Thesis

A novel haloalkaliphilic anoxygenic purple sulfur bacterium: initial studies of the physiology and regulation of carbon and nitrogen metabolism

Anoxygenic phototrophic purple sulfur bacteria belonging to the Ectothiorhodospiraceae inhabit alkaline saline to hypersaline eniviromnents, such as soda lakes. Mono Lake, located in California s Eastern Sierras (63 83 feet above sea level), is a saline ( -8% N aCl) and alkaline (-pH 1 0) meromictic lake. Enrichment cultures of sediment samples taken from the eastern shore of Mono Lake yielded a pure culture of Ectothiorhodospira strain MM2. Pigment analysis showed absorption maxima at 866 nm and 800 nm in whole cells, and 771 nm in methanol extracted cells indicating the presence of bacteriochlorophyll a. The major carotenoid present in strain MM2 was spirilloxanthin. Phylogenetic analyses of strain MM2 indicated that it groups within the genus Ectohiorhodo!>pira, and is most closely related to E. haloalkaliphila BN9902, the most alkaliphilic strains in this genus isolated to date. Strain MM2 grew at NaCl levels ranging from 0%-25%, with an optimum at 8% and good growth obtained at up to 20%, thus making it perhaps the most versatile halophilic member of the genus. Strain MM2 was able to grow photoautotrophically and several carbon sources supported good photoheterotrophic growth of this organism. In addition, strain MM2 was capable of aerobic chemoheterotrophic growth in the dark, a property described for only two other species in the Ectothiorhodospiracea. Our initial studies on the regulation of nitrogen metabolism in strain MM2 indicated that atmospheric dinitrogen, ammonia, glutamate, and glutamine could serve as sole nitrogen sources. Growth on low levels of ammonia ( 100 /-LM) and glutamate served to stimulate N2 fixation suggesting the presence of regulatory mechanisms employed by other anoxygenic phototrophic bacteria. Assays of crude extracts of strain MM2 indicated that fixed N2 or exogenous ammonia was incorporated into an organic form via the induction of two parallel pathways under nitrogen limitation conditions: (1) the GDH pathway, and (2) the GS/GOGAT pathway. To our knowledge this is the first report of a bacterium coordinating a dual system for regulating ammonia assimilation under both high and low fixed nitrogen conditions, and presents the question as to why this organism might require such a strategy. Significant GDH activities were also present in N2 versus ammoniagrown cells, suggesting that strain MM2 possesses a novel GDH enzyme with a low Krn for ammonia. All enzyme activities in strain MM2 were NADPHdependent. Experiments are currently in progress to identify and clone genes involved in nitrogen fixation and nitrogen metabolism in this haloalkaliphilic phototroph. A regulatory mechanism linking carbon and nitrogen metabolism as described recently for nonsulfur purple bacteria (Joshi and Tabita, 1996) may also be present in strain MM2. Physiological studies of wild type cells grown anaerobically in the dark in minimal media containing C02 and malate showed that large volumes of H2 gas were produced, suggesting induction of nitrogenase, or some other hydrogenase system as a redox balancing mechanism permitting growth under these conditions. Experiments are currently in progress to characterize this global regulatory phenomenon in Ectothiorhodospira strain MM2.

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