Lawrence Berkeley National Laboratory

In an effort to investigate effects of environmental stressors on bacteria that are found to co-exist in several DOE-contaminated sites, we studied the effect of salt stress on Geobacter metallireducens strain GS15. A comparison of these results with salt stress in Desulfovibrio vulgaris and Shewanella oneidensis will provide a better understanding of bioremediation by these bacteria under hypersaline conditions. Strain GS15 was grown in the defined LS4D medium at 30oC containing 10mM acetate and 10mM Fe-NTA. In these growth conditions, the minimum inhibitory concentration of NaCl on this organism was determined to be 100mM. To examine salt stress response of GS15 in detail, 100mM NaCl was added to a midlog phase culture and cells were harvested after 1, 2 and 4 hours for different analyses including Fe(III) reduction, transcriptomics and phospholipids fatty acid analysis (PLFA). In stressed cultures, after 4 hours of exposure to salt, only 7mM of Fe(II) was reduced compared to more than 9mM in non-stressed controls. Transcriptomic analysis revealed that after 4 hrs, the most up-regulated genes observed were those that encode heat shock proteins, such as Hsp90, Hsp 20, GrpE, DnaK, and DnaJ. These heat shock proteins perform protein repair functions which can be in demand during stress. Also up-regulated were genes encoding proteins in the large and small subunits of the ribosome. The most down-regulated genes included those encoding the, sodium ion symporter, some genes of cytochrome C family and flagellar proteins (flagellin, FlaG, and FlgJ). It was also observed that several proteins involved in the process of chemotaxis like CheA, CheR, CheY, CheX along with several histidine kinases and response regulators were downregulated. PLFA analysis of stressed cells was performed and compared to non-stressed cells, showing small shifts in relative levels of saturated and unsaturated fatty acids in response to salt stress. The results of this study suggests that presence of 100mM NaCl causes some perturbation in the cell machinery of strain GS15 and that cells are able to overcome this stress without fatal consequences in response to stress.