Lawrence Berkeley National Laboratory

Phenotype Microarray (PM) has been developed for the high throughput and rapid assessment of phenotypic responses of microbes to approximately 2,000 metabolites and chemicals under aerobic conditions. Previously in our lab, a method was developed for PM under anaerobic conditions. In the present work we describe a method of inoculum standardization of anaerobes to ensure repeatability of results between replicate runs. Our tests were conducted with the sulfate reducing bacterium Desulfovibrio vulgaris strain Hildenborough in a defined lactate sulfate medium. For optimization of results, several factors were tested that included growth phase of inoculum having the greatest capability for growth after inoculation, optimal centrifugation times at 6000 g for highest retention of cell pellet, optimal inoculum concentration of resuspended cells as determined by AODC which was compared to OD at 600nm and %T. Our results show that standardization was achieved as demonstrated by repeatability of growth data between biological replicates of D. vulgaris in the PM. The application of the anaerobic PM was tested in two different studies with a wild type DvH and a single crossover sensor histidine kinase mutant strain of D. vulgaris with a potentially interesting phenotype under salt stress. The differential expression patterns of wt D. vulgaris and the mutant strain of D. vulgaris were compared. Osmotic sensitivity to NaCl and KCl was increased in the mutant strain with inhibition of growth above 3% as compared to 6% and 5% with the wt. No protection of the mutant was conferred by the addition of osmoprotectant. In another test, the mutant strain was used for the novel application of PM technology to investigate phenotypic expression of an organism under stressed conditions. In this study, anaerobic PM of the mutant strain under osmotic stress was generated with 250mM NaCl vs 250mM KCl and compared with the expression pattern of the organism under non stressed conditions. The mu