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting. ResultsVCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR. ConclusionOverexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.

Structural Insights into Regulation of Vibrio Virulence Gene Networks.

One of the best studied aspects of pathogenic Vibrios are the virulence cascades that lead to the production of virulence factors and, ultimately, clinical outcomes. In this chapter, we will examine the regulation of Vibrio virulence gene networks from a structural and biochemical perspective. We will discuss the recent research into the numerous proteins that contribute to regulating virulence in Vibrio spp such as quorum sensing regulator HapR, the transcription factors AphA and AphB, or the virulence regulators ToxR and ToxT. We highlight how insights gained from these studies are already illuminating the basic molecular mechanisms by which the virulence cascade of pathogenic Vibrios unfold and contend that understanding how protein interactions contribute to the host-pathogen communications will enable the development of new antivirulence compounds that can effectively target these pathogens.

Vibrio cholerae high cell density quorum sensing activates the host intestinal innate immune response.

SUMMARYQuorum sensing fundamentally alters the interaction of Vibrio cholerae with aquatic environments, environmental hosts, and the human intestine. At high cell density, the quorum-sensing regulator HapR represses not only expression of cholera toxin and the toxin co-regulated pilus, virulence factors essential in human infection, but also synthesis of the Vibrio polysaccharide (VPS) exopolysaccharide-based matrix required for abiotic and biotic surface attachment. Here, we describe a feature of V. cholerae quorum sensing that shifts the host-pathogen interaction toward commensalism. By repressing pathogen consumptive anabolic metabolism and, in particular, tryptophan uptake, V. cholerae HapR stimulates host intestinal serotonin production. This, in turn, activates host intestinal innate immune signaling to promote host survival.

A proteome-wide screen to identify transcription factors interacting with the Vibrio cholerae rpoS promoter

We describe a proteomic approach to identify transcription factors binding to a target promoter. The method's usefulness was tested by identifying proteins binding to the Vibrio cholerae rpoS promoter in response to cell density. Proteins identified in this screen included the nucleoid-associated protein Fis and the quorum sensing regulator HapR.

Biochemical screening approach to identify regulatory DNA-binding proteins

The interaction between transcription factors, promoter elements and RNA polymerase is crucial to bacterial adaptation to physical and chemical changes in the environment. Transcription factor discovery has strongly relied on the isolation of regulatory mutants followed by biochemical confirmation. However, highly pleiotropic regulatory mutations can often be deleterious, unstable and difficult to select, or could directly or indirectly affect gene expression at multiple levels. Here, we describe a biochemical approach to identify trans-acting regulatory proteins independent of their cellular function that could be used as an alternative to genetic screens. The method consists in (i) incubating bacterial lysates with an immobilized DNA encompassing a promoter as defined by RNA-Seq data (ii) pull down of bound proteins and (iii) liquid chromatography tandem mass spectrometry (LC/MS/MS). We tested the usefulness of this approach by identifying proteins binding to the Vibrio cholerae rpoS promoter that drives the expression of the general stress response regulator RpoS. The approach identified several proteins binding to the rpoS promoter that included the factor for inversion stimulation (FIS), and the master quorum sensing regulator HapR. Binding of both purified proteins to the rpoS promoter was confirmed by electrophoresis mobility shift assays. The role of fis and hapR on rpoS expression was examined in strains containing a chromosomally-integrated rpoS-lacZ fusion. Deletion of fis had little effect while HapR appeared to enhance rpoS expression.

Expression of Hemolysin Is Regulated Under the Collective Actions of HapR, Fur, and HlyU in Vibrio cholerae El Tor Serogroup O1.

The biotype El Tor of serogroup O1 and most of the non-O1/non-O139 strains of Vibrio cholerae can produce an extracellular pore-forming toxin known as cholera hemolysin (HlyA). Expression of HlyA has been previously reported to be regulated by the quorum sensing (QS) and the regulatory proteins HlyU and Fur, but lacks the direct evidence for their binding to the promoter of hlyA. In the present work, we showed that the QS regulator HapR, along with Fur and HlyU, regulates the transcription of hlyA in V. cholerae El Tor biotype. At the late mid-logarithmic growth phase, HapR binds to the three promoters of fur, hlyU, and hlyA to repress their transcription. At the early mid-logarithmic growth phase, Fur binds to the promoters of hlyU and hlyA to repress their transcription; meanwhile, HlyU binds to the promoter of hlyA to activate its transcription, but it manifests direct inhibition of its own gene. The highest transcriptional level of hlyA occurs at an OD600 value of around 0.6–0.7, which may be due to the subtle regulation of HapR, Fur, and HlyU. The complex regulation of HapR, Fur, and HlyU on hlyA would be beneficial to the invasion and pathogenesis of V. cholerae during the different infection stages.

Molecular basis for the differential expression of the global regulator VieA in Vibrio cholerae biotypes directed by H-NS, LeuO and quorum sensing.

VieA is a cyclic diguanylate phosphodiesterase that modulates biofilm development and motility in Vibrio cholerae O1 of the classical biotype. vieA is part of an operon encoding the VieSAB signal transduction pathway that is nearly silent in V. cholerae of the El Tor biotype. A DNA pull-down assay for proteins interacting with the vieSAB promoter identified the LysR-type regulator LeuO. We show that in classical biotype V. cholerae, LeuO cooperates with the nucleoid-associated protein H-NS to repress vieSAB transcription. LeuO and H-NS interacted with the vieSAB promoter of both biotypes with similar affinities and protected overlapping DNA sequences. H-NS was expressed at similar levels in both cholera biotypes. In contrast, El Tor biotype strains expressed negligible LeuO under identical conditions. In El Tor biotype vibrios, transcription of vieSAB is repressed by the quorum sensing regulator HapR, which is absent in classical biotype strains. Restoring HapR expression in classical biotype V. cholerae repressed vieSAB transcription by binding to its promoter. We propose that double locking of the vieSAB promoter by H-NS and HapR in the El Tor biotype prior to the cessation of exponential growth results in a more pronounced decline in VieA specific activity compared to the classical biotype.

RNA-seq analysis identifies new genes regulated by the histone-like nucleoid structuring protein (H-NS) affecting Vibrio cholerae virulence, stress response and chemotaxis.

The histone-like nucleoid structuring protein (H-NS) functions as a transcriptional silencer by binding to AT-rich sequences at bacterial promoters. However, H-NS repression can be counteracted by other transcription factors in response to environmental changes. The identification of potential toxic factors, the expression of which is prevented by H-NS could facilitate the discovery of new regulatory proteins that may contribute to the emergence of new pathogenic variants by anti-silencing. Vibrio cholerae hns mutants of the El Tor biotype exhibit altered virulence, motility and environmental stress response phenotypes compared to wild type. We used an RNA-seq analysis approach to determine the basis of the above hns phenotypes and identify new targets of H-NS transcriptional silencing. H-NS affected the expression of 18% of all predicted genes in a growth phase-dependent manner. Loss of H-NS resulted in diminished expression of numerous genes encoding methyl-accepting chemotaxis proteins as well as chemotaxis toward the attractants glycine and serine. Deletion of hns also induced an endogenous envelope stress response resulting in elevated expression of rpoE encoding the extracytoplamic sigma factor E (σE). The RNA-seq analysis identified new genes directly repressed by H-NS that can affect virulence and biofilm development in the El Tor biotype cholera bacterium. We show that H-NS and the quorum sensing regulator HapR silence the transcription of the vieSAB three-component regulatory system in El Tor biotype V. cholerae. We also demonstrate that H-NS directly represses the transcription of hlyA (hemolysin), rtxCA (the repeat in toxin or RTX), rtxBDE (RTX transport) and the biosynthesis of indole. Of these genes, H-NS occupancy at the hlyA promoter was diminished by overexpression of the transcription activator HlyU. We discuss the role of H-NS transcriptional silencing in phenotypic differences exhibited by V. cholerae biotypes.

Quorum regulatory small RNAs repress type VI secretion in Vibrio cholerae

Type VI secretion is critical for Vibrio cholerae to successfully combat phagocytic eukaryotes and to survive in the presence of competing bacterial species. V. cholerae type VI secretion system genes are encoded in one large and two small clusters. In V. cholerae, type VI secretion is controlled by quorum sensing, the cell-cell communication process that enables bacteria to orchestrate group behaviours. The quorum-sensing response regulator LuxO represses type VI secretion genes at low cell density and the quorum-sensing regulator HapR activates type VI secretion genes at high cell density. We demonstrate that the quorum regulatory small RNAs (Qrr sRNAs) that function between LuxO and HapR in the quorum-sensing cascade are required for these regulatory effects. The Qrr sRNAs control type VI secretion via two mechanisms: they repress expression of the large type VI secretion system cluster through base pairing and they repress HapR, the activator of the two small type VI secretion clusters. This regulatory arrangement ensures that the large cluster encoding many components of the secretory machine is expressed prior to the two small clusters that encode the secreted effectors. Qrr sRNA-dependent regulation of the type VI secretion system is conserved in pandemic and non-pandemic V. cholerae strains.

A Highly Specific Cell-Based High-Throughput Screening Assay for Ligands of Cyclic Adenosine Monophosphate Receptor Protein in Gram-Negative Bacteria

Quorum sensing is a cell-cell communication process in bacteria that involves the production, release, and subsequent detection of chemical signal molecules called autoinducers. In Vibrio cholerae, multiple input signals activate the expression of the quorum sensing regulator HapR, which acts to repress the expression of virulence factors. We have shown that CRP, the cyclic adenosine monophosphate (cAMP) receptor protein, enhances quorum sensing by activating the biosynthesis of cholera autoinducer 1, the major signaling molecule that contributes to the activation of HapR. Thus, proquorum sensing CRP agonists could inhibit virulence and lead to new drugs to treat severe cholera. In this study, we show that expression of the quorum sensing-regulated luxCDABE operon can be used as a robust readout for CRP activity. Further, we describe and validate a highly specific cell-based luminescence high-throughput screening assay for proquorum sensing CRP ligands. A pilot screen of 9,425 compounds yielded a hit rate of 0.02%, one hit being cAMP itself. The Z' value for this assay was 0.76 and its coefficient of variance 8% for the positive control compound. To our knowledge, this is the first cell-based assay for ligands of the highly conserved CRP protein of Gram-negative bacteria. The use of this assay to screen large chemical libraries could identify lead compounds to treat cholera, as well as small molecules to probe ligand-receptor interactions in the CRP molecule.

Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease

Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ΔhapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.

NOD-Like Receptor Activation by Outer Membrane Vesicles from Vibrio cholerae Non-O1 Non-O139 Strains Is Modulated by the Quorum-Sensing Regulator HapR

Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses, and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. Small interfering RNA (siRNA)-mediated gene knockdown revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain-, leucine-rich repeat-containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum-sensing regulator HapR, which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum-sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs under infective conditions. This work thus identifies a new facet of how Vibrio affects host immune responses and defines a role for the quorum-sensing machinery in this process.

Differential response of Vibrio cholerae planktonic and biofilm cells to autoinducer 2 deficiency

The formation of biofilm communities enhances the persistence of Vibrio cholerae in aquatic environments. Biofilm production is repressed by the quorum-sensing regulator HapR in response to the accumulation of CAI-1 and AI-2. CAI-1 is the strongest input signal activating HapR, whereas the role of AI-2 remains ill-defined. In the present study, we show that a V. cholerae luxS (AI-2-defective) mutant made increased biofilm. Interestingly, cells in the biofilm were more responsive to AI-2 deficiency than cells from the planktonic population.

Cyclic AMP post-transcriptionally regulates the biosynthesis of a major bacterial autoinducer to modulate the cell density required to activate quorum sensing

In Vibrio cholerae, expression of the quorum sensing regulator HapR is induced by the accumulation of a major autoinducer synthesized by the activity of CqsA. Here we show that the cAMP–cAMP receptor protein complex regulates cqsA expression at the post-transcriptional level. This conclusion is supported by the analysis of cqsA–lacZ fusions, the ectopic expression of cqsA in Δcrp mutants and by Northern blot analysis showing that cqsA mRNA is unstable in Δcrp and Δcya (adenylate cyclase) mutants. Addition of cAMP to the culture of a Δcya mutant restored cqsA mRNA stability and cholera autoinducer 1 production. Lowering intracellular cAMP levels by addition of d-glucose increased the cell density required to activate HapR. These results indicate that cAMP acts as a quorum modulator.

The Extracellular Nuclease Dns and Its Role in Natural Transformation of Vibrio cholerae

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification.

The Cyclic AMP Receptor Protein Modulates Colonial Morphology in Vibrio cholerae

Inactivation of the quorum-sensing regulator HapR causes Vibrio cholerae El Tor biotype strain C7258 to adopt a rugose colonial morphology that correlates with enhanced biofilm formation. V. cholerae mutants lacking the cyclic AMP (cAMP) receptor protein (CRP) produce very little HapR, which results in elevated expression of Vibrio exopolysaccharide (vps) genes and biofilm compared to the wild type. However, Deltacrp mutants still exhibited smooth colonial morphology and expressed reduced levels of vps genes compared to isogenic hapR mutants. In this study we demonstrate that deletion of crp and cya (adenylate cyclase) converts a rugose DeltahapR mutant to a smooth one. The smooth DeltahapR Deltacrp and DeltahapR Deltacya double mutants could be converted back to rugose by complementation with crp and cya, respectively. CRP was found to enhance the expression of VpsR, a strong activator of vps expression, but to diminish transcription of VpsT. Ectopic expression of VpsR in smooth DeltahapR Deltacrp and DeltahapR Deltacya double mutants restored rugose colonial morphology. Lowering intracellular cAMP levels in a DeltahapR mutant by the addition of glucose diminished VpsR expression and colonial rugosity. On the basis of our results, we propose a model for the regulatory input of CRP on exopolysaccharide biosynthesis.

The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae

Vibrio cholerae is the causative agent of cholera, which continues to be a major public health concern in Asia, Africa and Latin America. The bacterium can persist outside the human host and alternates between planktonic and biofilm community lifestyles. Transition between the different lifestyles is mediated by multiple signal transduction pathways including quorum sensing. Expression of the Zn-metalloprotease haemagglutinin (HA)/protease is subject to a dual regulation which involves the quorum-sensing regulator HapR and the cAMP receptor protein. In a previous study, we observed that a mutant defective in the cAMP-receptor protein (CRP) expressed lower levels of HapR. To further investigate the role of CRP in modulating HapR and other signal transduction pathways, we performed global gene expression profiling of a Deltacrp mutant of El Tor biotype V. cholerae. Here we show that CRP is required for the biosynthesis of cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected, the Deltacrp mutant produced more cholera toxin and enhanced biofilm. Expression of flagellar genes, reported to be affected in DeltahapR mutants, was diminished in the Deltacrp mutant. However, an epistasis analysis indicated that cAMP-CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. cholerae to colonize the small intestine and cause disease. These genes included ompU, ompT and ompW encoding outer-membrane proteins, the alternative sigma factor sigma(E) required for intestinal colonization, and genes involved in anaerobic energy metabolism. Our results indicate that CRP plays a crucial role in the V. cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. cholerae in the human host and the environment.

The quorum sensing regulator HapR downregulates the expression of the virulence gene transcription factor AphA in Vibrio cholerae by antagonizing Lrp‐ and VpsR‐mediated activation

HapR is a quorum sensing-regulated transcription factor that represses the virulence cascade in Vibrio cholerae by binding to a specific site centred at -71 in the aphA promoter, ultimately preventing activation of the tcpPH promoter on the Vibrio pathogenicity island. In an effort to elucidate the mechanism by which HapR represses aphA expression, we identified two transcriptional regulators, Lrp and VpsR, both of which activate the aphA promoter. Lrp, the leucine-responsive regulatory protein, binds to a region between -136 and -123 in the promoter to initiate aphA expression. VpsR, the response regulator that controls biofilm formation, binds to a region between -123 and -73 to activate aphA expression. HapR represses aphA expression by antagonizing the functions of both of these activators. The HapR binding site at -71 lies downstream of the Lrp binding site and overlaps the VpsR binding site. HapR binding thus directly blocks access of VpsR to the promoter. A naturally occurring point mutation in the aphA promoter (G-77T), which has previously been shown to prevent HapR binding, also prevents VpsR binding. In the absence of HapR, either Lrp or VpsR is capable of achieving nearly full expression of the aphA promoter, but when present together their effects are to some degree additive. The aphA promoter is also negatively autoregulated and an AphA binding site is centred at -20. The results here provide a model for the dual activation of the aphA promoter by Lrp and VpsR as well as its dual repression by HapR and AphA.

Transcriptional regulation of Vibrio cholerae hemagglutinin/protease by the cyclic AMP receptor protein and RpoS.

Vibrio cholerae secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HA/protease), which is encoded by hapA and displays a broad range of potentially pathogenic activities. Production of HA/protease requires transcriptional activation by the quorum-sensing regulator HapR. In this study we demonstrate that transcription of hapA is growth phase dependent and specifically activated in the deceleration and stationary growth phases. Addition of glucose in these phases repressed hapA transcription by inducing V. cholerae to resume exponential growth, which in turn diminished the expression of a rpoS-lacZ transcriptional fusion. Contrary to a previous observation, we demonstrate that transcription of hapA requires the rpoS-encoded sigma(s) factor. The cyclic AMP (cAMP) receptor protein (CRP) strongly enhanced hapA transcription in the deceleration phase. Analysis of rpoS and hapR mRNA in isogenic CRP+ and CRP- strains suggested that CRP enhances the transcription of rpoS and hapR. Analysis of strains containing hapR-lacZ and hapA-lacZ fusions confirmed that hapA is transcribed in response to concurrent quorum-sensing and nutrient limitation stimuli. Mutations inactivating the stringent response regulator RelA and the HapR-controlled AphA regulator did not affect HA/protease expression. Electrophoretic mobility shift experiments showed that pure cAMP-CRP and HapR alone do not bind the hapA promoter. This result suggests that HapR activation of hapA differs from its interaction with the aphA promoter and could involve additional factors.