Identification of non-coding RNAs in environmental vibrios.

The discovery of non-coding RNA (ncRNA) has been mainly limited to laboratory model systems and human pathogenic bacteria. In this study, we begin to explore the ncRNA diversity in four recently sequenced environmental Vibrio species (Vibrio alginolyticus 40B, Vibrio communis 1DA3, Vibrio mimicus VM573 and Vibrio campbellii BAA-1116) by performing in silico searches using Infernal and Rfam for the identification of putative ncRNA-encoding genes. This search method resulted in the identification of 31-38 putative ncRNA genes per species and the total ncRNA catalogue spanned an assortment of regulatory mechanisms (riboswitches, cis-encoded ncRNAs, trans-encoded ncRNAs, modulators of protein activity, ribonucleoproteins, transcription termination ncRNAs and unknown). We chose to experimentally validate the identifications for V. campbellii BAA-1116 using a microarray-based expression profiling strategy. Transcript hybridization to tiled probes targeting annotated V. campbellii BAA-1116 intergenic regions revealed that 21 of the 38 predicted ncRNA genes were expressed in mid-exponential-phase cultures grown in nutrient-rich medium. The microarray findings were confirmed by testing a subset of three highly expressed (6S, tmRNA and TPP-2) and three moderately expressed (CsrB, GcvB and purine) ncRNAs via reverse transcription PCR. Our findings provide new information on the diversity of ncRNA in environmental vibrios while simultaneously promoting a more accurate annotation of genomic intergenic regions.