Regulation of osmolyte synthesis in the moderate halophile Halobacillus halophilus

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The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus copes with the salinity in its environment by the intracellular production of compatible solutes in molar concentrations. Interestingly, it switches its osmolyte strategy from glutamate/glutamine at moderate to proline at high salinities and we have obtained evidence for a two-step process involved in sensing salinity with chloride and glutamate as key players. Here, we will address the molecular basis of sensing and responding to changing salinities using subcellular systems. The glutamine synthetase activity in whole cells was characterized as a chloride and salt sensor and the responsible enzyme (GlnA2) was identified and purified. We will address the mode of sensing and activation by analyzing purified GlnA2 with biochemical, biophysical, molecular and genetic tools. The genes involved in salinity-dependent proline biosynthesis were also identified and their expression was shown to be stimulated to a great extent by glutamate. In the second part of the project we will focus on the mechanism of glutamate-activated gene expression. Furthermore, the regulation of production of proline biosynthetic proteins as well as their enzymatic activity will be studied.