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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

Department of Cell and Molecular Biology,¹ Department of Surface Biotechnology, Biomedical Center, Uppsala University, Uppsala, Sweden²

Received 16 December 2003/ Returned for modification 27 January 2004/ Accepted 23 May 2004

Mutations in the translation elongation factor G (EF-G) make Salmonella enterica serovar Typhimurium resistant to the antibiotic fusidic acid. Fus^r 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. Fus^r mutants have a reduced rate of transcription of hemA, a gene whose product catalyzes the first committed step in heme biosynthesis. Hypersensitivity of Fus^r 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 Fus^r 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 Fus^r mutants. Sequestration of divalent iron in the growth media decreases the sensitivity of Fus^r mutants to oxidative stress. Taken together, these results suggest that Fus^r mutants are hypersensitive to oxidative stress because their low levels of heme reduce both catalase activity and respiration capacity. The sensitivity of Fus^r 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.

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