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Defined Anaerobic Growth Medium for Studying Candida albicans Basic Biology and Resistance to Eight Antifungal Drugs. Raluca Dumitru, 2004.The polymorphic fungus Candida albicans is one of the most versatile opportunistic pathogens in humans . Many organs of the human body are potential targets for infection by this pathogen, but infection is commonly localized in the gastrointestinal tract, an environment providing anaerobic growth conditions . We describe a chemically defined anaerobic growth medium for four strains of Candida albicans (A72, SC5314, MEN, and 10261) . It is a defined liquid glucose-phosphate-proline growth medium supplemented with oleic acid, nicotinic acid, and ammonium chloride . The cells did not require or respond to added ergosterol . Oleic acid and nicotinic acid are growth factors which are required only for the anaerobic growth of C . albicans . An important technical feature of this study was the use of anaerobically grown inocula to study anaerobic growth . Anaerobically, the cells grew exclusively as mycelia at 25, 30, and 37°C . The doubling time at 30°C was ca . 20 h . The cells did not produce farnesol and did not respond to exogenous farnesol, and they were resistant to the highest tested levels of amphotericin B and four of the azole antifungals . We suggest that the anaerobic growth of C . albicans may contribute to the trailing end point phenomenon and the resistance of C . albicans biofilms to antifungal drugs . Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression. Tal Zusman, 2002.To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene . The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid . The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L . pneumophila . Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain . Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion . However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii . To determine the involvement of the relA gene product in expression of L . pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains . Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain . Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained . We concluded that RelA is dispensable for intracellular growth of L . pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L . pneumophila icm/dot gene expression . Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies. F. G. Healy, 2002.The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism . Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase . Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family . It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog . The ORF was mutated in S . acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A . Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A . Comparisons of electrospray mass spectra as well as 1H- and 13C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A . The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts . TxtC produced in E . coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form . Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide . Biofilm Formation by Hyperpiliated Mutants of Pseudomonas aeruginosa. Poney Chiang, 2003.Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation . Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation . Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment . Biodegradation of the Nitramine Explosive CL-20. Sandra Trott, 2003.The cyclic nitramine explosive CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) was examined in soil microcosms to determine whether it is biodegradable . CL-20 was incubated with a variety of soils . The explosive disappeared in all microcosms except the controls in which microbial activity had been inhibited . CL-20 was degraded most rapidly in garden soil . After 2 days of incubation, about 80% of the initial CL-20 had disappeared . A CL-20-degrading bacterial strain, Agrobacterium sp . strain JS71, was isolated from enrichment cultures containing garden soil as an inoculum, succinate as a carbon source, and CL-20 as a nitrogen source . Growth experiments revealed that strain JS71 used 3 mol of nitrogen per mol of CL-20 .
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