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Fusidic Acid-Resistant Mutants of Salmonella enterica Serovar Typhimurium Have Low Levels of Heme and a Reduced Rate of Respiration and Are Sensitive to Oxidative Stress. Mirjana Macvanin, 2004.Mutations in the translation elongation factor G (EF-G) make Salmonella enterica serovar Typhimurium resistant to the antibiotic fusidic acid . Fusr mutants are hypersensitive to oxidative stress and rapidly lose viability in the presence of hydrogen peroxide . We show that this phenotype is associated with reduced activity of two catalase enzymes, HPI (a bifunctional catalase-hydroperoxidase) and HPII (a monofunctional catalase) . These catalases require the iron-binding cofactor heme for their activity . Fusr mutants have a reduced rate of transcription of hemA, a gene whose product catalyzes the first committed step in heme biosynthesis . Hypersensitivity of Fusr mutants to hydrogen peroxide is abolished by the presence of  -aminolevulinic acid, the precursor of heme synthesis, in the growth media and by the addition of glutamate or glutamine, amino acids required for the first step in heme biosynthesis . Fluorescence measurements show that the level of heme in a Fusr mutant is significantly lower than it is in the wild type . Heme is also an essential cofactor of cytochromes in the electron transport chain of respiration . We found that the rate of respiration is reduced significantly in Fusr mutants . Sequestration of divalent iron in the growth media decreases the sensitivity of Fusr mutants to oxidative stress . Taken together, these results suggest that Fusr mutants are hypersensitive to oxidative stress because their low levels of heme reduce both catalase activity and respiration capacity . The sensitivity of Fusr mutants to oxidative stress could be associated with loss of viability due to iron-mediated DNA damage in the presence of hydrogen peroxide . We argue that understanding the specific nature of antibiotic resistance fitness costs in different environments may be a generally useful approach in identifying physiological processes that could serve as novel targets for antimicrobial agents .
Genomic Organization and Molecular Characterization of SM1, a Temperate Bacteriophage of Streptococcus mitis. Ian R. Siboo, 2003.The direct binding of Streptococcus mitis to human platelets is mediated in part by two proteins (PblA and PblB) encoded by a lysogenic bacteriophage (SM1) . Since SM1 is the first prophage of S . mitis that has been identified and because of the possible role of these phage-encoded proteins in virulence, we sought to characterize SM1 in greater detail . Sequencing of the SM1 genome revealed that it consisted of 34,692 bp, with an overall G+C content of 39 mol% . Fifty-six genes encoding proteins of 40 or more amino acids were identified . The genes of SM1 appear to be arranged in a modular, life cycle-specific organization . BLAST analysis also revealed that the proteins of SM1 have homologies to proteins from a wide variety of lambdoid phages . Bioinformatic analyses, in addition to N-terminal sequencing of the proteins, led to the assignment of possible functions to a number of proteins, including the integrase, the terminase, and two major structural proteins . Examination of the phage structural components indicates that the phage head may assemble using stable multimers of the major capsid protein, in a process similar to that of phage r1t . These findings indicate that SM1 may be part of a discrete subfamily of the Siphoviridae that includes at least phages r1t of Lactococcus lactis and SF370.3 of Streptococcus pyogenes . Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture. Katsunori Mizoguchi, 2003.The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture . Following the addition of bacteriophage PP01, E . coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h-1 and by 3 orders of magnitude at a lower dilution rate (0.327 h-1) . However, the appearance of a series of phage-resistant E . coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture . The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared . Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E . coli O157:H7 to escape PP01 infection . One of the escape mutants of E . coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h-1 and persisted until the end of the experiment (  200 h) . Mu mutant cells could coexist with bacteriophage PP01 in batch culture . Concentrations of the Mu cells and bacteriophage PP01 increased together . The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture . Thus, coevolution of phage and E . coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture .
Antimicrobial Effects of Mustard Flour and Acetic Acid against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Serovar Typhimurium. Min-Suk Rhee, 2003.This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C . Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount) . Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (106 to 107 CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically . The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was S . enterica serovar Typhimurium (1 day) < E . coli O157:H7 (3 days) < L . monocytogenes (9 days) . The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C . There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%) . Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone . The reduction of E . coli O157:H7 and L . monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone . Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E . coli O157:H7 and L . monocytogenes . These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid .
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