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Isolation and Characterization of Burkholderia cenocepacia Mutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability. Kate L. Farmer, 2004.The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin . To isolate mutants which do not produce this siderophore, we mutagenized B . cenocepacia with the transposon mini-Tn5Tp . Two nonfluorescent mutants were identified which were unable to produce pyochelin . In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter . The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl . Sulfate uptake assays confirmed that both mutants were defective in sulfate transport . Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin . Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth . We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis . These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions . The Active Component of the Bioemulsifier Alasan from Acinetobacter radioresistens KA53 Is an OmpA-Like Protein. Amir Toren, 2002.The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein . Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity . The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp . In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli . Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E . coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex . In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca . 80 µg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein . E . coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity . The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan .
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