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Identification of the L-Aspartate Transporter in Bacillus subtilis. Graciela Lorca, 2003.YveA of Bacillus subtilis, a putative transporter of the amino acid/polyamine/organocation (APC) superfamily, is shown to mediate uptake of both L-aspartate and L-glutamate as well as having sensitivity to L-aspartate hydroxamate . This 14 TMS protein is the primary aspartate uptake system in B . subtilis and serves as the prototype for a new family within the APC superfamily . Novel 4'-Substituted Stavudine Analog with Improved Anti-Human Immunodeficiency Virus Activity and Decreased Cytotoxicity. Ginger E. Dutschman, 2004.The antiviral drug 2',3'-didehydro-3'-deoxythymidine (D4T; also know as stavudine and Zerit), which is used against human immunodeficiency virus (HIV), causes delayed toxicity (peripheral neuropathy) in long-term use . After examining a series of 2',3'-didehydro-3'-deoxy-4'-substituted thymidine (4'-substituted D4T) analogs, 4'-ethynyl D4T was found to have a fivefold-better antiviral effect and to cause less cellular and mitochondrial toxicity than D4T . The antiviral activity of this compound can be reversed by dThd but not by dCyd . The compound acted synergistically with ß-L-2',3'-deoxy-3'-thiacytidine (also known as lamivudine) and ß-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (also known as elvucitabine) and additively with 2',3'-dideoxyinosine (also known as didanosine and Videx) and 3'-azido-3'-deoxythymidine (also known as Retovir and zidovudine) against HIV . 4'-Ethynyl D4T is phosphorylated by purified human thymidine kinase 1 (TK-1) from CEM cells with a faster relative Vmax and a lower Km value than D4T . The efficiency of TK-1 in the phosphorylation of 4'-ethynyl D4T is fourfold better than that of D4T . While D4T is broken down by the catabolic enzyme thymidine phosphorylase, the level of breakdown of 4'-ethynyl D4T was below detection . Since 4'-ethynyl D4T has increased anti-HIV activity and decreased toxicity and interacts favorably with other currently used anti-HIV drugs, it should be considered for further development as an anti-HIV drug . Synchronous Effects of Temperature, Hydrostatic Pressure, and Salinity on Growth, Phospholipid Profiles, and Protein Patterns of Four Halomonas Species Isolated from Deep-Sea Hydrothermal-Vent and Sea Surface Environments. Jonathan Z. Kaye, 2004.Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30°C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts) . The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30°C, 0.1 MPa, and 4% total salts . Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures . Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions . The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses . Phospholipid profiles obtained at 30°C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure . Profiles of cytosolic and membrane proteins of Halomonas axialensis and H . hydrothermalis performed at 30°C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two . The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments . Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE. Akihiko Kosugi, 2002.engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y . Tamaru and R . H . Doi, J . Bacteriol . 181:3270-3276, 1999) . The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins . Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA . Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface . This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C . cellulovorans . When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall . However, rEngEs without SLH domains lost their ability to bind to cell walls . When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE . However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions . These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin . Coordinate Regulation of the Escherichia coli Formate Dehydrogenase fdnGHI and fdhF Genes in Response to Nitrate, Nitrite, and Formate: Roles for NarL and NarP. Henian Wang, 2003.Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons . To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions . Complementary patterns of gene expression were seen . Optimal fdhF-lacZ expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression . In contrast, fdnG-lacZ expression was induced 25-fold in the presence of intermediate to high nitrate concentrations . Consistent with prior reports, NarL was able to induce fdnG-lacZ expression . However, NarP could not induce expression; rather, it functioned as an antagonist of fdnG-lacZ expression under low-nitrate conditions (i.e., it was a negative regulator) . Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP . The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing . Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon . Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis. Erh-Min Lai, 2003.The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease . To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry . We identified 38 B . subtilis spore proteins, 12 of which are known coat proteins . We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively . In addition, we initiated a study of coat proteins in B . anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins . We also queried the unfinished B . anthracis genome for potential coat proteins . Our analysis suggests that the B . subtilis and B . anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins . Nonetheless, a significant number of coat proteins are probably unique to each species . These results should accelerate efforts to develop B . anthracis detection methods and understand the ecological role of the coat .
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