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.

In recent years, genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress; however, the current method of deletion construction via marker exchange mutagenesis does not allow for easy selection of multiple sequential gene deletions because of the low number of selectable markers now available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation of D. vulgaris, an in-frame markerless deletion system is being developed based on the upp-encoded uracil phosphoribosyltransferase as an element for a counterselection strategy. In wild-type D. vulgaris, growth is inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene is resistant to 5-FU. The introduction of a plasmid containing the wild-type upp gene expressed constitutively from the aph(5')-III promoter (the promoter for the kanamycin resistance gene in Tn5) into the upp deletion strain restored sensitivity to 5-FU. This observation is the basis for the establishment of a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion does not contain an antibiotic cassette, multiple gene deletions can be generated in a single strain using this method. To construct such a markerless deletion for the R-subunit (DVU1703) of a type I restriction-modification system, Gateway Technology methods (Invitrogen) are being used. A destination vector containing the constitutively expressed wild-type upp gene has been constructed and is available for generating deletion vectors. Its use is reported here.

The dissimilatory sulfate-reducing deltaproteobacterium Desulfovibrio vulgaris Hildenborough (ATCC 29579) was chosen by the research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status. Here, we describe a collection of genetic constructs generated by ENIGMA that are available to the research community.