Dissimilatory metal-reducing microorganisms play an important role in the natural cycling of organic matter and minerals in aquatic sediments, submerged soils, and subsurface environments and can be important agents for the bioremediation of both organic and metal contamination (13
). Molecular analyses (16S ribosomal DNA) have revealed that dissimilatory metal-reducing microorganisms in the genus Geobacter
are prominent members of the microbial community in a diversity of environments in which dissimilatory metal reduction is either naturally occurring or artificially stimulated (22
species are obligate anaerobes belonging to the delta subdivision of the Proteobacteria
. These organisms have the ability to completely oxidize organic compounds to carbon dioxide with either humic substances or Fe(III) as the sole electron acceptor (15
). Other metals which can serve as electron acceptors for Geobacter
species include Mn(IV), U(IV), Co(III), and Tc(VII). Several Geobacter
species can also reduce nitrate and fumarate. The organic compounds oxidized by Geobacter
species invariably include acetate and other short-chain fatty acids. In addition, some Geobacter
species are capable of completely oxidizing monoaromatic compounds such as benzoate, phenol, p
-cresol, and toluene to carbon dioxide with Fe(III) as the electron acceptor.
Little is known about the biochemical pathways that couple the oxidation of organic compounds to the reduction of metals in Geobacter species or about the regulation of these processes. Because many electron carriers will nonspecifically reduce metals and humic substances in vitro, it has been difficult to use biochemical studies to determine which of the numerous redox active proteins present in Geobacter species are actually involved in the reduction of metals and humic substances in vivo. It may therefore be easier to deduce the physiological roles of redox active proteins and enzyme complexes via a genetic approach—gene disruption followed by phenotypic analysis. Until now, the lack of a genetic system for Geobacter species has prevented the application of this type of approach to the study of the physiology of these organisms.
Here we report the development of a genetic system for Geobacter sulfurreducens. G. sulfurreducens
, which was isolated from hydrocarbon-contaminated soil (7
), has all of the important metabolic features of Geobacter
species, including the ability to oxidize monoaromatic compounds (15
). Furthermore, G. sulfurreducens
also has the capacity to grow with fumarate serving as the sole electron acceptor, a property which is essential for the generation of mutants that are defective in the transfer of electrons to metals and humic substances.
Preliminary studies have suggested that G. sulfurreducens
might have genes for nitrogen fixation (4
). The ability to fix nitrogen may be required for Geobacter
to compete successfully in petroleum-contaminated subsurface environments which are carbon rich but contain little fixed nitrogen (4
). Methods for genetically manipulating G. sulfurreducens
were developed as part of a study assessing the capacity of this organism to fix nitrogen. In this study, the targeted disruption of a G. sulfurreducens
homolog of the nifD
gene, a gene required for nitrogen fixation by other microorganisms (9
), was found to eliminate the ability of G. sulfurreducens
to grow in a medium devoid of fixed nitrogen. The ability of G. sulfurreducens
to grow in this medium was restored when a functional copy of the gene was reintroduced in trans
. These results indicate that G. sulfurreducens
fixes nitrogen in a manner similar to that of other nitrogen-fixing microorganisms. The genetic techniques described herein should be applicable to the study of other aspects of G. sulfurreducens
physiology and should make it possible to take full advantage of the information present in the forthcoming sequence of the genome of this environmentally significant organism.