Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia

• Published in: Microbiology (Society for General Microbiology) Vol. 155; no. 6; pp. 2029 - 2039
• Main Authors: Loutet, Slade A, Bartholdson, S. Josefin, Govan, John R. W, Campopiano, Dominic J, Valvano, Miguel A
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01-06-2009; Society for General Microbiology
• Subjects: Amino Acid Sequence; Anti-Bacterial Agents - pharmacology; Bacteriology; Biological and medical sciences; Burkholderia; Burkholderia cepacia - drug effects; Burkholderia cepacia - enzymology; Burkholderia cepacia - genetics; Drug Resistance, Bacterial; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Complementation Test; Microbiology; Miscellaneous; Molecular Sequence Data; Mutagenesis, Insertional; Polymyxin B - pharmacology; Recombinant Fusion Proteins - biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Bacterial - analysis; RNA, Bacterial - genetics; Uridine Diphosphate Glucose Dehydrogenase - genetics; Uridine Diphosphate Glucose Dehydrogenase - metabolism; Resistance; Antibiotic; Enzyme; Cyclic peptides; Polypeptide; Bacteria; Glucose 1-dehydrogenase; Oxidoreductases; Burkholderia; Antibacterial agent; Dehydrogenase; Polymyxin B
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.027607-0

1 Department of Microbiology and Immunology, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada
2 Centre for Infectious Diseases, University of Edinburgh Medical School, Edinburgh EH16 4SB, UK
3 School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK
4 Department of Medicine, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada
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 ugd 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.
Correspondence Miguel A. Valvano mvalvano{at}uwo.ca
Abbreviations: APs, antimicrobial peptides; Ara4N, 4-amino-4- deoxy - L -arabinose; CF, cystic fibrosis; DDM, dodecylmaltoside; pmB, polymyxin B; Ugd, UDP-glucose dehydrogenase
Supplementary methods and tables of strains and primers are available with the online version of this paper.