Abstract

1. Sulfate Reduction and the Sodium Gradient in Desulfovibrio vulgaris Hildenborough Background Future Plans References Δnqr1 Growth Model System E.C. Drury and J.D. Wall Abstract • nqr1: • Construction of a deletion mutant for the decaheme cytochrome to investigate its role of electron transfer specifically in Sulfate reduction. • Disruption of the sodium gradient to further elucidate the connection between the Na+ gradient to the NQR complex. • Continued growth studies in fermentation, electron donors and acceptors, and media to determine the extent of the role of the NQR complex in different growth conditions. • Sulfate Permease: • Construction of deletion mutants and physiological studies for (DVU0053, DVU0279, and DVU1999) to investigate possible link with the NQR complex and the observed phenotype of impaired sulfate reduction. The anaerobic sulfate-reducing bacteria (SRB) have the ability to reduce sulfate and some environmental contaminants. Ion gradients are necessary for nutrient acquisition and energy generation. Here, the sodium gradient is being investigated for its potential involvement in sulfate metabolism. The NQR complex of six proteins has been suggested to play a role in generating or maintaining a sodium motive force coupled to primary metabolism in marine bacteria. Orthologous operons have been identified in Desulfovibrio vulgaris Hildenborough and Desulfovibrio desulfuricans G20, and the possible influence of this complex on sulfate reduction, the characteristic energy pathway of these bacteria, was explored. The predicted NQR operon in D. vulgaris is orthologous to a complex encoding a sodium-dependent NADH quinone oxidoreductase, studied in Vibrio alginolyticus and Rhodobacter capsulatus. A deletion mutant of nqr1 (VIMSS208298, DVU2792), the second gene in the operon, in D. vulgaris was constructed in the ATCC#29579 strain, as well the derivative strain lacking the 203 kb megaplasmid. The Δnqr1 mutant was impaired in its ability to reduce sulfate, although its growth was comparable to wild type with sulfite or thiosulfate as the terminal electron acceptor. The transmembrane NQR complex is being investigated for its role in electron transfer for sulfate reduction and its role in maintaining the sodium gradient for sulfate transport. Figure 1. WT and Δnqr1D. vulgaris grown on Lactate and Sulfate/Sulfite • 20% inhibition of maximum growth on Sulfate (SO42-), comparison of wild type vs. Δnqr1. • Similar growth pattern (as above) was shown for the wild type and Δnqr1 with Pyruvate as the electron donor and Sulfate or Sulfite as the electron acceptor. • Significantly larger discrepancy observed between wild type and Δnqr1 in growth with Hydrogen as the electron donor and Sulfate as the electron acceptor. Figure 2. Δnqr1D. vulgaris subcultured to Lactate and Sulfate/Sulfite Table. 1. Mutants constructed for this work • A subculture was performed after growing the Δnqr1 mutant in Sulfate and Sulfite media (previous graph) to determine if the slowed growth phenotype on Sulfate was overcome after reaching exponential stage or having been grown on Sulfite. • Growth on Sulfite by the Δnqr1 was unaffected regardless of the previous growth medium. Figure 3. Δnqr1D. vulgaris grown on Lactate and Thiosulfate Figure 4. Proposed Model of NQR System a. Δ : constructed by marker replacement, Kanamycin resistance. b. K.O. : constructed by plasmid insertion. • http://www.microbesonline.org/ • M. Hayashi, et al. (2001) Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus. Biochim Biophys Acta. 1505(1):37-44 • Morita, Y., et al. 1998. NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli. Antimicrob. Agents Chemother. 42:1778-1782.- Thank you to Dr. Tomofusa Tsuchiya for the E. coli mutants for the NorM studies. 49kb 12kb nqr1: The NQR complex, a NADH quinone oxidoreductase, is predicted to be implicated in electron transport and potentially pumping Na+. The first gene in the predicted operon, dhcA, is a decaheme cytochrome. The last gene is a predicted membrane-associated lipoprotein. The rest of the genes are predicted to form the NQR complex. Growth on Thiosulfate (with Lactate) by the Δnqr1 mutant showed little difference from the growth of the wild type.