Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

Abstract

Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio "sRNA core."

FIGURE 1.

V. splendidus genome expression levels along chromosomes. (Top) Schematic circular diagrams of V. splendidus chromosomes I and II. Keys to the chromosomal circular diagrams (outside to inside): scale (in nucleotides): + strand CDS (red), − strand CDS (blue), position of sRNAs (dark green), r5′UTRs (light green), asRNAs (orange), log₂ of coverage per nucleotide, averaged on a window of 10,000 for the + strand (olive green) and for the − strand (purple). The position of the origin of replication for both chromosomes is indicated (nt 0). (Bottom) Linear representation of log₂ coverage (expressed as read numbers per a sliding window of 10,000 nt) along both chromosomes. The arrows indicate the sense of propagation of the replication forks from the origin of replication to the terminus of replication.

FIGURE 2.

Visualization interface and models for the three kinds of sRNA candidates. (A) We used the Artemis sequence editor tool to check the regulatory RNA candidates manually. We added two tracks to the Artemis classical view of genomic information: the ln of read coverage track (top) and the BamView of the mapped reads (middle). Forward sequences and reverse sequences are above and under the line, respectively. (White boxes) CDSs; (blue boxes) annotated proteins; (pink boxes) cis-encoded r5′UTRs; (orange boxes) cis-encoded asRNAs; (salmon boxes) trans-encoded sRNAs. Models for parameter definition are diagrammatically outlined for each category of regulatory RNAs under the Artemis window. See Materials and Methods and Results sections for details. (B) Schematic representation of the different steps in script execution. r5′UTR, sRNA, and asRNA computational workflows are depicted, respectively, by the left, middle, and right pathways. Most of the steps are managed using one or more S-MART tools. (Nb reads) Number of reads; (d) distance below which two clusters of overlapping reads are merged.

FIGURE 3.

t44 is an r5′UTR. (A) An Artemis window showing expression of the t44 sRNA (pink box) as the 5′ UTR of genes VS_2353 and VS_2352. The read cluster corresponding to the 5′-UTR part of the transcript (t44) is highlighted in salmon. (Yellow boxes) ARNold prediction of Rho-independent transcription terminators (see Materials and Methods). (Blue boxes) Annotated CDS. The orange line underneath indicates the position of the oligonucleotide probe used for the Northern blot. (B) Northern blot of t44. Total RNA samples were prepared from different time points during growth of V. splendidus in marine salt medium at 20°C and submitted to electrophoresis on an agarose gel before transfer to a Hybond N⁺ membrane. The culture OD₆₀₀ for each sample is indicated above each lane. The membrane was hybridized with a [γ-³²P]ATP-labeled oligonucleotide probe (Supplemental Table S2; Panel A for the position of the probe). The approximate size of the bands (determined by comparison with the position of RNA markers of known size) is indicated on the left.

FIGURE 4.

Expression analysis of sRNAs by Northern blots. Total RNA samples were prepared from different time points during growth of V. splendidus in marine salt medium and submitted to electrophoresis on an 8% acrylamide gel before transfer to a Hybond N⁺ membrane. The culture OD₆₀₀ for each sample is indicated above each lane, and the growth curve is presented underneath. Membranes were probed with [γ-³²P]-labeled specific oligonucleotides for each candidate sRNA (Supplemental Table S2). The name of the sRNA is indicated on the right as well as the size of the transcript visualized in the RNA-seq. The right-most column indicates the presence of a transcription terminator predicted by ARNold (see Materials and Methods). (On the left) The approximate size determined by comparison with the migration of RNAs of known sizes. 4.5S RNA and tmRNA were used as loading controls.

FIGURE 5.

Confirmation of the existence of sRNAs by RT-PCR. Total RNA samples were prepared as described from cells taken from three time points of growth. The corresponding OD₆₀₀ is indicated above each lane. RNA samples were used as templates for reverse transcription and amplification by PCR (see Materials and Methods) with specific forward and reverse primers (Supplemental Table S2), and the amplification products were subjected to agarose gel electrophoresis together with known size DNA markers (Fermentas). The sizes are indicated on the left side of each panel. (Top panel) RNA samples were checked for complete removal of genomic DNA, using primers specific to 4.5S RNA and tmRNA. When reverse transcriptase (RT) was omitted, no product could be detected. For each sRNA to be tested, a control was run with no template added (labeled -, on top of the lane). Each gel picture is labeled with the name of the tested sRNA.

FIGURE 6.

A fourth copy of CsrB in V. splendidus. (A) Visualization of the genomic context of CsrB2 (left) and CsrB4 (right) on the Artemis viewer. A graph representing the ln of the coverage is visible on the top window. Bam reads are visible below. (Salmon) The extent of both CsrBs. (Blue arrows) The two possible sizes of CsrB2. (Yellow boxes) Putative transcription terminator as predicted by ARNold. (B) Expression of the four CsrBs during growth of V. splendidus. The Northern blots were carried out as described in Figure 4. (C) Secondary structure of the four CsrBs as predicted by Mfold (see Materials and Methods for details). (Orange) The loops containing the CsrA binding motifs (AGGA or AGGGA).

FIGURE 7.

Two examples of putative asRNAs in V. splendidus. (A) Visualization of the genomic context of Vsr285 on the Artemis viewer. Bam reads are visible on the top window and are highlighted in salmon. (Orange box) The Vsr285 encoding gene. (Yellow boxes) Putative transcription terminators, predicted by ARNold. (Bright pink) The r5′UTR of VS_II1110 encoding GuaC. (B) Analysis of expression of Vsr285 during growth of V. splendidus. Northern blotting was carried out as in Figure 4. (C) Vsr65 could be part of a type I toxin–antitoxin system. (Left) Genomic context of Vsr65 (orange box), transcribed antisense of VS_0930 (blue box). (Right) TMHMM profile of VS_0930 (see Materials and Methods) and amino acid sequence of the peptide. (Red) The two predicted transmembrane domains.