# Presentation: 351042

##       Sulfate Reduction and the Sodium Gradient in 

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**      ****Sulfate Reduction and the Sodium Gradient in **
**	****	****	****	****	****	****	****	****	****	****	****	****	****	****	****	*****Desulfovibrio vulgaris *****Hildenborough**

- 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 Δ*nqr1* 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.

-   _[http://www.microbesonline.org/](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.

_*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.

-    _*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.

-   20% inhibition of maximum growth on Sulfate (SO42-), comparison of wild type vs. Δ*nqr1*.
-   Similar growth pattern (as above) was shown for the wild type and Δ*nqr1* with Pyruvate as the electron donor and Sulfate or Sulfite as the electron acceptor.
-   Significantly larger discrepancy observed between wild type and Δ*nqr1* in growth with Hydrogen as the electron donor and Sulfate as the electron acceptor.

- A subculture was performed after growing the Δ*nqr1 *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 1. _WT and __Δ__*nqr1*__ __*D. vulgaris*__ grown on Lactate and Sulfate/Sulfite_

- Figure 2. _Δ__*nqr1*__ __*D. vulgaris*__ subcultured to Lactate and Sulfate/Sulfite_

- Figure 4. _Proposed Model of NQR System_

- Table. 1. _Mutants constructed for this work_

- Figure 3. _Δ__*nqr1*__ __*D. vulgaris*__ grown on Lactate and Thiosulfate_

- E.C. Drury and J.D. Wall

- Growth on Thiosulfate (with Lactate) by the Δ*nqr1* mutant showed little difference from the growth of the wild type.

- a.  Δ :  constructed by marker replacement, Kanamycin resistance.
- b.  K.O. :  constructed by plasmid insertion.