Regulatory targets of quorum sensing in Vibrio cholerae: evidence for two distinct HapR-binding motifs

Abstract

The quorum-sensing pathway in Vibrio cholerae controls the expression of the master regulator HapR, which in turn regulates several important processes such as virulence factor production and biofilm formation. While HapR is known to control several important phenotypes, there are only a few target genes known to be transcriptionally regulated by HapR. In this work, we combine bioinformatic analysis with experimental validation to discover a set of novel direct targets of HapR. Our results provide evidence for two distinct binding motifs for HapR-regulated genes in V. cholerae. The first binding motif is similar to the motifs recently discovered for orthologs of HapR in V. harveyi and V. vulnificus. However, our results demonstrate that this binding motif can be of variable length in V. cholerae. The second binding motif shares common elements with the first motif, but is of fixed length and lacks dyad symmetry at the ends. The contributions of different bases to HapR binding for this second motif were demonstrated using systematic mutagenesis experiments. The current analysis presents an approach for systematically expanding our knowledge of the quorum-sensing regulon in V. cholerae and other related bacteria.

Figure 1.

Alignment of predicted Motif 1 (A) and Motif 2 (B) binding sites in intergenic regions of V. cholerae. Genes for which predicted binding sites are on the complementary strand are indicated with gray shading. Loci labeled in orange indicate the first of two divergently transcribed genes that contain a HapR binding site in the intergenic region. The 22-bp-binding regions are shown for each binding site. While the predicted binding site length is seen to be variable for Motif 1 genes, Motif 2-binding sites show very weak conservation beyond 16 bp. The alignment demonstrates the distinctions between the two proposed motifs in terms of concluding sequences and binding site lengths.

Figure 2.

HapR binds to promoters containing either Motif 1- or Motif 2-binding sites. HapR's ability to bind to various promoters containing either of the two consensus binding sites was determined by EMSA in the presence of poly(dI–dC). Due to differences in HapR-binding affinity, two different sets of HapR protein concentrations were used, as indicated by the color of the wedges above the gel shifts. Solid wedges represent four times more protein than unfilled wedges. Since Motif 1 does not have a set length, promoters containing Motif 1-binding sites of varying lengths were tested, and the length of the putative binding site for each promoter is indicated. Consensus binding sequences that fall between two divergently transcribed genes are labeled with both flanking gene locus numbers. The double bands of vc1181 resulted from the nonspecific labeled PCR products of the vc1181 promoter region.

Figure 3.

HapR regulates expression from promoters containing either Motif 1 or Motif 2. Promoter-lux fusions were constructed and transformed into WT and hapR-deleted strains. Fold induction by HapR was determined as the ratio between bioluminescence readings from the reporter fusions in WT/ΔhapR. Negative fold induction indicates repression. Each bar represents three data points, and error bars represent standard deviations. Divergently transcribed genes with HapR binding sites located in between are indicated with boxes.

Figure 4.

Point mutations in the Motif 2-consensus-binding site alter HapR binding affinity as predicted by the PSWM. (A) EMSAs were performed on a 60-bp region of the vca0880 promoter that contains the Motif 2-binding site as well as the same region with the binding site deleted. (B) Sequence logo for Motif 2-binding sites reflecting the V. cholerae-specific PSWM derived after one iteration of the procedure outlined in Figure S1. (C) HapR-binding affinity was determined by quantifying the amount of bound and unbound DNA and calculating the ratio of bound/unbound. The effect of various point mutations in the binding site on HapR-binding affinity was determined by EMSAs over a range of HapR concentrations.