Lawrence Berkeley National Laboratory

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.