Lawrence Berkeley National Laboratory

Identification and analysis of small non-coding RNAs (sRNAs) in D. vulgaris and other metal-reducing bacteria are essential for uncovering novel regulatory mechanisms involved in processes critical to the DOE such as stress response, environmental adaptation, and contaminant remediation. Previous work by the 'Environmental Stress Pathway Project' has enhanced our systems knowledge of D. vulgaris via valuable transcriptional and proteomic profiles, however regulation by sRNAs could not be detected in those experiments. In an effort to identify sRNAs in D. vulgaris, a strategy for cloning total RNA ranging in size from 20-200 nt was employed. Following addition of directional aptamer sequences, cDNAs were produced and cloned for sequencing. Sequence analysis of a small portion of the resulting cDNA library yielded two identical ~;65 nt sRNA clones (Dv-sRNA2) possessing complementary sequence to the RBS of open reading frame (ORF) DVU0678. While DVU0678 is adjacent to the Dv-sRNA2 gene, the ORF is transcribed from the opposite chromosomal strand. Northern analysis specialized for sRNAs verified the expression of Dv-sRNA2 as an individual transcript under anaerobic lactate/sulfate growth (LS4D medium). These data suggest that when Dv-sRNA2 is transcribed, translation of DVU0678 will be inhibited. DVU0678 has been annotated to encode a putative 34 amino acid protein unique to D. vulgaris strains Hildenborough and DP4, hampering our abilities to discern the role of DVU0678 in the cell. Further sequence analysis of the Dv-sRNA DNA locus by 'PromScan' identified a putative sigma54-recognition site (97percent probability) 43 nt upstream of the predicted sRNA transcriptional start site and therefore suggests that Dv-sRNA2 may be member of the sigma54 regulon. A perfect stem-loop terminator was also identified 26 nt downstream of the Dv-sRNA2 DNA sequence. Current analysis is underway to ascertain the expression profile for this sRNA as well as the effect over-expression has on the physiology and transcriptional response of D. vulgaris under multiple environmental conditions.