title: Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia.
abstract: Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugd(BCAL2946), ugd(BCAM0855) and ugd(BCAM2034), all of which were individually inactivated. Only inactivation of ugd(BCAL2946) resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugd(BCAL2946) or ugd(BCAM0855) but not ugd(BCAM2034) was expressed from plasmids. The growth of a conditional ugd(BCAL2946) mutant, created in the Deltaugd(BCAM0855) background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugd(BCAL2946) or ugd(BCAM0855) expressed in trans, but not by ugd(BCAM2034). Biochemical analysis of the purified, recombinant forms of Ugd(BCAL2946) and Ugd(BCAM0855) revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD(+). Purified Ugd(BCAM2034) showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugd(BCAL2946) was 5.4- and 135-fold greater than that of ugd(BCAM0855) and ugd(BCAM2034), respectively. Together, these data indicate that the combined activity of Ugd(BCAL2946) and Ugd(BCAM0855) is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugd(BCAL2946), is required for polymyxin B resistance.
authors.affiliation: Department of Microbiology and Immunology, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Slade A
authors.initials: SA
authors.lastname: Loutet
authors.affiliation: School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK.|Centre for Infectious Diseases, University of Edinburgh Medical School, Edinburgh EH16 4SB, UK.
authors.forename: S Josefin
authors.initials: SJ
authors.lastname: Bartholdson
authors.affiliation: Centre for Infectious Diseases, University of Edinburgh Medical School, Edinburgh EH16 4SB, UK.
authors.forename: John R W
authors.initials: JRW
authors.lastname: Govan
authors.affiliation: School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK.
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Campopiano
authors.affiliation: Department of Medicine, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada.|Department of Microbiology and Immunology, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016956:Burkholderia cepacia / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D024881:Drug Resistance, Bacterial; D015870:Gene Expression; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011112:Polymyxin B / Q000494:pharmacology*; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D014533:Uridine Diphosphate Glucose Dehydrogenase; D011112:Polymyxin B