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Transcriptome Analysis of the Response of Pseudomonas aeruginosa to Hydrogen Peroxide. Marco Palma, 2004.Pseudomonas aeruginosa must often overcome a high concentration of oxidants to successfully infect the human host . We report here the results of a transcriptome profiling comparing cells treated with H2O2 and untreated controls . The data indicate that the early response of P . aeruginosa to H2O2 consists of an upregulation of protective mechanisms and a downregulation of primary metabolism . In Vitro Effects of Resveratrol on the Viability and Infectivity of the Microsporidian Encephalitozoon cuniculi. José Leiro, 2004.Microsporidians of the genus Encephalitozoon are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments . The present study investigated the viability and infectivity of spores of Encephalitozoon cuniculi that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine . RESV at 50 µM showed significant sporicidal activity, and at 10 to 50 µM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line . At 10 µM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability . These results suggest that RESV may be useful in the treatment of Encephalitozoon infections . In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli. Stephan Gruenewald, 2004.Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin . Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches . Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule . In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases . E . coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4'-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes . D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis . Coexpression of the corresponding genes in E . coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment . Furthermore, the cyclic dipeptide is stable and not toxic to E . coli and is secreted into the culture medium without the need for any additional factors . Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature . By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter . As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals . Redefining the Role of psr in ß-Lactam Resistance and Cell Autolysis of Enterococcus hirae. Frédéric Sapunaric, 2003.The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the ß-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of Enterococcus hirae was investigated by disruption or substitution of the corresponding pbp5 and psr genes by Campbell-type recombination . The resulting modifications were confirmed by hybridization and PCR . The low susceptibility of E . hirae to ß-lactams was confirmed to be largely dependent on the presence of PBP5 . However, against all expectations, inactivation of psr in ATCC 9790 or complementation of R40 cells with psr did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates . These results indicated that the psr gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in ß-lactam resistance or in the regulation of cell autolysis in E . hirae . Function and Regulation of the Formate Dehydrogenase Genes of the Methanogenic Archaeon Methanococcus maripaludis. Gwendolyn E. Wood, 2003.Methanococcus maripaludis is a mesophilic species of Archaea capable of producing methane from two substrates: hydrogen plus carbon dioxide and formate . To study the latter, we identified the formate dehydrogenase genes of M . maripaludis and found that the genome contains two gene clusters important for formate utilization . Phylogenetic analysis suggested that the two formate dehydrogenase gene sets arose from duplication events within the methanococcal lineage . The first gene cluster encodes homologs of formate dehydrogenase  (FdhA) and ß (FdhB) subunits and a putative formate transporter (FdhC) as well as a carbonic anhydrase analog . The second gene cluster encodes only FdhA and FdhB homologs . Mutants lacking either fdhA gene exhibited a partial growth defect on formate, whereas a double mutant was completely unable to grow on formate as a sole methanogenic substrate . Investigation of fdh gene expression revealed that transcription of both gene clusters is controlled by the presence of H2 and not by the presence of formate .
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