title: Insertional inactivation of branched-chain alpha-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses.
abstract: Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain alpha-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12 degrees C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.
authors.affiliation: Department of Microbiology and Immunology, A T Still University of Health Sciences, Kirksville, MO 63501, USA. vsingh@atsu.edu
authors.forename: Vineet K
authors.initials: VK
authors.lastname: Singh
authors.forename: Dipti S
authors.initials: DS
authors.lastname: Hattangady
authors.forename: Efstathios S
authors.initials: ES
authors.lastname: Giotis
authors.forename: Atul K
authors.initials: AK
authors.lastname: Singh
authors.forename: Neal R
authors.initials: NR
authors.lastname: Chamberlain
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Stuart
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Wilkinson
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000235:genetics*; D000468:Alkalies / Q000633:toxicity; D000818:Animals; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics*; D002087:Butyrates / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D003080:Cold Temperature; D015169:Colony Count, Microbial; D003470:Culture Media / Q000737:chemistry; D004847:Epithelial Cells / Q000382:microbiology; D005227:Fatty Acids / Q000032:analysis*; D005260:Female; D017353:Gene Deletion; D006801:Humans; D008099:Liver / Q000382:microbiology; D008560:Membrane Fluidity; D051379:Mice; D008813:Mice, Inbred ICR; D050296:Microbial Viability; D016254:Mutagenesis, Insertional; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress; D013154:Spleen / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology*; D014050:Toluene / Q000633:toxicity; D014774:Virulence
chemical_list: D000468:Alkalies; D001426:Bacterial Proteins; D002087:Butyrates; D003470:Culture Media; D005227:Fatty Acids; D016877:Oxidants; D014050:Toluene; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); C019475:2-methylbutanoic acid