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Role for Phosphoglucomutase in Vibrio fischeri-Euprymna scolopes Symbiosis. Cindy R. DeLoney, 2002.Vibrio fischeri, a luminescent marine bacterium, specifically colonizes the light organ of its symbiotic partner, the Hawaiian squid Euprymna scolopes. In a screen for V . fischeri colonization mutants, we identified a strain that exhibited on average a 10-fold decrease in colonization levels relative to that achieved by wild-type V . fischeri . Further characterization revealed that this defect did not result from reduced luminescence or motility, two processes required for normal colonization . We determined that the transposon in this mutant disrupted a gene with high sequence identity to the pgm (phosphoglucomutase) gene of Escherichia coli, which encodes an enzyme that functions in both galactose metabolism and the synthesis of UDP-glucose . The V . fischeri mutant grew poorly with galactose as a sole carbon source and was defective for phosphoglucomutase activity, suggesting functional identity between E . coli Pgm and the product of the V . fischeri gene, which was therefore designated pgm . In addition, lipopolysaccharide profiles of the mutant were distinct from that of the parent strain and the mutant exhibited increased sensitivity to various cationic agents and detergents . Chromosomal complementation with the wild-type pgm allele restored the colonization ability to the mutant and also complemented the other noted defects . Unlike the pgm mutant, a galactose-utilization mutant (galK) of V . fischeri colonized juvenile squid to wild-type levels, indicating that the symbiotic defect of the pgm mutant is not due to an inability to catabolize galactose . Thus, pgm represents a new gene required for promoting colonization of E . scolopes by V . fischeri . Biochemical Characterization of a ß-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate. James A. Coker, 2003.A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil . It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall . Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species . A gene (bgaS) encoding a family 2 ß-galactosidase was cloned from this organism into an Escherichia coli host . Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C) . To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E . coli LacZ counterpart, which was purified for comparison . The His tag additions reduced the specific activities of both ß-galactosidases but did not alter the other characteristics of the enzymes . Kinetic studies using o-nitrophenyl-ß-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ ß-galactosidases . The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C . Comparisons of family 2 ß-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle . Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties . Phylogenetic Diversity of Nitrogenase (nifH) Genes in Deep-Sea and Hydrothermal Vent Environments of the Juan de Fuca Ridge. Mausmi P. Mehta, 2003.The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid . Dissolved N2 gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid . In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid . A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal . Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers . Other nifH genes unique to the vent samples include proteobacteria and divergent Archaea . All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity) . These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater .
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