HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplementary material All Versions of this Article: mic.0.027607-0v1 155/6/2029    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Google Scholar Articles by Loutet, S. A. Articles by Valvano, M. A. PubMed PubMed Citation Articles by Loutet, S. A. Articles by Valvano, M. A. Agricola Articles by Loutet, S. A. Articles by Valvano, M. A.

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

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

Supplementary methods and tables of strains and primers are available with the online version of this paper.

This article has been cited by other articles:

				X. Ortega, A. Silipo, M. S. Saldias, C. C. Bates, A. Molinaro, and M. A. Valvano Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide: TRUNCATION OF THE CORE OLIGOSACCHARIDE LEADS TO INCREASED BINDING AND SENSITIVITY TO POLYMYXIN B J. Biol. Chem., August 7, 2009; 284(32): 21738 - 21751. [Abstract] [Full Text] [PDF]