Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation

Abstract

Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼ 47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.

Keywords: RNA-seq; Vibrio cholerae; biofilm; quorum sensing; sRNA.

Fig. 1.

Expression profiling and TSS mapping of wild-type and luxO D47E V. cholerae. (A) Venn diagram of differentially expressed genes in the wild-type and luxO D47E V. cholerae C6706 strains at LCD (OD₆₀₀ of 0.1) and HCD (OD₆₀₀ of 2.0). Numbers of cDNA reads for annotated genes were compared to wild-type cells at HCD (OD₆₀₀ of 2.0). Statistically significant genes (P < 0.05) that changed >1.5-fold are shown. (B) Identification and classification of TSSs in V. cholerae. A total of 7,240 TSSs were identified by dRNA-seq and classified according to their genomic locations (Top). A, antisense; I, internal; O, orphan; P, primary; S, secondary. (C) Length distribution of 5′ UTRs in V. cholerae. For each primary and secondary TSS, the distance to the cognate translation initiation site was calculated and the frequency of each 5′ UTR length was plotted. (D) Consensus motif for V. cholerae promoters. DNA sequences from −40 to +1 upstream of the 7,240 TSSs were analyzed for conserved sequence elements using the MEME tool.

Fig. 2.

Expression analysis of 3′ UTR-derived sRNAs. (A) sRNAs with dedicated promoters. Total RNA was obtained at the designated times during growth from wild-type, luxO D47E, and Δhfq V. cholerae strains. Northern blots were probed for six 3′ UTR-derived sRNAs. The genomic locations and relative orientations are shown above the gels. Genes are shown in black; sRNAs are shown in red or gray. Arrows and scissors indicate TSSs and processing sites, respectively. Filled triangles indicate bands derived from TSSs; open triangles indicate bands derived from processing. The 5S rRNA served as the loading control. (B) sRNAs derived from transcript processing. The designations are the same as in A except that the promoters are shared between the sRNA and the mRNA. The mRNAs undergo ribonucleolytic cleavage to yield the sRNAs.

Fig. 3.

Identification and expression of the VqmR sRNA. (A) dRNA-seq data for the vqmR-vqmA locus. Shown are cDNA reads mapping to the vqmR and vqmA genes in V. cholerae. Blue bars indicate expression in the TEX (−) samples; red bars show expression in the TEX (+) samples. (Upper) cDNA reads mapping to the plus strand of the V. cholerae genome. (Middle) Schematic representation of the vqmR-vqmA genomic arrangement. (Bottom) cDNA reads mapping to the minus strand of the V. cholerae genome. Strains and growth conditions are indicated on the Right. (B) Production of VqmR in V. cholerae. Total RNA obtained at the designated ODs for wild-type, luxO D47E, and Δhfq V. cholerae strains was probed for VqmR on Northern Blots. A DNA-marker (M) is provided on the Left. The 5S rRNA served as the loading control. (C) Sequence alignment of vqmR genes. The sequences, including the promoter regions, of vqmR genes from different vibrio species were aligned. The −10 box, the TSS, and the conserved sequences R1 and R2 are denoted. Dotted arrows indicate the rho-independent terminator element. Mutations introduced into the vqmR promoter (used in EMSA studies; SI Appendix, Fig. S5C) are indicated above the alignment. Vch, V. cholerae; Vfu, Vibrio furnissii; Van, Vibrio anguillarum; Vha, Vibrio harveyi; Vpa, Vibrio parahaemolyticus; Vvu, Vibrio vulnificus; Val, Vibrio alginolyticus. (D) Production of VqmR in different vibrios. VqmR was assessed by Northern blot for the indicated vibrio species using a probe for conserved region R2. Triangles indicate full-length VqmR. The 5S rRNA served as the loading control.

Fig. 4.

VqmA activates vqmR transcription. (A) Total RNA was obtained for wild-type/pctr, ΔvqmA/pctr, and ΔvqmA/pvqmA V. cholerae strains at the indicated times during growth. The Northern blot was probed for VqmR. The 5S rRNA served as loading control. (B) GFP production from a vqmR transcriptional reporter was measured in E. coli carrying the indicated plasmids following 12 h growth in LB with 0.2% (final concentration) glucose (black bars) or 0.2% (final concentration) l-arabinose (gray bars). Error bars represent SD of three replicates. (C) Electrophoretic mobility shift assay (EMSA) showing that VqmA protein binds the vqmR promoter sequence. Migration of the [³²P] end-labeled vqmR promoter fragment in the absence and presence of increasing concentrations of purified VqmA::3XFLAG protein (indicated by the black triangle above the gel) was determined by native polyacrylamide gel electrophoresis and autoradiography. The open triangle indicates free DNA; filled triangle indicates DNA in complex with VqmA::3XFLAG. A negative control consisting of a mutated version of the vqmR promoter is shown in SI Appendix, Fig. S5C.

Fig. 5.

The VqmR mRNA targets. (A) Expression of VqmR-controlled target mRNAs was measured in wild-type and ΔvqmR V. cholerae strains. The strains carried either a control plasmid (pctr) or a plasmid harboring the vqmR gene (pvqmR). RNA was monitored using qRT-PCR. Expression in the wild-type strain was set to 1. Error bars represent SD of three replicates. (B) Secondary structure of VqmR. The secondary structure of VqmR was derived from SI Appendix, Fig. S6. Conserved regions R1 and R2 are indicated in red. (C) VqmR regulation of target mRNAs. E. coli harboring plasmids carrying the eight genes denoted on the x axis each fused to gfp were cotransformed with a control plasmid (pctr) or the indicated VqmR-expressing plasmids. gfp and vqmR transcription were driven by constitutive promoters. Strains were grown in LB for 8 h and GFP production was measured. GFP levels in strains carrying the control plasmid (pctr) were set to 1. Error bars represent SD of three replicates.

Fig. 6.

Base pairing of VqmR with vc1063 and vpsT. (A) Predicted base pairing between VqmR region R1 and vc1063 mRNA. VqmR region R1 is indicated in blue and the vc1063 Shine Dalgarno sequence is boxed. Mutations introduced in B are indicated with arrows. (B) VqmR repression of vc1063 requires region R1. Vc1063::GFP and Vc1063*::GFP (G-10C) were measured using Western blot. RNAPα served as the loading control. VqmR (PvqmR) and VqmR C63G (PvqmR*) were probed on Northern blot. The 5S rRNA served as loading control. (C) Predicted base pairing between VqmR region R2 and vpsT mRNA. Region R2 is indicated in red and the vpsT Shine Dalgarno sequence is boxed. Mutations introduced in D are indicated with arrows. (D) VqmR repression of vpsT requires region R2. VpsT::GFP and VpsT*::GFP (G-17C) were measured using Western blot. RNAPα served as the loading control. VqmR (PvqmR) and VqmR C94G (PvqmR*) were probed on Northern blot. The 5S rRNA served as loading control.

Fig. 7.

VqmR represses biofilm formation in V. cholerae. (A) Western blot analysis of VpsT::3XFLAG. Wild-type, ΔvqmR, ΔvqmA, ΔluxO, and ΔhapR single mutants and ΔluxO vqmR and ΔhapR vqmR double mutant V. cholerae strains were grown to OD₆₀₀ of 1.0 and examined for VpsT::3XFLAG production using an anti-FLAG antibody. RNAPα served as the loading control. Changes in VpsT::3XFLAG protein production are indicated below the figure (WT was set to 100%). (B) Production of VpsL::GFP. Wild-type, ΔvqmR, and ΔvqmA V. cholerae strains carrying a vpsL::gfp transcriptional reporter on a plasmid were grown for 6 h and GFP production was measured. Error bars represent SD of three replicates. (C) Biofilm production of vqmR-expressing strains. Biofilm formation of the ΔhapR V. cholerae strain constitutively expressing the fluorescent mKate2 protein was assayed in the presence of a control plasmid (Top, pctr), a plasmid carrying vqmR (Middle, pvqmR), or a plasmid carrying VqmR C94G (pvqmR*) that does not base pair with vpsT (Bottom). Biofilms were imaged following a 24-h incubation at 30 °C. (Scale bar: 5 µm.) The inlays show the colony morphologies of the indicated strains grown on agar plates and incubated for 24 h at 30 °C. (D) Model of VqmR activity in V. cholerae. Transcription of vqmR is activated by VqmA. VqmR directly regulates at least eight target mRNAs including vpsT. VpsT, in turn, acts as an activator of VpsL production and biofilm formation.