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Identifying Regulatory Targets of the Small RNA MtlS in Vibrio cholerae

In the facultative pathogen Vibrio cholerae, MtlS is a small RNA (sRNA) transcribed antisense to the 5′ untranslated region of mtlA, which codes for the transporter of the sugar alcohol mannitol. MtlS works in cis as a translational repressor of mtlA and it is postulated that the sRNA allows the bacteria to respond rapidly to changes in environmental carbon sources. Because bacterial sRNAs can target multiple genes, we hypothesized that MtlS may regulate other genes in addition to mtlA. Previous work in the lab used RNA target‐prediction programs and mass spectrometry‐based proteomics to identify additional MtlS targets. The results identified 6 potential targets: VCA1045 (mtlA), VCA0820, VCA1046, VC1560, VC1899, and VC2683. The objective of this study was to determine if the putative targets were post‐transcriptionally regulated by MtlS. We constructed gfp translational reporter fusions of the candidate genes in a V. cholerae ΔmtlS strain carrying either a control vector or a plasmid ectopically expressing mtlS. An mtlA‐GFP fusion served as a positive control and exhibited the expected down‐regulation of gfp expression when mtlS was expressed. We have tested reporter fusions to all the candidate genes for regulation by MtlS by monitoring total cell fluorescence and western blot analysis. In an experiment done in duplicate in the presence of MtlS, during stationary phase growth we observed 20% down‐regulation of VC1899, encoding a protein with homology to nucleic acid‐binding proteins. In an experiment done in duplicate, we have also observed 30% logarithmic and stationary phase downregulation of VCA1046, encoding the mannitol dehydrogenase mtlD, in the presence of MtlS. Results based on another experiment conducted in duplicate indicate 20% logarithmic and stationary phase down‐regulation of VC1560, encoding a protein with homology to a catalase‐peroxidase, in the presence of MtlS. Tests with VC2683, encoding a putative cystathionine gamma‐synthase, have been inconclusive, as both down‐regulation and up‐regulation by MtlS has been observed; further experiments will need to be conducted with the VC2683‐gfp fusion. The VCA0820‐gfp fusion does not exhibit any fluorescence, in the presence or absence of MtlS. This work positively contributes to the notion that a single sRNA can have multiple regulatory targets. Ultimately, this work will expand our knowledge on MtlS's role in helping V. cholerae adapt quickly to changes in environmental carbon sources and on sRNAs in general.