In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Center for Biotechnology Information (NCBI) has established the dbSNP database [S.T.Sherry, M.Ward and K. Sirotkin (1999) Genome Res., 9, 677-679]. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. The complete contents of dbSNP can also be downloaded in multiple formats via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/.

The Rnf complex is a respiratory enzyme that catalyzes oxidation of reduced ferredoxin to the reduction of NAD, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic, acetogenic bacteria the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, , which enabled us to delete the genes and to test their role. The mutant did not grow on H+CO, neither did it produce acetate or ATP from H+CO, and ferredoxin:NAD oxidoreductase activity as well as Na translocation was also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low energy substrates such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO reduction. The electron bifurcating hydrogenase of reduces ferredoxin and indeed, addition of H to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex which renders the Rnf complex essential also for growth on low energy substrates. Ferredoxin and NAD are key electron-carriers in anaerobic bacteria, but energetically they are not equivalent since the redox-potential of ferredoxin is lower than that of the NADH/NAD couple. We describe by mutant studies in that the main function of Rnf is to energeticallylink cellular pools of ferredoxin and NAD When ferredoxin>NADH, exergonic electron flow from ferredoxin to NAD generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH>ferredoxin, the Rnf works in reverse. This reaction is essential for growth on low energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO reduction. Our studies put a new perspective on the cellular function of the membrane-bound, ion-translocating Rnf complex widespread in bacteria.

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