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In Vitro Activity of Anidulafungin against Selected Clinically Important Mold Isolates. Zekaver Odabasi, 2004.In this study, we evaluated the in vitro activity of anidulafungin against selected mold isolates . Anidulafungin showed promising activity against Bipolaris spicifera, Exophiala jeanselmei, Fonsecaea pedrosoi, Madurella mycetomatis, Penicillium marneffei, Phialophora verrucosa, Pseudallescheria boydii, Sporothrix schenckii, and Wangiella dermatitidis. Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of D-Erythorbic Acid in Recombinant Pichia pastoris. Tuomas Salusjärvi, 2004.A D-erythorbic acid-forming soluble flavoprotein, gluconolactone oxidase (GLO), was purified from Penicillium cyaneo-fulvum strain ATCC 10431 and partially sequenced . Peptide sequences were used to isolate a cDNA clone encoding the enzyme . The cloned gene (accession no . AY576053) exhibits high levels of similarity with the genes encoding other known eukaryotic lactone oxidases and also with the genes encoding some putative prokaryotic lactone oxidases . Analysis of the coding sequence of the GLO gene indicated the presence of a typical secretion signal sequence at the N terminus of GLO . No other targeting or anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria . Experimental evidence, including the N-terminal sequence of mature GLO and data on glycosylation and localization of the enzyme in native and recombinant hosts, supports this analysis . The GLO gene was expressed in Pichia pastoris, and recombinant GLO was produced by using the strong methanol-induced AOX1 promoter . In order to evaluate the suitability of purified GLO for production of D-erythorbic acid, we immobilized it on N-hydroxysuccinimide-activated Sepharose and found that the immobilized GLO retained full activity during immobilization but was rather unstable under reaction conditions . Our results show that both soluble and immobilized forms of GLO can, in principle, be used for production of D-erythorbic acid from D-glucono-  -lactone or (in combination with glucose oxidase and catalase) from glucose . We also demonstrated the feasibility of glucose-D-erythorbic acid fermentation with recombinant strains coexpressing GLO and glucose oxidase genes, and we analyzed problems associated with construction of efficient D-erythorbic acid-producing hosts .
barS1, a Gene for Biosynthesis of a  -Butyrolactone Autoregulator, a Microbial Signaling Molecule Eliciting Antibiotic Production in Streptomyces Species.Noriyasu Shikura, 2002.From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M1 and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the -butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S . virginiae . The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [Mr, 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA . The deduced BarS1 protein is weakly homologous to ß-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase . The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis . Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product . In the
 barS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost . The production of virginiamycin by the
barS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S . virginiae and that the failure of virginiamycin production was a result of the loss of VB production .
The Flavoenzyme Ferredoxin (Flavodoxin)-NADP(H) Reductase Modulates NADP(H) Homeostasis during the soxRS Response of Escherichia coli. Adriana R. Krapp, 2002.Escherichia coli cells from strain fpr, deficient in the soxRS-induced ferredoxin (flavodoxin)-NADP(H) reductase (FPR), display abnormal sensitivity to the bactericidal effects of the superoxide-generating reagent methyl viologen (MV) . Neither bacteriostatic effects nor inactivation of oxidant-sensitive hydrolyases could be detected in fpr cells exposed to MV . FPR inactivation did not affect the MV-driven soxRS response, whereas FPR overexpression led to enhanced stimulation of the regulon, with concomitant oxidation of the NADPH pool . Accumulation of a site-directed FPR mutant that uses NAD(H) instead of NADP(H) had no effect on soxRS induction and failed to protect fpr cells from MV toxicity, suggesting that FPR contributes to NADP(H) homeostasis in stressed bacteria . The Activator of GntII Genes for Gluconate Metabolism, GntH, Exerts Negative Control of GntR-Regulated GntI Genes in Escherichia coli. Ryouichi Tsunedomi, 2003.Gluconate is one of the preferred carbon sources of Escherichia coli, and two sets of gnt genes (encoding the GntI and GntII systems) are involved in its transport and metabolism . GntR represses the GntI genes gntKU and gntT, whereas GntH was previously suggested to be an activator for the GntII genes gntV and idnDO-gntWH. The helix-turn-helix residues of the two regulators GntR and GntH exhibit extensive homologies . The similarity between the two regulators prompted analysis of the cross-regulation of the GntI genes by GntH . Repression of gntKU and gntT by GntH, as well as GntR, was indeed observed using transcriptional fusions and RNA analysis . High GntH expression, from cloned gntH or induced through 5-ketogluconate, was required to observe repression of GntI genes . Two GntR-binding elements were identified in the promoter-operator region of gntKU and were also shown to be the target sites of GntH by mutational analysis . However, the GntI genes were not induced by gluconate in the presence of enhanced amounts of GntH, whereas repression by GntR was relieved by gluconate . The repression of GntI genes by GntH is thus unusual in that it is not relieved by the availability of substrate . These results led us to propose that GntH activates GntII and represses the GntI genes in the presence of metabolites derived from gluconate, allowing the organism to switch from the GntI to the GntII system . This cross-regulation may explain the progressive changes in gnt gene expression along with phases of cell growth in the presence of gluconate . Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a Dehalococcoides Species. Jianzhong He, 2003.A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen . To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich . After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors . PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process . Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 µmol liter-1day-1, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate . The half-saturation constant (KS) for VC was 5.8 µM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE . Similar VC dechlorination rates were observed at temperatures between 22 and 30°C, and negligible dechlorination occurred at 4 and 35°C . Reductive dechlorination in medium amended with ampicillin was strictly dependent on H2 as electron donor . VC-dechlorinating cultures consumed H2 to threshold concentrations of 0.12 ppm by volume . 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population . These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors .
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