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Bactericidal Activity of Glycinecin A, a Bacteriocin Derived from Xanthomonas campestris pv . glycines, on Phytopathogenic Xanthomonas campestris pv . vesicatoria Cells. Huy Thang Pham, 2004.The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv . glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group (S . G . Heu et al., Appl . Environ . Microbiol . 67:4105-4110, 2001) . In the present study, we aimed at determining the glycinecin A-induced cause of death . Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient . Glycinecin A treatment also induced leakage of potassium ions from X . campestris pv . vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner . Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A . These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria . Differential Spectrum of Mutations That Activate the Escherichia coli bgl Operon in an rpoS Genetic Background. Sudha Moorthy, 2002.The bgl promoter is silent in wild-type Escherichia coli under standard laboratory conditions, and as a result, cells exhibit a ß-glucoside-negative (Bgl-) phenotype . Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS . Mutations that confer a Bgl+ phenotype arise spontaneously at a detectable frequency . Transposition of DNA insertion elements within the regulatory locus, bglR, constitutes the major class of activating mutations identified in laboratory cultures . The rpoS-encoded  S, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions . In an attempt to see if the rpoS status of the cell influences the nature of the mutations that activate the bgl promoter, we analyzed spontaneously arising Bgl+ mutants in rpoS+ and rpoS genetic backgrounds . We show that the spectrum of activating mutations in rpoS cells is different from that in rpoS+ cells . Unlike rpoS+ cells, where insertions in bglR are the predominant activating mutations, mutations in hns make up the majority in rpoS cells . The physiological significance of these differences is discussed in the context of survival of natural populations of E . coli .
Molecular Characterization and Quantitative Analysis of Superoxide Dismutases in Virulent and Avirulent Strains of Aeromonas salmonicida subsp . salmonicida. A. Dacanay, 2003.Aeromonas salmonicida subsp . salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry . The mechanisms by which A . salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis . We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB . The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs . The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs . Two enzymes with activities consistent with both these ORFs were identified by inhibition of O2--catalyzed tetrazolium salt reduction in both gels and microtiter plate assays . The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria . The regulatory sequences upstream of putative sodA were consistent with these differences . We could not identify other SOD isozymes such as sodC either functionally or through data mining . Levels of SOD were significantly higher in virulent than in avirulent strains of A . salmonicida subsp . salmonicida strain A449 when cultured in vitro and in vivo . Involvement of a Protein Tyrosine Kinase in Production of the Polymeric Bioemulsifier Emulsan from the Oil-Degrading Strain Acinetobacter lwoffii RAG-1. David Nakar, 2003.The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster . This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis . The two gene products, Wzc and Wzb were overexpressed and purified . Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity . Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc . Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a Vmax of 12 µmol of PNPP min-1 mg-1 and a Km of 8 mM PNPP at 30°C . The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production . Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus . Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency . Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively . Neither of these mutants contained cell-associated emulsan . However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide . A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation .
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