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

Abstract

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.

FIG. 1.

RNase protection assay analysis of hapA expression. V. cholerae strains were grown in TSB medium to an OD₆₀₀ of 2 (deceleration phase). At this point, 10-ml aliquots were withdrawn for total-RNA extraction (lanes 2 to 6) and the remaining culture was further incubated for 16 h (late stationary phase) (lane 7 to 11). Lanes: 1, intact probe (RNase-minus control); 2 and 7, C6706; 3 and 8 C6709-1; 4 and 9, KSK394 (crp); 5 and 10; N16961 (hapR); 6 and 11; DSM-V491 (rpoS). MW, molecular size in base pairs.

FIG. 2.

Transcription of rpoS, hapR, and crp in wild-type and isogenic regulatory mutants. V. cholerae strains were grown in TSB to an OD₆₀₀ of 2, and RT-PCR was conducted as described in Materials and Methods. (A) Lanes: 1, 3, and 5, C6706 (crp⁺); 2, 4, and 6, KSK394 (crp). (B) Lanes: 1, 3, and 5 C6709-1 (rpoS⁺); 2, 4, and 6, DMS-V491 (rpoS). The mRNA detected in each lane is written below each panel. (C) Real-time RT-PCR. RNA extracted from three cultures of C6706 and KSK394 was analyzed for the relative expression of rpoS and hapR (targets) with recA mRNA as reference.

FIG. 3.

Glucose repression does not require CRP. Strains AJB2 (A) and AJB3 (B) containing a hapA-lacZ transcriptional fusion inserted in the hapA locus in a crp⁺ (AC-V66) and crp (KSK394) background, respectively, were grown in TSB medium to an OD₆₀₀ of 1. Cultures were divided in half and further incubated for 4-h with (▪) and without (□) glucose. Samples were taken hourly for enzyme assays and OD₆₀₀ determinations. Each value represents the average of three independent cultures. Error bars indicate the standard deviation of the mean.

FIG. 4.

Correlation between glucose repression and growth stimulation. (A and B) Strain AJB2 was grown to an OD₆₀₀ of 2, and the culture was divided in half. Glucose was added to one half (B), and the other half was used as a control (A). Samples were taken at 1-h intervals for β-Galactosidase activity (▪) and OD₆₀₀ readings (□). (C) Strain AJB29 containing a rpoS-lacZ transcriptional fusion was grown in TSB to an OD₆₀₀ of 1, and the culture was divided in half. Half was supplemented with glucose (▪), and half was used as a control (□). Relative activities in panels A and B refer to the Miller units at the time of glucose supplementation. Final Miller units reported are the mean of three independent cultures. Error bars indicate the standard deviation of the mean.

FIG. 5.

Effect of a nutritional downshift on hapA promoter activity. (A) V. cholerae AJB2 was grown to an OD₆₀₀ of 0.5. Half of the culture was centrifuged, and the cells were resuspended in 0.25 × TSB (▪); the remaining cells were kept in 1 × TSB (□). Cultures were analyzed for β-galactosidase activity at different time points. (B) Overnight cultures of strain AJB26 (hapR-lacZ) (open bars) and AJB2 (hapA-lacZ) (shaded bars) were diluted in TSB medium of different strengths and incubated at 37°C until late stationary phase. Cultures were analyzed for hapR- and hapA-driven β-galactosidase expression. The final Miller units reported represent the mean of three independent cultures. Error bars indicate the standard deviation of the mean.

FIG. 6.

Western blot analysis of HA/protease expression in V. cholerae mutants. Supernatants from V. cholerae strains grown to saturation in TSB medium were concentrated in Centricon-10 centrifugal filters (Amicon Bioseparations), subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide) and transferred to a polyvinylidene difluoride membrane. Lanes: 1, C7258; 2, AJB19; 3, N16961; 4, AJB216; 5, AC-V66; 6, AJB231; 7, DSM-V491; 8, pure HA/protease.

FIG. 7.

DNA binding assays. (A) His₆-HapR. DIG-labeled DNA fragments (10 ng) were incubated with no protein (lane 1), 64 ng of protein (lane 2), 128 ng of protein (lane 3), no protein (lane 4), 64 ng of protein (lane 5), 128 ng of protein (lane 6), no protein (lane 7), and 64 ng of protein (lane 8) (B) His₆-CRP. DIG-labeled DNA fragments (10 ng) were incubated with no protein (lane 1), 50 ng of protein and cAMP (lane 2), 50 ng of protein (lane 3), 500 ng of protein and cAMP (lane 4), 500 ng of protein (lane 5), no protein (lane 6), no protein with cAMP (lane 7), 500 ng of protein and cAMP (lane 8), and 500 ng of protein (lane 9). The concentration of cAMP was 500 μM.

FIG. 8.

Regulatory interactions involved in hapA transcription regulation. The hapA gene is transcribed in response to two concurrent environmental signals: high cell density and nutrient limitation. Nutrient limitation leads to entry of bacteria into the stationary phase, with enhanced transcription of rpoS and high levels of σ^S. At high cell density in the stationary phase, HapR is expressed and, in combination with an unidentified factor (X) and σ^S, activates the transcription of hapA. Glucose represses hapA transcription by inducing cells to resume exponential growth, blocking the pathway to accumulation of σ^S. The cAMP-CRP complex enhances the transcription of hapA by positively influencing the transcription of rpoS and hapR. CRP could increase HapR levels by acting on luxO or hapR. HapR represses the expression of AphA, required for the production of CT and TCP. In the absence of AphA, pva, encoding penicillin V amidase, is expressed.