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Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli. Dong Soo Hwang, 2004.Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine . However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed . Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time . Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography . The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance . Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments . The Staphylococcus aureus cidAB Operon: Evaluation of Its Role in Regulation of Murein Hydrolase Activity and Penicillin Tolerance. Kelly C. Rice, 2003.Recent studies have shown that expression of the Staphylococcus aureus lrgAB operon inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing . It was proposed that the lrgAB gene products function in a manner analogous to an antiholin, inhibiting a putative holin from transporting murein hydrolases out of the cell . In the present study the cidAB operon was identified and characterized based on the similarity of the cidA and cidB gene products to the products of the lrgAB operon . Zymographic and quantitative analyses of murein hydrolase activity revealed that mutation of the cidA gene results in decreased extracellular murein hydrolase activity compared to that of S . aureus RN6390, the parental strain . Complementation of cidA expression restored the wild-type phenotype, indicating that expression of the cidAB operon has a positive influence on extracellular murein hydrolase activity . The cidA mutant also displayed a significant decrease in sensitivity to the killing effects of penicillin . However, complementation of the cidA defect did not restore penicillin sensitivity to wild-type levels . Reverse transcriptase PCR also revealed that cidAB is maximally expressed during early exponential growth, opposite of what was previously observed for lrgAB expression . Based on these results, we propose that the cidAB operon encodes the holin-like counterpart of the lrgAB operon and acts in a manner opposite from that of lrgAB by increasing extracellular murein hydrolase activity and increasing sensitivity to penicillin-induced killing . Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures of Saccharomyces cerevisiae. Antonius J. A. van Maris, 2003.Pyruvate decarboxylase-negative (Pdc-) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth . This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol . To test this hypothesis and to eliminate the C2 requirement of Pdc- S . cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA . The addition of L-carnitine to growth media did not restore growth of a Pdc- strain on glucose, indicating that the C2 requirement was not solely due to the inability of S . cerevisiae to synthesize this compound . The S . cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde . Overexpression of GLY1 enabled a Pdc- strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA . Fractionation studies revealed a cytosolic localization of threonine aldolase . The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated . These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis . The Pdc- GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations . Like any other Pdc- strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess . Remarkable Diversity of Phototrophic Purple Bacteria in a Permanently Frozen Antarctic Lake. Elizabeth A. Karr, 2003.Although anoxygenic photosynthesis is thought to play an important role in the primary productivity of permanently frozen lakes in the Antarctic dry valleys, the bacterial communities responsible for this metabolism remain uncharacterized . Here we report the composition and activity of phototrophic purple bacteria in Lake Fryxell, Antarctica, as determined by analysis of a photosynthesis-specific gene, pufM . The results revealed an extensive diversity and highly stratified distribution of purple nonsulfur bacteria in Lake Fryxell and showed which phylotypes produced pufM transcripts in situ . Enrichment cultures for purple bacteria yielded two morphotypes, each with a pufM signature identical to signatures detected by environmental screening . The isolates also contained gas vesicles, buoyancy structures previously unknown in purple nonsulfur bacteria, that may be necessary for these organisms to position themselves at specific depths within the nearly freezing water column .
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