INTRODUCTION {#s0}
============

Vibrio cholerae is a Gram-negative bacterium that causes the diarrheal disease cholera ([@B1]). Estimated to claim 3 to 5 million victims every year, cholera is endemic in regions of Asia and sub-Saharan Africa ([@B1][@B2][@B4]). Localized epidemics are also frequent; half a million cases have been attributed to the current outbreak in Yemen ([@B2]). Although notorious as a pathogen of humans, V. cholerae is native to aquatic environments ([@B5]). In this situation, the organism proliferates by colonizing crustaceans and other biota in their habitat ([@B5][@B6][@B10]). In particular, chitinous surfaces provide a substrate for biofilm formation and nutrients ([@B5]). Humans encounter V. cholerae following the ingestion of contaminated food or water ([@B1]). In response, the bacterium produces mucin-degrading enzymes and upregulates motility ([@B5]). This facilitates penetration of the intestinal mucosa ([@B5]). The subsequent attachment of V. cholerae cells to the intestinal epithelium requires toxin-coregulated pili (TCP) and accessory colonization factors (ACF) ([@B11][@B12][@B13]). Ultimately, disease results from the production of factors including cholera toxin (CTX), repeats in toxin (RTX), and hemolysin (HlyA) ([@B42], [@B63][@B64][@B65]).

Unsurprisingly, the expression of V. cholerae genes for quorum sensing, host colonization, and toxin production/export is precisely regulated ([@B5]). Most notably, an AraC/XylS family transcription factor called ToxT directly regulates the transcription of *ctxAB*, *acfAD*, and genes encoding the TCP ([@B14]). Production of ToxT is induced in the intestine and is codependent on two OmpR family regulators, ToxR and TcpP, which respond to extracellular signals that include osmolarity, pH, and bile ([@B15][@B16][@B17]). Genes encoding outer membrane porins OmpT and OmpU are also regulated by ToxR in a pathway that permits initial sensing of bile and subsequent resistance ([@B18]). Together, the aforementioned gene regulatory events comprise the ToxR regulon. Transcription factors with targets overlapping the ToxR regulon include VpsT, AphA, AphB, and the cyclic AMP (cAMP) receptor protein (CRP) ([@B19][@B20][@B24]). Best studied in Escherichia coli, CRP can activate transcription by binding targets centered either 41.5 or 61.5 bp upstream from a transcription start site ([@B25]). Since CRP binds DNA in response to the intracellular availability of cAMP, genes are controlled in response to nutrient availability ([@B25][@B26][@B28]). As such, CRP plays an integral role in the utilization of alternative carbon sources ([@B29], [@B30]). Hence, many E. coli genes that are differentially regulated in the intestine are controlled by CRP, including CTX-related toxins in pathogenic E. coli strains ([@B26], [@B31]). In V. cholerae, CRP is known to influence the ToxR regulon; CRP directly inhibits *tcpP* expression and activates the transcription of *ompT* ([@B22], [@B32], [@B33]). Remarkably, despite being a global regulator of transcription, direct control by CRP has only been demonstrated for seven V. cholerae genes ([@B22], [@B31], [@B33][@B34][@B38]). Furthermore, gene regulation by CRP during colonization of a host intestinal tract has never been studied. In this work, we have used chromatin immunoprecipitation (ChIP) coupled with DNA sequencing (ChIP-seq) to map the distribution of CRP across the V. cholerae genome. We show substantial overlap between the ToxR regulon and control of additional virulence factors not regulated by the ToxR system. Focusing on one such target, encoding RTX and its export system, we show that CRP is essential for specific induction of gene expression during intestinal colonization. Examination of additional CRP target genes reveals that similar effects are widespread.

RESULTS {#s1}
=======

Genome-wide distribution of CRP and RNA polymerase in Vibrio cholerae*.* {#s1.1}
------------------------------------------------------------------------

We used ChIP-seq to map global DNA binding by CRP and the RNA polymerase σ^70^ subunit in V. cholerae strain N16961 grown to mid-log phase in M9 minimal medium supplemented with 1% (wt/vol) fructose ([@B39]). The strain, isolated from a Bangladeshi patient in 1971, comprises 3,885 genes borne on two circular chromosomes of 2,961,146 bp (chromosome I) and 1,072,314 bp (chromosome II) ([@B39]). The binding profiles of CRP and σ^70^ are shown in [Fig. 1a](#fig1){ref-type="fig"}. In each plot, genes are illustrated by mauve lines ([Fig. 1a](#fig1){ref-type="fig"}, first and second tracks), σ^70^ binding in blue ([Fig. 1a](#fig1){ref-type="fig"}, third track), and CRP binding in orange ([Fig. 1a](#fig1){ref-type="fig"}, fourth track). We identified 497 binding peaks for σ^70^ and 119 binding peaks for CRP. The σ^70^ peaks were not distributed equitably; chromosome II accounts for 27% of the V. cholerae genome but aligned with 40% of the σ^70^ binding peaks. To assess the validity of our data, we examined the DNA sequence attributes of each peak. Hence, we used MEME (Multiple Em for Motif Elicitation) to identify sequence motifs associated with σ^70^ or CRP binding. The most statistically significant DNA motif associated with each group of peaks is shown in [Fig. 1b](#fig1){ref-type="fig"}. As expected, MEME recovered significant motifs matching the sequence of a housekeeping bacterial promoter (*E* = 4.5 × 10^−29^) ([Fig. 1b](#fig1){ref-type="fig"}, top) and the palindromic CRP binding sequence (*E* = 2.5 × 10^−2^) ([Fig. 1b](#fig1){ref-type="fig"}, bottom). For all peaks, we determined the distance to the nearest start codon and sorted these distances into 100-bp bins. The distribution of peaks among the bins is illustrated in [Fig. 1c](#fig1){ref-type="fig"}; σ^70^ most frequently binds the 100 bp preceding the 5′ end of a gene, while CRP binds further upstream. Of the 119 CRP binding peaks, 67 colocated with binding of σ^70^ ([Fig. 1c](#fig1){ref-type="fig"}, inset). We also compared our data with existing compendiums of the V. cholerae transcriptome ([@B40], [@B41]). Briefly, Papenfort and coworkers used differential RNA-seq to map transcription start sites (TSS) in V. cholerae ([@B40]). There was significant overlap with our data; 81% of the σ^70^ binding peaks matched a TSS (*P* = 1.3 × 10^−302^) ([Fig. 1d](#fig1){ref-type="fig"}). Thus, the combined data describe sigma factor preference, promoter sequence, and sites of transcription initiation for the majority of V. cholerae transcription units. In a separate study, Fong and Yildiz used DNA microarrays to detect changes in RNA levels resulting from *crp* deletion ([@B41]). Again, the overlap was significant, and 52 of the 119 CRP binding peaks identified a differentially expressed gene (*P* = 2.1 × 10^−9^) ([Fig. 1e](#fig1){ref-type="fig"}). Note that greater overlap of the CRP binding and gene regulatory data is not expected; many CRP-controlled promoters are active only under specific conditions, and most transcriptome changes will result from indirect effects of CRP ([@B26][@B27][@B30]).

![Global analysis of CRP and σ^70^ binding in Vibrio cholerae. (a) Genome-wide distribution of CRP and the RNA polymerase σ^70^ subunit in Vibrio cholerae strain N16961. Plots are shown for the two N16961 chromosomes. In each plot, the tick mark at the 12 o'clock position represents the first base pair (bp) of the chromosome and subsequent tick marks are spaced by 0.5 Mbp. In each plot, the first and second tracks (mauve lines) show the positions of genes, the third track (blue) is the σ^70^ binding profile, and the fourth track (orange) is the CRP binding profile. (b) DNA sequence motifs recovered from CRP and σ^70^ binding peaks. Top, DNA sequence motif identified by MEME present in DNA sequences associated with σ^70^ binding; bottom, DNA sequence motif generated from CRP binding peaks. (c) Locations of CRP and RNA polymerase binding peaks with respect to genes. Histogram depicts the distances between ChIP-seq binding peaks and the nearest 5′ end of a gene; data for CRP binding are in orange, and data for σ^70^ binding are in blue. Each binding peak was allocated to a series of 100-bp bins. Inset, Venn diagram that illustrates the number of overlapping CRP and σ^70^ binding peaks. (d) Overlap between σ^70^ DNA binding and transcription start sites. The Venn diagram illustrates numbers of overlapping σ^70^ binding sites (blue) and transcription start sites (green) ([@B40]). A σ^70^ binding peak centered within 50 bp of a transcription start was considered to overlap. To generate the *P* value, we used the chi-square test. To generate values for the expected overlap between the data sets, assuming no correlation, we randomized the positions of the σ^70^ peaks. (e) Overlap between CRP binding and CRP-regulated genes. The Venn diagram illustrates overlap between genes adjacent to CRP binding peaks (orange) and genes that were differently expressed in the absence of CRP (mauve) ([@B41]). To generate the *P* value, we used the chi-square test. To generate values for the expected overlap between the data sets, assuming no correlation, we randomly selected 831 genes from the V. cholerae genome and determined the number that were adjacent to CRP binding peaks.](mbo0041839690001){#fig1}

Expression of the *rtxBDE* operon is repressed by CRP. {#s1.2}
------------------------------------------------------

Many V. cholerae genes involved in pathogenicity were targeted by CRP ([Table 1](#tab1){ref-type="table"}). We focused our attention on the gene cluster responsible for the cytotoxic activity of V. cholerae ([@B42]). The region comprises two operons called *rtxHCA* and *rtxBDE*. The *rtxA* gene encodes RTX toxin, while *rtxH* and *rtxC* encode a hypothetical protein and an acyltransferase, respectively. The divergent *rtxBDE* operon encodes components of the toxin secretion system. [Figure 2a](#fig2){ref-type="fig"} shows binding of CRP and σ^70^ to DNA between the two operons. The sequence of the intergenic region is shown in [Fig. 2b](#fig2){ref-type="fig"}. Two putative CRP sites (orange and underlined) overlap the center of the CRP ChIP-seq binding peak ([Fig. 2b](#fig2){ref-type="fig"}, asterisk). To confirm binding, we purified V. cholerae CRP for use in DNase I footprinting assays. The footprinting data are consistent with CRP binding to both of the putative sites ([Fig. 2c](#fig2){ref-type="fig"}). To identify promoters of *rtxHCA* and *rtxBDE* transcription, we examined our ChIP-seq data for σ^70^ and the TSS mapping data of Papenfort et al. ([@B40]). However, these were poorly informative; the σ^70^ binding levels were low and Papenfort et al. identified only a single intergenic promoter for *rtxHCA*. Indeed, the TSS data suggest that the region is prone to spurious intragenic transcription; the *rtxHCA* and *rtxBDE* genes contain a total of 21 internal promoters ([@B40]). To map canonical promoters in the *rtxHCA*-*rtxBDE* intergenic region, we used two approaches; mRNA primer extension and *in vitro* transcription. The results of the primer extension analysis are shown in [Fig. 2d](#fig2){ref-type="fig"}. We were unable to derive any extension products from *rtxBDE* transcripts, but a single 201-nucleotide extension product was generated from the *rtxHCA* transcript. The corresponding TSS aligns perfectly with the *rtxHCA* TSS identified by Papenfort et al. (labeled P*rtxH* in [Fig. 2b](#fig2){ref-type="fig"}) ([@B40]). A promoter −10 element is appropriately positioned upstream, and deletion of this sequence abolishes mRNA production (see [Fig. S1a](#figS1){ref-type="supplementary-material"} in the supplemental material). Since both RNA-seq and primer extension failed to identify promoters for *rtxBDE*, we reasoned that the operon must be repressed *in vivo*. Hence, the *rtxBDE* intergenic region was cloned upstream from the λ*oop* terminator in plasmid pSR to create a template for *in vitro* transcription. The result of the experiment is shown in lane 1 of [Fig. 2e](#fig2){ref-type="fig"}. The control RNAI transcript is derived from the plasmid replication origin, and further transcripts could originate within the cloned intergenic DNA. Truncation of the *rtxBDE* intergenic region did not prevent synthesis of the additional RNAs ([Fig. 2e](#fig2){ref-type="fig"}, lane 2). Hence, the transcripts originate downstream from the truncation site marked by the inverted triangle in [Fig. 2b](#fig2){ref-type="fig"}. We made derivatives of the truncated DNA template with point mutations in all potential −10 hexamers. The mutations are illustrated in [Fig. 2b](#fig2){ref-type="fig"}. Two pairs of mutations, −29G −28G and −12G −11G, each prevent the production of a different transcript ([Fig. 2e](#fig2){ref-type="fig"}, lanes 3 to 5). The mutations have similar effects *in vivo* ([Fig. S1b](#figS1){ref-type="supplementary-material"}). We conclude that transcription originates from the promoters labeled P1*rtxB* and P2*rtxB* in [Fig. 2b](#fig2){ref-type="fig"}. Neither CRP site is appropriately positioned to activate P*rtxH*, P1*rtxB*, or P2*rtxB*. However, it has previously been shown that pairs of CRP binding sites upstream from promoters can repress transcription ([@B43]). Thus, we created derivatives of the intergenic region where the CRP sites were inactivated by the point mutations shown in [Fig. 2b](#fig2){ref-type="fig"}. We also altered the sites to match the consensus for CRP binding. The various DNA fragments were cloned in the appropriate orientation upstream from *lacZ* in plasmid pRW50T. The resulting DNA constructs were moved into V. cholerae strain N16961 by conjugation. Promoter activity was inferred by measuring β-galactosidase activity in lysates of the exconjugants. The data show that P*rtxH* activity is unaltered by any of the mutations ([Fig. 2f](#fig2){ref-type="fig"}). Conversely, the poorly active *rtxBDE* promoters have higher levels of activity when the CRP sites are mutated ([Fig. 2g](#fig2){ref-type="fig"}, stippled bar). Consistent with our observations, Fong and Yildiz reported repression of *rtxBDE* by CRP in their transcriptome analysis ([@B41]).

10.1128/mBio.00966-18.2

\(a\) Transcription from P*rtxH* requires identified promoter elements. (i) The results of primer extension assays that detect the *rtxH* transcript derived from plasmid pRW50T carrying the *rtxH* (panel ii, top) or truncated *rtxH*.1 (panel ii, bottom) DNA fragment. (ii) P*rtxH* is highlighted blue and CRP binding sites are shown in orange. (b)** **P1*rtxB* and P2*rtxB* make similar contributions to *rtxB* transcription. The figure shows β-galactosidase activity measurements for lysates of V. cholerae cells transformed with pRW50T derivatives carrying the full-length *rtxB* regulatory region (*rtxB*), a truncated derivative lacking CRP binding sites (*rtxB*.1), or versions of the truncated fragment with indicated promoter mutations. (c) Effects of *crp* and *tcpA* on zebrafish larva colonization. (i) Images from multiple zebrafish larvae colonized with the indicated V. cholerae strains. All strains were transformed with plasmid pMW-GFP to facilitate visualization of bacteria. (ii) Quantified fluorescence from multiple microscopy images. (d) Raw gel images. Download FIG S1, PDF file, 0.4 MB.

Copyright © 2018 Manneh-Roussel et al.

2018

Manneh-Roussel et al.

This content is distributed under the terms of the

Creative Commons Attribution 4.0 International license

.

###### 

CRP binding peaks identified by ChIP-seq in the V. cholerae strain N16961 genome

  -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Genome\         Peak center[^a^](#ngtab1.1){ref-type="table-fn"}   Site center[^b^](#ngtab1.2){ref-type="table-fn"}   Site *P* value[^c^](#ngtab1.3){ref-type="table-fn"}   Sequence[^d^](#ngtab1.4){ref-type="table-fn"}   Gene(s) close to\                                        CRP\
  component                                                                                                                                                                                                                   CRP binding sites[^e^](#ngtab1.5){ref-type="table-fn"}   regulated[^f^](#ngtab1.6){ref-type="table-fn"}
  --------------- -------------------------------------------------- -------------------------------------------------- ----------------------------------------------------- ----------------------------------------------- -------------------------------------------------------- ------------------------------------------------
  Chromosome I    55771                                              55796.5                                            3.5E−03                                               `AGTGACTAAGCGTACA`                              23Sa1                                                    ND

                  99871                                              99790.5                                            8.3E−03                                               `TGTTACGAATATTACA`                              *glpE*\<(VC0103)                                         Yes

                  134815                                             134755.5                                           5.6E−03                                               `TTTGTTTTGGATCGAT`                              VC0142a\<\>VC0143                                        Yes

                  150732                                             150753.5                                           3.2E−07                                               `TGAGATTCAAATCACA`                              VC0159\<\>16Sb                                           Yes

                  177320                                             177327.5                                           3.2E−03                                               `CATAATCTGTATCAAA`                              VC0175                                                   Yes

                  242521                                             242505.5                                           7.1E−03                                               `TAAGGTTTAAGCCATT`                              (VC0237)                                                 No

                  249227                                             249187.5                                           9.8E−03                                               `TTTGAAGGATGGCGTT`                              (VC0242)                                                 No

                  264229                                             264210.5                                           5.1E−03                                               `TTAAATATGTATCATA`                              VC0258\>\<VC0259                                         No

                  274582                                             NA[^g^](#ngtab1.7){ref-type="table-fn"}            NA                                                    NA                                              (VC0269)                                                 No

                  294153                                             294127.5                                           7.6E−03                                               `TGTAGGTGATATCTCA`                              VC0284                                                   No

                  454607                                             454517.5                                           3.4E−03                                               `TGTGTTGTTGCTCAAT`                              VC0423\<\>VC0424                                         Yes

                  516492                                             516436.5                                           1.7E−03                                               `TGACAGTAATATCACT`                              VC0485\<\>VC0486                                         No

                  522621                                             522571.5                                           5.3E−03                                               `AGTCCATTTGCTCACA`                              VC0489                                                   No

                  527963                                             527988.5                                           1.1E−03                                               `AGTTATTTTTTTCACT`                              VC0493\<\>VC0494                                         No

                  569841                                             569895.5                                           4.9E−04                                               `AACGATTTTCCTCATA`                              VC0537\<\>VC0538                                         Yes

                  657711                                             657766.5                                           5.2E−05                                               `TGTGACTCCCTTCGCA`                              VC0621                                                   No

                  676093                                             676049.5                                           2.4E−03                                               `AATGATATAAATCCAA`                              *[ompU]{.ul}*\<\>*greA*                                  Yes

                  707829                                             707908.5                                           7.3E−03                                               `GCCGCTTGGCATCACA`                              VC0661\<\>VC0662                                         No

                  712256                                             712206.5                                           1.6E−03                                               `TGCAATCTAAGTCATT`                              VC0665                                                   No

                  713923                                             714007.5                                           1.1E−03                                               `TTAGAATTTAATCGTA`                              VC0666                                                   No

                  748263                                             748338.5                                           4.1E−03                                               `GGCGAGATTACGCGTA`                              VC0699\<\>VC0700                                         No

                  756779                                             756854.5                                           8.4E−07                                               `TGTGATAAAAGTCACT`                              VC0706                                                   No

                  762509                                             762522.5                                           1.8E−03                                               `TCTGACAATTATCTCG`                              VC0713                                                   Yes

                  788515                                             788501.5                                           6.9E−03                                               `TGTGAAATTTCACAAG`                              VC0734                                                   No

                  815345                                             815346.5                                           3.6E−07                                               `TGTGATATGATTCACA`                              *engA*                                                   Yes

                  818079                                             818158.5                                           2.6E−03                                               `GGTTAATTAAGTCGCA`                              VC0765                                                   Yes

                  819917                                             819936.5                                           1.0E−02                                               `CGTCCGCAATATCAAA`                              VC0766\<\>VC0767                                         No

                  880295                                             880357.5                                           5.3E−03                                               `TATGAGAAAGATAAAA`                              (VC0821)                                                 No

                  888697                                             888746.5                                           2.1E−03                                               `TGCAATTAAGTTCTCA`                              [*tcpI*\<\>*tcpP*]{.ul}                                  Yes

                  894874                                             894817.5                                           7.9E−03                                               `TATTATTGGATTCATT`                              (VC0833)                                                 No

                  904634                                             NA                                                 NA                                                    NA                                              (VC0842)                                                 Yes

                  906219                                             NA                                                 NA                                                    NA                                              [*acfA*\<\>*acfD*]{.ul}                                  No

                  911066                                             911128.5                                           3.1E−03                                               `TATGATGAAAAACATT`                              VC0845\>\<VC0846                                         No

                  936058                                             936026.5                                           1.5E−03                                               `AAAGAGCTAAATCGTT`                              (VC0870)                                                 No

                  999018                                             999093.5                                           9.6E−03                                               `CTTGGTTGTTTTCAAT`                              VC0932\<\>VC0934                                         Yes

                  1011835                                            1011745.5                                          2.4E−03                                               `AGTGAGCTTGCCCAAG`                              (VC0947)                                                 No

                  1037560                                            1037566.5                                          1.2E−03                                               `TTCGACGCATTTCAAA`                              VC0972                                                   Yes

                  1054013                                            1054031.5                                          1.4E−05                                               `CGTGATTTTTGTCGCG`                              *tppB*\<\>*rfaH*                                         No

                  1061159                                            1061198.5                                          1.5E−06                                               `GGTGATTAGGATCACA`                              *nagA*\<\>VC0995                                         No

                  1090145                                            1090112.5                                          5.6E−04                                               `TGTGATGTTTGGCATC`                              VC1021                                                   No

                  1100204                                            1100264.5                                          4.0E−05                                               `TGTGATGCAAATCGAT`                              VC1034                                                   Yes

                  1139471                                            1139535.5                                          4.0E−03                                               `TCTGATTATTTTCAAG`                              VC1073                                                   No

                  1174954                                            1174946.5                                          6.7E−06                                               `TGTGGTTTATGTCACA`                              VC1104                                                   No

                  1198758                                            1198847.5                                          6.0E−05                                               `TGTGAGCTGTGGCACT`                              VC1130\<\>VC1131                                         No

                  1212539                                            1212568.5                                          4.7E−03                                               `AGAGGCGAAATTCATT`                              VC1142\<\>*clpS*                                         Yes

                  1224786                                            1224787.5                                          4.2E−06                                               `TGTGATACTGGTCTCA`                              VC1152\<\>*tfoX*                                         No

                  1382925                                            1382894.5                                          5.9E−03                                               `TGTGAGAATTGTTAAT`                              VC1301                                                   Yes

                  1396772                                            1396717.5                                          1.2E−03                                               `ATTGATGTCACTCAAA`                              VC1313\<\>VC1314                                         Yes

                  1408936                                            1408937.5                                          3.8E−03                                               `TTTTAACTGGTTCACA`                              VC1323\<\>VC1325                                         Yes

                  1549042                                            1549038.5                                          6.9E−03                                               `TGTGCAATTTGTCTGA`                              *rtxB*\<\>*rtxH*                                         Yes

                  1568164                                            1568072.5                                          5.5E−03                                               `TATGAAAATGATGATA`                              *[ctxA]{.ul}*                                            No

                  1683652                                            1683636.5                                          2.0E−03                                               `AGTGATGGGGTTAACA`                              VC1571\<\>VC1572                                         No

                  1703584                                            1703620.5                                          6.4E−03                                               `TAATAAAAATGTCACA`                              VC1592                                                   No

                  1741600                                            1741668.5                                          4.7E−05                                               `TGTGATACGCTTCTCG`                              VC1620\<\>VC1621                                         Yes

                  1776678                                            1776642.5                                          4.8E−03                                               `AGTGATTTATCACTAA`                              VC1649\<\>VC1650                                         No

                  1789510                                            1789532.5                                          1.9E−05                                               `TATGACCAGTATCGCA`                              VC1656\<\>VC1658                                         No

                  1903470                                            1903498.5                                          8.0E−04                                               `TTTGAGTTAATTCAAT`                              (VC1736)                                                 Yes

                  1919651                                            1919579.5                                          6.2E−03                                               `TGTGCTAAATACAACG`                              (VC1771)                                                 Yes

                  1922932                                            NA                                                 NA                                                    NA                                              (VC1773)                                                 No

                  1967295                                            1967278.5                                          2.1E−06                                               `CGAGATCTAAATCACA`                              VC1825\<\>VC1826                                         No

                  1984776                                            1984846.5                                          3.3E−04                                               `TGAGAACTTTGTCAAA`                              VC1844                                                   Yes

                  1990074                                            1990055.5                                          4.4E−03                                               `GTCGAGACCACTCATA`                              VC1851                                                   No

                  1994054                                            1994088.5                                          5.4E−03                                               `ATTAATAAAAATCAAA`                              *[ompT]{.ul}*\<\>*dinG*                                  Yes

                  2004839                                            2004771.5                                          2.9E−03                                               `TTTTAACAAAGTCACA`                              VC1864\<\>VC1865                                         Yes

                  2055395                                            2055442.5                                          6.2E−03                                               `CATCAAATTTTTCACA`                              VC1904\<\>VC1905                                         Yes

                  2059077                                            2059085.5                                          9.7E−03                                               `TGCCACGCAACGCTCA`                              *cysB*\<\>VC1909                                         Yes

                  2168387                                            2168407.5                                          4.2E−03                                               `TTTGAGGAATTCCGCT`                              VC2013                                                   Yes

                  2190666                                            2190734.5                                          5.0E−03                                               `TGTGCGAATGTTAACA`                              VC2035                                                   No

                  2193110                                            2193078.5                                          1.1E−05                                               `AACGATATAAATCACA`                              VC2036\<\>VC2037                                         Yes

                  2374476                                            2374498.5                                          3.0E−05                                               `TGTGAGCTTTATCATG`                              VC2219\<\>VC2220                                         No

                  2433743                                            2433742.5                                          1.1E−06                                               `GGTGATTAAAATCACA`                              VC2278\<\>VC2279                                         No

                  2457608                                            2457631.5                                          1.3E−06                                               `AGCGATTAAGATCACA`                              VC2303\<\>VC2305                                         No

                  2537389                                            2537396.5                                          4.6E−03                                               `TGTGAATTCGGTGAAA`                              *gltB*                                                   No

                  2550352                                            2550386.5                                          8.7E−03                                               `TGTTACTGGTATAACA`                              (VC2385)                                                 No

                  2551356                                            2551299.5                                          3.5E−03                                               `AGTGATAAAAGTGAAG`                              (VC2386)                                                 No

                  2558608                                            2558615.5                                          3.7E−03                                               `GATGAATTTATTCATC`                              VC2390                                                   Yes

                  2610382                                            2610307.5                                          9.8E−03                                               `GCTGATTCGCGTCTTG`                              VC2435\<\>*tolC*                                         No

                  2653838                                            2653780.5                                          7.5E−04                                               `CGCGAGTCTCTTCAAA`                              VC2473                                                   Yes

                  2667326                                            2667406.5                                          8.8E−03                                               `TAATATTCACGTCAAA`                              VC2486                                                   No

                  2699390                                            2699329.5                                          1.5E−03                                               `GGTGATGGTCGCCACT`                              *pyrB*                                                   No

                  2743349                                            2743361.5                                          8.1E−04                                               `ATCGCGTCACATCACA`                              VC2561\<\>*cpdB*                                         No

                  2787939                                            2787903.5                                          3.0E−04                                               `TGAGATAAACCCCACA`                              VC2618                                                   Yes

                  2845246                                            2845280.5                                          5.7E−07                                               `TGTGATTTTCATCACG`                              VC2677                                                   No

                  2864757                                            2864763.5                                          9.2E−04                                               `ATAGATAAAACTCTCA`                              VC2698\<\>*aspA*                                         Yes

                  2933468                                            2933432.5                                          7.5E−04                                               `TTTGATTATCATCAAC`                              16sg                                                     ND

                  2936869                                            2936904.5                                          3.1E−04                                               `TTCGATACCAAGCACA`                              23Sh                                                     ND

                                                                                                                                                                                                                                                                                       

  Chromosome II   12067                                              12085.5                                            5.4E−08                                               `TGTGATCCGAATCACT`                              VCA0012\<\>VCA0013                                       Yes

                  86364                                              86274.5                                            5.8E−03                                               `GTCGAAATTCGCCACA`                              VCA0076                                                  No

                  99016                                              98927.5                                            2.0E−07                                               `TGTGATCTTTATCACT`                              VCA0089                                                  No

                  114856                                             114864.5                                           8.6E−03                                               `TTTAATAGATTTCTCA`                              VCA0104\<\>VCA0105                                       No

                  152867                                             152849.5                                           7.4E−04                                               `TGTGATTGATGTGGCA`                              VCA0138                                                  No

                  181749                                             181688.5                                           2.5E−03                                               `TGAGAAAGCATTCAAA`                              VCA0164\<\>VCA0165                                       No

                  217815                                             217798.5                                           6.0E−03                                               `TGTTATAAAAACCAAT`                              (VCA0200)                                                No

                  237015                                             237049.5                                           6.6E−03                                               `TAAGAATTATTTTACA`                              *hlyB*\<\>*hlyA*                                         No

                  247246                                             247185.5                                           7.5E−03                                               `TTGGCATAGCATCACA`                              VCA0224\<\>VCA0225                                       Yes

                  267292                                             267253.5                                           3.7E−03                                               `TGATAGGTAGATCACC`                              VCA0246\<\>VCA0247                                       No

                  300413                                             300391.5                                           8.4E−03                                               `TGCCCTATCTATCAAA`                              VCA0281                                                  Yes

                  334916                                             334914.5                                           4.3E−03                                               `ATTGACAGCTATCTAA`                              (VCA0334)                                                No

                  458259                                             458254.5                                           3.8E−03                                               `CGTGATTAAAAACGTC`                              VCA0523                                                  Yes

                  481906                                             481918.5                                           2.0E−03                                               `TTTCATAAAAGTCACG`                              VCA0544\<\>VCA0545                                       Yes

                  492167                                             492243.5                                           6.9E−07                                               `TGTGATTGGAATCACT`                              VCA0554\<\>VCA0556                                       No

                  564616                                             NA                                                 NA                                                    NA                                              (VCA0628)                                                Yes

                  598381                                             598370.5                                           4.9E−04                                               `GTTGACAACAGTCACA`                              (VCA0662)\<\>VCA0663                                     No

                  630430                                             630499.5                                           1.5E−03                                               `AATGATAGATAACACA`                              VCA0691                                                  Yes

                  687485                                             687472.5                                           3.3E−03                                               `CGTGATCGACATTAAA`                              (VCA0742)\>VCA0743                                       No

                  741821                                             741822.5                                           7.8E−06                                               `TGTGCTTTACATCACT`                              VCA0801                                                  Yes

                  784413                                             784364.5                                           2.1E−05                                               `TGTGATGCCGCTCGCA`                              VCA0840                                                  Yes

                  785337                                             785352.5                                           5.1E−04                                               `TTTGAACTTAGTCATT`                              VCA0843                                                  Yes

                  801056                                             801043.5                                           1.7E−05                                               `TGTGAAATGGCTCGCA`                              VCA0849                                                  Yes

                  832501                                             832514.5                                           6.2E−03                                               `TGCGACCTTGATTAAC`                              VCA0880                                                  Yes

                  849892                                             849906.5                                           3.0E−03                                               `GTTGACGCCTTTCTCA`                              VCA0896                                                  Yes

                  870862                                             870876.5                                           1.1E−03                                               `AATGATCAGGGGCAAA`                              VCA0917\<\>VCA0919                                       Yes

                  874452                                             874411.5                                           4.1E−03                                               `TATAAATCAAATCATT`                              VCA0923                                                  Yes

                  897315                                             897352.5                                           4.8E−06                                               `AGCGAGCCAAATCACA`                              VCA0945\<\>VCA0946                                       Yes

                  902411                                             902377.5                                           6.6E−03                                               `TGAAACACTTACCACT`                              VCA0952                                                  Yes

                  930851                                             NA                                                 NA                                                    NA                                              (VCA0982)\<\>VCA0983                                     No

                  963517                                             963536.5                                           8.2E−04                                               `TGTTAAGCAAATCGCA`                              VCA1013\<\>VCA1015                                       No

                  994614                                             994588.5                                           2.5E−05                                               `CATGACACAGGTCACA`                              VCA1043\<\>(VCA1044)                                     Yes

                  1015957                                            1015902.5                                          2.0E−05                                               `TTTGACCATTATCACA`                              VCA1063                                                  No
  -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Center of peak for CRP binding in ChIP-seq assays.

Center of binding site identified by FIMO (Find Individual Motif Occurrences) using DNA motif recovered from the ChIP-seq data by MEME (Multiple Em for Motif Elicitation).

*P* value assigned to each site by FIMO describing the significance of the match to the motif generated by MEME.

DNA sequence of site identified by FIMO.

Parentheses indicate that the CRP site is located within that gene. Pairs of arrows represent divergent (\<\>) or convergent (\>\<) genes. Single arrows indicate that gene pairs are in the same orientation on either the forward (\>) or reverse (\<) strand. Gene identification numbers are shown unless an alternative name for the gene is provided in the genome annotation or the wider literature. Genes regulated by ToxR or ToxT are underlined.

CRP-regulated genes described by Fong and Yildiz ([@B41]). ND, not detected: genes encoding stable rRNA species were not included in the transcriptome analysis and so no change in transcription could be detected.

NA, not applicable.

![Repression of the *rtxBDE* operon by CRP. (a) The intergenic region between *rtxBDE* and *rtxHCA* is associated with CRP but not σ^70^. The graphs illustrate ChIP-seq data for CRP (orange) and σ^70^ (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 P1*rtxB* 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 σ^70^ 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 P*rtxH* 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 P*rtxH*. (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 P1*rtxB* and P2*rtxB*.](mbo0041839690002){#fig2}

Expression of the *rtxBDE* operon responds to nutrient availability in a CRP-dependent manner. {#s1.3}
----------------------------------------------------------------------------------------------

The ability of CRP to bind DNA *in vivo* is regulated by nutrient availability. Hence, CRP binds to target sites when cells are grown in M9 minimal medium but binding is reduced in lysogeny broth (LB) and abolished upon the addition of glucose ([@B25][@B26][@B28]). As such, repression of *rtxBDE* should be relieved in rich medium. To test this, we used strain N16961 carrying the *rtxB*::*lacZ* fusions on pRW50T. The various strains were grown in M9 minimal medium, LB broth, or LB broth supplemented with 0.4% glucose. As expected, β-galactosidase activity due to the *rtxB*::*lacZ* fusion increased in LB broth and rose further upon the addition of glucose ([Fig. 3](#fig3){ref-type="fig"}, compare solid orange bars). Furthermore, inactivation of the CRP sites had a reduced effect in LB broth and no effect when glucose was present ([Fig. 3](#fig3){ref-type="fig"}, compare solid and stippled orange bars for each growth condition). Importantly, the sizes of changes in gene expression observed were similar to data for other CRP-regulated promoters ([@B31]). We confirmed that the observed gene expression was due to P1*rtxB* and P2*rtxB*. Hence, mutation of the promoter −10 elements greatly reduced *lacZ* expression ([Fig. 3](#fig3){ref-type="fig"}, open bars).

![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.](mbo0041839690003){#fig3}

CRP plays an important role during colonization of an aquatic host. {#s1.4}
-------------------------------------------------------------------

Although largely overlooked as a vector for V. cholerae, a recent study found that up to 87% of fish species were colonized by the bacterium in certain localities ([@B7]). Indeed, it has been suggested that colonization of fish has sustained the epidemicity of cholera in India ([@B8]). Since fish and humans have similar gut mucosa, the former have emerged as a model to study intestinal colonization ([@B9], [@B10]). The zebrafish larva model is particularly useful; bacteria are added to saltwater solutions in which larvae are free swimming and colonization follows without intervention ([@B10]). Given that changes in nutrient availability are associated with host colonization, we examined the role of CRP in this process. [Figure 4a](#fig4){ref-type="fig"} shows representative images of zebrafish larvae infected with V. cholerae strain E7946 or derivatives. Further images for each strain are shown in [Fig. S1c](#figS1){ref-type="supplementary-material"}. All strains express green fluorescent protein to facilitate their visualization. Infections due to the wild-type strain are disseminated throughout the intestinal tract ([Fig. 4a](#fig4){ref-type="fig"}, left). Conversely, infections caused by cells lacking CRP or TCP are limited to the upper intestinal tract and fail to colonize the midintestine and posterior intestine ([Fig. 4a](#fig4){ref-type="fig"}, middle and right). Quantification of fluorescence in microscopy images revealed 3-fold reductions for the Δ*crp* and Δ*tcpA* strains relative to the amount in the wild type ([Fig. S1c](#figS1){ref-type="supplementary-material"}). We also monitored survival of the larvae during incubation with the bacteria ([Fig. 4b](#fig4){ref-type="fig"}). All larvae infected with wild-type V. cholerae were dead by the end of the time course ([Fig. 4b](#fig4){ref-type="fig"}, black line). Conversely, infections caused by strains lacking CRP or TCP were not usually fatal ([Fig. 4b](#fig4){ref-type="fig"}, orange and blue lines). Hence, both CRP and TCP are important for colonization of fish ([@B22]).

![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](#figS1){ref-type="supplementary-material"} 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.](mbo0041839690004){#fig4}

Induction of the *rtxBDE* operon during host colonization is mediated by CRP. {#s1.5}
-----------------------------------------------------------------------------

We next considered the possibility that transcription from the *rtxBDE* promoters might be triggered during colonization of the intestinal tract. To test this, zebrafish larvae were colonized with either wild-type or Δ*crp* derivatives of V. cholerae carrying *rtxB*::*lacZ* fusions in plasmid pRW50T. After colonization, planktonic bacteria were recovered from the water and larvae were sacrificed to release the intestinal bacteria. The levels of β-galactosidase expression were then determined from lysates of the two populations. The data obtained for wild-type V. cholerae are shown in the left panel of [Fig. 4c](#fig4){ref-type="fig"}. Low *rtxBDE* expression was measured for planktonic V. cholerae. However, *rtxBDE* expression increased substantially during colonization of the larval intestinal tract. As expected, this increase in expression required P1*rtxB* and P2*rtxB* ([Fig. 4c](#fig4){ref-type="fig"}, compare orange and open bars). In cells lacking CRP, the expression of *rtxBDE* was uncoupled from host colonization. Hence, high *rtxBDE* expression was measured in planktonic as well as intestinal populations ([Fig. 4c](#fig4){ref-type="fig"}, right). Note that the differences in gene expression observed are not due to the different colonization properties of the Δ*crp* strain; deregulation of *rtxBDE* occurs in planktonic populations rather than within the larvae. Furthermore, any differences in bacterial cell numbers were accounted for by normalization.

CRP modulates the expression of many V. cholerae genes during host colonization. {#s1.6}
--------------------------------------------------------------------------------

We reasoned that other CRP-targeted promoters would lose the ability to differentiate between aquatic environments and the host intestinal tract when CRP was absent. To test this, the promoters of the following five genes were selected, using our ChIP-seq data as a guide: *tolC* (encoding an outer membrane channel important for bile tolerance), *acfA* (encoding accessory colonization factor A), *acfD* (encoding accessory colonization factor D), *nudF* (encoding a pyrophosphatase), and *hlyA* (encoding hemolysin) ([@B44]). The promoter region of each gene was cloned upstream from *lacZ* in plasmid pRW50T, and the β-galactosidase activity was determined ([Fig. 5a](#fig5){ref-type="fig"}). The data show that all of the genes were expressed at different levels in planktonic (solid bars) and intestinal (striped bars) populations. The experiment was repeated in cells lacking Δ*crp* ([Fig. 5b](#fig5){ref-type="fig"}). The expression of all genes was rendered insensitive to host colonization ([Fig. 5b](#fig5){ref-type="fig"}, compare solid and striped bars).

![CRP couples the expression of many V. cholerae genes to host colonization. Results of β-galactosidase assays done using lysates of bacterial cells grown planktonically in E3 medium or obtained from the zebrafish intestinal tract. Strains are indicated and were transformed with derivatives of pRW50T encoding different LacZ fusions. Significant differences between levels of β-galactosidase activity in planktonic and intestinal populations were observed in all cases for wild-type cells (*P* values determined using a two-tailed Student's *t* test were 0.0014, 0.0011, 0.0003, 0.0005, and 0.0037 for the *tolC*, *acfA*, *acfD*, *nudF*, and *hlyA* promoters, respectively). For cells lacking CRP, a significant, albeit much smaller difference was only apparent for the *acfD* promoter (*P* = 0.0036).](mbo0041839690005){#fig5}

DISCUSSION {#s2}
==========

The ability of V. cholerae to persist in environmental reservoirs, colonize the intestinal tract, and cause disease requires careful coordination of gene expression ([@B5]). This process is best characterized for key virulence factors that collectively reside in the ToxR regulon ([@B5], [@B12], [@B14]). In this paper, we have investigated the role of CRP. We show that CRP targets five of the nine ToxR regulon transcription units. Hence, we identified binding sites for CRP adjacent to *ompU*, *acfA*, and *acfD*, in addition to the known targets *ompT* and *tcpPH*. Other genes involved in V. cholerae pathogenicity were also targets for CRP. These included *rtxBDE*, *tolC*, and *hlyA*. Previous transcriptome analysis led to speculation that CRP may modulate the expression of V. cholerae virulence factors in response to host colonization ([@B33]). Here, we have tested this prediction using the zebrafish larva colonization model ([@B9]). For all genes examined, differential expression between planktonic and intestinal populations required CRP. We have paid particularly close attention to genes encoding the RTX toxin export machinery. Our data are consistent with repression of *rtxBDE* by CRP that is relieved within the intestinal tract and other nutrient-rich environments. Previous host colonization studies support our model. In particular, Mandlik and colleagues previously monitored global transcription in V. cholerae using RNA-seq ([@B45]). Their data demonstrate induction of *rtxBDE* within the intestinal tracts of both mice and rabbits. Furthermore, the same study detected repression of *rtxBDE* in M9 minimal medium compared to its expression in LB broth. Similarly, Boardman et al. noted repression of *rtxBDE* in nutrient-poor environments ([@B46]). We argue that CRP mediates these effects directly by binding sites upstream from the *rtxBDE* promoters ([Fig. 2](#fig2){ref-type="fig"} [to](#fig3){ref-type="fig"} [4](#fig4){ref-type="fig"}). Curiously, while the RTX toxin export machinery is expressed only upon nutrient upshift, the divergent *rtxHCA* genes that encode the RTX toxin appear constitutively transcribed ([Fig. 2](#fig2){ref-type="fig"}). Indeed, posttranscriptional control by the VqmR small RNA (sRNA) has previously been shown to regulate RTX toxin expression ([@B45]). We speculate that this allows the system to exist in a poised state so that toxin export is only triggered within a host organism.

Our data show that CRP is required for control of V. cholerae genes in addition to the *rtxBDE* operon in response to the intestinal environment ([Fig. 5](#fig5){ref-type="fig"}). Hence, CRP is integral to the regulatory network that controls V. cholerae lifestyle switching. This is intriguing, given that oral rehydration therapies (ORT) used to treat the effects of cholera contain glucose ([@B26], [@B47]). Hence, it is possible that ORT modification could be used to modulate the ability of V. cholerae to colonize a host and cause disease ([@B26], [@B47]). Complete dissection of the role CRP plays during V. cholerae lifestyle switching should provide an evidence base for any such ORT modification. In this regard, our findings provide an important starting point for further studies.

MATERIALS AND METHODS {#s3}
=====================

Strains, plasmids, and oligonucleotides. {#s3.1}
----------------------------------------

V. cholerae strains N16961 and E7946 are described by Heidelberg et al. and Miller et al., respectively ([@B39], [@B48]). The Δ*crp* derivative of E7946 was constructed by MuGENT using PCR oligonucleotides listed in [Table S1](#tabS1){ref-type="supplementary-material"} in the supplemental material ([@B49]). The E. coli K-12 strains JCB387 and DH5α are described by Page et al. ([@B50]) and Taylor et al. ([@B51]) and were used for general cloning and conjugation, respectively. Plasmid pRW50T was constructed by excision of the DNA fragment comprising *cynX* from pRW50 ([@B52]) using NheI and BstEII. The *oriT* region was amplified from plasmid RK2 ([@B53]) in such a way that *oriT* was flanked by NheI and BstEII restriction sites to facilitate ligation at the locus from which *cynX* was excised. Plasmid pDCRP-Vc is a derivative of pDCRP ([@B54]) that encodes the CRP protein of V. cholerae rather than that of E. coli. More-detailed descriptions of strains and plasmids, along with sequences of oligonucleotides, are provided in [Table S1](#tabS1){ref-type="supplementary-material"}.

10.1128/mBio.00966-18.3

Strains, plasmids, and oligonucleotides. Download TABLE S1, DOCX file, 0.03 MB.

Copyright © 2018 Manneh-Roussel et al.

2018

Manneh-Roussel et al.

This content is distributed under the terms of the

Creative Commons Attribution 4.0 International license

.

ChIP and DNA sequencing. {#s3.2}
------------------------

Immunoprecipitations with monoclonal anti-CRP and anti-σ^70^ antibodies (Neoclone, Madison, WI) were done as described by Haycocks et al. ([@B26]) using lysates of strain N16961 ([@B39]). Lysates were prepared from mid-log-phase cells cultured in M9 minimal medium supplemented with 1% (wt/vol) fructose. Libraries were prepared using immunoprecipitated protein-DNA complexes immobilized with protein A-Sepharose. DNA fragments were then given blunt ends, poly(A) tails, and bar codes. This was done using an NEB quick blunting and ligation kit, the Klenow fragment (5′-3′ exo-; NEB), and NEXTflex chromatin immunoprecipitation-DNA sequencing (ChIP-seq) barcodes (Bioo Scientific). Following elution of complexes from the protein-A Sepharose, cross-links were reversed, and bar-coded libraries were amplified by PCR. The number of PCR cycles was determined empirically for each library. After amplification, the library concentration was quantified using Qubit analysis and real-time PCR. Equimolar library concentrations were pooled and sequenced using an Illumina MiSeq instrument.

Bioinformatics. {#s3.3}
---------------

The Fastq files obtained after DNA sequencing were converted into Fastq Sanger format, using FastqGroomer, and aligned to GenBank reference sequences (accession numbers [AE003852.1](https://www.ncbi.nlm.nih.gov/nuccore/AE003852.1) and [AE003853.1](https://www.ncbi.nlm.nih.gov/nuccore/AE003853.1)) using BWA (Burroughs-Wheeler Aligner) for Illumina. The reference sequences correspond to chromosome I and chromosome II, respectively, of V. cholerae strain N16961. The resulting SAM (Sequence Alignment Map) files were converted to BAM (Binary Alignment Map) format using SAM-to-BAM. For each experiment, coverage per base was determined using multiBamSummary. Subsequent processing was done using R. Data were normalized to the same average read depth, and mean coverage per base was determined for each pair of biological replicates. Signals due to nonspecifically immunoprecipitated DNA present in a mock experiment were subtracted from the final binding profiles. To select peaks for CRP or σ^70^ binding, we used Artemis to generate a coverage plot and selected peaks. The peak centers were set as the center of the region passing the cutoff rounded to the nearest integer. Peaks for CRP and σ^70^ were defined as overlapping if the peak centers were within 250 bp of each other.

Proteins. {#s3.4}
---------

The V. cholerae CRP protein was expressed in E. coli strain M182Δcrp and purified using cAMP-agarose as described previously ([@B54]). The V. cholerae RNA polymerase was purified using a method derived from that of Burgess and Jendrisak ([@B55]). Briefly, V. cholerae strain N16961 was grown to mid-log phase in 8 liters of LB medium. Cells were harvested by centrifugation and resuspended in 100 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM MgCl~2~, 0.1 mM dithiothreitol \[DTT\], 2 mM EDTA, 1 mM 2-mercaptoethanol, 5% glycerol, 0.2% Triton X-100, and 0.25 mg/ml lysozyme). One protease inhibitor cocktail tablet (Roche) was added per 20 ml of buffer. Cell lysis and DNA shearing were done using four 30-s pulses, at 20% output, with a Misonix, Inc., XL2020 tip sonicator. Lysates were cleared by centrifugation at 39,000 × *g* for 45 min at 4°C. Following filtration (0.45-µm filter), polymin P and ammonium sulfate precipitations were done as described in Burgess and Jendrisak ([@B55]). Precipitated protein was resuspended in TGED buffer (10 mM Tris-HCl, pH 7.9, 5% glycerol, 0.1 mM EDTA, and 0.1 mM DTT) containing 100 mM NaCl and passed through a HiPrep heparin FF column (GE Healthcare). The column was washed with 0.1 M NaCl TGED, and RNA polymerase was eluted in TGED using a gradient to 1 M NaCl. RNA polymerase-containing fractions were pooled and protein precipitated using ammonium sulfate. After resuspension in TGED, RNA polymerase was further purified using a Mono Q HR column (GE Healthcare). Column washing and protein elution were as described in the previous step. RNA polymerase-containing fractions were pooled and dialyzed against −80°C storage buffer (TGED, 0.1 M NaCl, 50% glycerol).

DNase I footprinting and *in vitro* transcription. {#s3.5}
--------------------------------------------------

For electrophoretic mobility shift assay (EMSA) experiments, DNA fragments were prepared using PCR as described by Shimada et al. ([@B56]), with oligonucleotides listed in [Table S1](#tabS1){ref-type="supplementary-material"}. Protein binding and subsequent electrophoresis were done as described by Chintakayala et al. ([@B57]). For footprinting experiments, DNA fragments were prepared as described by Grainger et al. ([@B58]). Protein binding, DNA digestion, and electrophoresis were done as described by Singh and Grainger ([@B59]). Briefly, DNA fragments were labeled at one end using \[γ-^32^P\]ATP and T4 polynucleotide kinase and used at a final concentration of \~10 nM in footprinting reactions. All reaction mixtures contained an excess of herring sperm DNA (12.5 µg ml^−1^) as a nonspecific competitor. Our *in vitro* transcription assays were done as described by Haycocks et al. ([@B26]). DNase I-digested DNA and *in vitro*-generated RNA transcripts were analyzed on 6% DNA sequencing gels (Molecular Dynamics). The results were visualized using a Fuji phosphor screen and Bio-Rad Molecular Imager FX. Raw gel images are in [Fig. S1d](#figS1){ref-type="supplementary-material"}.

Primer extension assays. {#s3.6}
------------------------

Transcript start sites were mapped by primer extension, as described previously ([@B59]), using RNA purified from a V. cholerae strain carrying the appropriately oriented *rtxBDE-rtxHCA* intergenic region cloned in pRW50T. The 5′-end-labeled primer D49724, which anneals downstream from the HindIII site in pRW50, was used in all experiments. Primer extension products were analyzed on denaturing 6% polyacrylamide gels calibrated with size standards derived from M13mp18 phage DNA sequencing reactions. Gels were visualized using a Fuji phosphor screen and Bio-Rad Molecular Imager FX.

β-Galactosidase assays. {#s3.7}
-----------------------

β-Galactosidase assays using lysates of liquid V. cholerae cultures were done as described previously ([@B26]) following the protocol of Miller ([@B60]). For experiments with zebrafish larva, colonization by V. cholerae was first instigated as described below. E3 medium was prepared as a 1 liter 50× stock containing 14.6 g NaCl, 0.65 g KCl, 2.20 g CaCl~2~, 4.05 g MgSO~4~, and 23.85 g HEPES adjusted to pH 7. A 1× dilution was prepared using ddH2O. Following infection, the larvae were euthanized with 2.5 mg/ml tricaine and the E3 medium was agitated to resuspended bacteria that had sunk to the bottom of the well. The V. cholerae-containing E3 medium was then transferred to a sterile bijou and the larvae to a sterile 1.5-ml dolphin microcentrifuge tube. Fish were washed with E3 medium by gentle pipetting to remove residual bacteria. Larvae were then homogenized to release bacterial cells using a hand-held motorized homogenizer, and E3 was added so that the homogenate had a volume similar to that of the isolated medium. Two drops each of toluene and 1% (wt/vol) sodium deoxycholate were added to each sample, and the resulting cell lysates were assayed for β-galactosidase activity. To normalize for cell numbers, 0.5 µl of each cell suspension was diluted in 1.5 ml of E3 medium prior to cell lysis. One hundred microliters of this suspension was spread on LB agar plates containing 5 µg/ml tetracycline, 100 µg/ml streptomycin, 50 µg/ml spectinomycin, and 40 µg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β-[d]{.smallcaps}-galactopyranoside). This allowed for confident selection of V. cholerae cells containing pRW50T derivatives that were enumerated by counting the number of colonies formed after overnight incubation at 37°C. All assay values are the means of the results of three independent experiments with a standard deviation equivalent to \<10% of the mean β-galactosidase activity.

Zebrafish larva colonization and survival assays. {#s3.8}
-------------------------------------------------

Adult zebrafish were kept at pH 7.5 and 26°C in a recirculating tank system, with light/dark cycles of 14/10 h, at the University of Birmingham aquatic facility. Zebrafish care, breeding, and experimentation were done according to the Animal (Scientific Procedures) Act 1986 ([@B61]) under home office project license 40/3681. Zebrafish embryos derived from the wild-type AB strain ([@B62]) were harvested in petri dishes containing water from the fish system. After harvesting, between 50 and 60 embryos were transferred to 90-mm petri dishes containing 25 ml of E3 medium supplemented with 0.03% (vol/vol) methylene blue and 0.02 mg/ml 1-phenyl 2-thiourea. The embryos were incubated at 32°C for 4 days with light/dark cycles of 14/10 h. The incubation medium was regularly replaced to minimize microbial contamination. On day 3 of the incubation, required strains of V. cholerae were streaked to generate single colonies that were used to inoculate 5 ml of M9 minimal medium. The resulting cultures were incubated overnight at 37°C with shaking. One milliliter of the overnight culture was transferred to 5 ml of fresh M9 minimal medium the following day. The resulting culture was incubated at 37°C with shaking until mid-log phase. Cells were then harvested by centrifugation and washed three times with 5 ml of E3 buffer by sequential resuspension and centrifugation. After washing, the cells were resuspended in 5 ml of E3 medium, the optical density was determined, and 10^6^ cells were transferred into each well of a 24-well cell culture plate. The larvae were sedated by adding 166 µl of 40-mg/ml tricaine to the petri dish. Five larvae were then transferred to each well of the culture plate, which was incubated at 30°C overnight. Death was determined by loss of movement and heartbeat in opaque larvae that had settled at the bottom of the well.

Microscopy. {#s3.9}
-----------

Zebrafish embryos colonized with V. cholerae were imaged using a Zeiss Axio Observer Z1 microscope with 10× objective for fluorescence and differential interference contrast. Prior to visualization, embryos were immobilized in 0.4% low-melting-point agarose in E3 buffer and 160 µg/ml tricaine. Imaging was done at 32°C and humidity maintained at 80% using an OkoLab stage. The ImageJ image processing package (NIH) software was used to visualize the images and merge the fields.

Data availability. {#s3.10}
------------------

DNA sequencing reads are stored in ArrayExpress under accession number E-MTAB-6472. Genome Browser files ([Data Set S1](#dataS1){ref-type="supplementary-material"}[to](#dataS2 dataS3 dataS4 dataS5){ref-type="supplementary-material"}[S6](#dataS6){ref-type="supplementary-material"}) and instructions ([Text S1](#textS1){ref-type="supplementary-material"}) are provided in the supplemental material.

10.1128/mBio.00966-18.4

N16961 chromosome I GenBank file. Download DATA SET S1, TXT file, 6 MB.

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10.1128/mBio.00966-18.5

N16961 chromosome II GenBank file. Download DATA SET S2, TXT file, 2.2 MB.

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Manneh-Roussel et al.

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10.1128/mBio.00966-18.6

CRP ChIP-seq Artemis graph file for N16961 chromosome I. Download DATA SET S3, TXT file, 17.5 MB.

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Manneh-Roussel et al.

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10.1128/mBio.00966-18.7

CRP ChIP-seq Artemis graph file for N16961 chromosome II. Download DATA SET S4, TXT file, 6.4 MB.

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Manneh-Roussel et al.

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10.1128/mBio.00966-18.8

RNA polymerase σ^70^ ChIP-seq Artemis graph file for N16961 chromosome I. Download DATA SET S5, TXT file, 10.7 MB.

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2018

Manneh-Roussel et al.

This content is distributed under the terms of the

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10.1128/mBio.00966-18.9

RNA polymerase σ^70^ ChIP-seq Artemis graph file for N16961 chromosome II. Download DATA SET S6, TXT file, 4.2 MB.

Copyright © 2018 Manneh-Roussel et al.

2018

Manneh-Roussel et al.

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10.1128/mBio.00966-18.1

Instructions for viewing ChIP-seq data in the Artemis genome browser. Download TEXT S1, PDF file, 0.6 MB.

Copyright © 2018 Manneh-Roussel et al.

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Manneh-Roussel et al.

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**Citation** Manneh-Roussel J, Haycocks JRJ, Magán A, Perez-Soto N, Voelz K, Camilli A, Krachler A-M, Grainger DC. 2018. cAMP receptor protein controls *Vibrio cholerae* gene expression in response to host colonization. mBio 9:e00966-18. <https://doi.org/10.1128/mBio.00966-18>.

We thank Jenny Ritchie, Brendan Wren, Joe Wade, Chris Thomas, Rachel Kettles, and Doug Browning for advice and support.

This work was funded by BBSRC grant number BB/N005961/1 awarded to D.C.G. and A.-M.K. and NIH grant number AI055058 to A.C. The Islamic Development Bank supported J.M.-R. with the award of a Ph.D. scholarship.

[^1]: Present address: Andrés Magán, Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

[^2]: J.M.-R. and J.R.J.H. made equal contributions to this work.
