cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization

Repression of the rtxBDE operon by CRP. (a) The intergenic region between rtxBDE and rtxHCA is associated with CRP but not σ⁷⁰. The graphs illustrate ChIP-seq data for CRP (orange) and σ⁷⁰ (blue) binding to the rtx locus. Data have been smoothed in a 100-bp window. Genes are depicted by mauve arrows and labeled. (b) Sequence of the rtxBDE-rtxHCA gene regulatory region. The DNA sequence between rtxBDE and rtxHCA is shown. The center of the CRP binding peak identified in our ChIP-seq analysis is indicated by an asterisk. Putative CRP sites (orange) are underlined and labeled. The rtxH transcription start site (+1) is underlined and further highlighted by a bent arrow. The associated promoter −10 element is similarly colored and underlined. Two transcription start sites for rtxB are also labeled in the same way. The 5′ end of the rtxB.1 DNA fragment (see the legend to panel e) is indicated by an inverted black triangle. Point mutations used to inactivate CRP binding sites or promoter −10 elements are shown in red. (c) DNase I footprint of CRP binding to the rtxBDE-rtxHCA gene regulatory region. Results of a DNase I footprinting experiment using the rtxBDE intergenic region and purified V. cholerae CRP. The experiment is calibrated with a Maxam-Gilbert GA sequencing ladder, and positions relative to the P1rtxB transcription start site (+1) are labeled. The triangle indicates the addition of CRP at concentrations of 175, 350, 700, 1,400, 2,100, or 2,800 nM. The positions of the predicted CRP binding sites are shown by orange boxes. (d) Primer extension analysis of rtxH and rtxB promoter-derived transcripts. The gel shows arbitrary Sanger sequencing reactions for calibration (lanes 1 to 4) and primer extension products for rtxB (lanes 5 and 6) or rtxH (lanes 7 and 8) promoter-derived transcripts. (e) Transcripts derived from the rtxBDE intergenic region in vitro. The gel shows transcripts generated by V. cholerae RNA polymerase σ⁷⁰ holoenzyme using the rtxBDE intergenic region, cloned in plasmid pSR, as a DNA template. The RNAI transcript is derived from the plasmid replication origin and serves as an internal control. The rtxB.1 derivative contains a truncated version of the rtxBDE intergenic region. The site of the truncation is marked in panel b. Mutations introduced to disrupt potential −10 hexamers are noted above the gel and are also shown in panel b. (f) Activity of PrtxH is not affected by CRP. Results of a β-galactosidase assay done using lysates of N16961 cells transformed with derivatives of the lacZ reporter plasmid, pRW50T, where lacZ expression is controlled by PrtxH. (g) Expression of rtxB is repressed by CRP. Results of a β-galactosidase assay done using lysates of N16961 cells transformed with derivatives of the lacZ reporter plasmid, pRW50T, where lacZ expression is controlled by P1rtxB and P2rtxB.

Nutrient availability controls rtxBDE expression in a CRP-dependent manner. Results of β-galactosidase assays done using lysates of N16961 cells transformed with derivatives of the lacZ reporter plasmid, pRW50T, carrying different rtxB::lacZ fusions. Cells were grown in M9 minimal medium, LB, or LB supplemented with 0.4% (vol/vol) glucose.

CRP is required for efficient host colonization and dependent induction of rtxBDE. (a) Colonization of the zebrafish larva intestinal tract by V. cholerae strain E7946 and derivatives. The three panels show representative fluorescence microscopy images overlaid on light microscopy images of zebrafish larvae colonized with the indicated V. cholerae strains. All bacterial strains were transformed with plasmid pMW-GFP and express green fluorescent protein (GFP) to facilitate detection. Further images are shown in Fig. S1c in the supplemental material. (b) Survival of zebrafish larvae following infection with V. cholerae strain E7946 and derivatives. (c) Expression of rtxBDE is induced by zebrafish larva colonization. Results of a β-galactosidase assay done using lysates of bacterial cells growing planktonically in E3 medium or obtained from the zebrafish intestinal tract. Strains are indicated and were transformed with derivatives of pRW50T encoding different rtxB::lacZ fusions.