Halobacterium halobium is a very unusual microorganism that grows optimally in nearly saturated brine. Although H. halobium usually generates energy for growth by oxidative phosphorylation, it has the capacity for anaerobic growth by photophosphorylation. For photophosphorylation, the light-driven proton pumping activity of the purple membrane protein, bacteriorhodopsin, is necessary. Phototrophic growth also requires medium supplemented with retinal, the purple membrane chromophore, biosynthesis of which requires oxygen. H. halobium induces the synthesis of bacteriorhodopsin and also the intracellular organelles called gas vesicles when grown under limiting oxygen and high light intensity. Gas vesicles allow cells to float to the surface of the culture, thus, increasing the availability of oxygen and light. We have recently determined that the induction of both purple membrane and gas vesicles occurs at the transcriptional level. Thus, we have in hand a very interesting system for studying the molecular mechanisms of genetic regulation in H. halobium. We already have the necessary tools to carry out the proposed study, including cloned purple membrane (bop) and gas vesicle (gvp) genes, regulatory mutants, and assays for both gas vesicle and purple membrane content and mRNA levels. The following studies are planned: 1) We will re-examine the parameters that regulate gas vesicle and purple membrane synthesis, including the role of oxygen and light as well as other environmental and nutritional parameters. 2) We will also investigate the mechanism of coordinate synthesis of the purple membrane opsin and chromophore. 3) other H. halobium genes exhibiting similar modes of regulation will be isolated by differential hybridization techniques. 4) The promoter-regulatory region of bop, gvp and other coordinately regulated genes will be examined in the wild-type and regulatory mutants by in vivo footprinting experiments under various growth conditions. 5) Selectable shuttle vectors will be constructed for transfer of genes between H. halobium and E. coli. Important long-term goals would be to understand the precise biophysical mechanisms of gene regulation operating in the extremely saline cytoplasm of H. halobium. Knowledge about genetic regulation is important to health for many reasons such as the fact that many diseases result from mutation of normal regulatory properties.
