LExEn: Genetic and Physiological Analysis of the Determinants of Halophillism in Halomonas Elongata

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It is generally accepted that life on Earth originated in sea water. Although primordial cells arose in an environment in which Na + is the most abundant cation, organisms have evolved to maintain the cytoplasmic concentration of Na + much lower than the external Na + concentration. The potential energy generated by unequal distribution of Na + across the cytoplasmic membrane is used to drive diverse processes, such as nutrient uptake, cell motility, and the electrical activity of neurons. NaCl at high concentrations is toxic to most organisms, and therefore, high salinity is one example of an extreme environment. There are a limited number of organisms which require high concentrations of NaCl that would be inhibitory to most other organisms. Species which are dependent on high concentrations of NaCl are called halophiles. Although it is generally believed that halophiles require elevated concentrations of Na + to drive nutrient uptake, this has not been proven conclusively. One of the most halophilic eubacteria is Halomonas elongata, which requires at least 0.5 M NaCl. The research project is testing the hypotheses that H. elongata requires high concentrations of Na + because some of its energy-dependent reactions, such as transport of nutrients or the synthesis of ATP, are driven by a Na + gradient. This organism is uniquely suitable for the analysis of the determinants of halophilism because it can grow in a simple defined salts medium and, as a member of the g-division of the Proteobacteria (which includes E. coli), it is amenable to genetic analysis. This research has the following specific aims: 1) Determine the Na+ requirement of the transport system for glycine betaine in Halomonas elongata. This transporter is chosen for analysis because it has already been expressed in functional form in E. coli, making it possible to test whether its activity has similar requirements for Na + in the non-halophilic background of E. coli as in the halophilic background of H. elongata. 2) Determine whether the transport systems for glucose and amino acids have high Na + requirement in H. elongata. 3) Clone the gene for the glucose transporter of H. elongata and carry out comparative studies of the Na + requirement of the product of this gene in H. elongata and in E. coli. These studies have the practical benefit that they will contribute to the development of H. elongata and other halophiles for the use of detoxification of environmental pollutants in highly saline areas.