Development of a genetic system for Geobacter sulfurreducens

Members of the genus Geobacter are the dominant metal-reducing microorganis ms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic sys tem was developed for Geobacter sulfurreducens, The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it a t high efficiency were established. A protocol for the introduction of fore ign DNA into G. sulfurreducens by electroporation was also developed, Two c lasses of broad-host-range vectors, IncQ and pBBR1, were found to be capabl e of replication in G. sulfurreducens. Ln particular, the IncQ plasmid pCD3 42 was found to be a suitable expression vector for this organism, When the information and novel methods described above were utilized, the nifD gene of G, sulfurreducens was disrupted by the single-step gene replacement met hod, Insertional mutagenesis of this key gene in the nitrogen fixation path way impaired the ability of G, sulfurreducens to grow in medium lacking a s ource of fixed nitrogen. Expression of the nifD gene in trans complemented this phenotype, This paper constitutes the first report of genetic manipula tion of a member of the Geobacter genus.