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Published online ahead of print on 21 April 2009 as doi:10.1099/mic.0.027607-0
Microbiology 2009;155:2029.

Microbiology (2009), DOI 10.1099/mic.0.027607-0
© 2009 Society for General Microbiology
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Microbiology 0 (2009), mic.0.027607; DOI  10.1099/mic.0.027607-0
© 2009 Society for General Microbiology

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

S. A Loutet1, S. J. Bartholdson2, J. R. W. Govan2, D. J. Campopiano2 and M. A. Valvano1,3

1 University of Western Ontario;
2 University of Edinburgh

ABSTRACT

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalyzed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugdBCAL2946, ugdBCAM0855 and ugdBCAM2034, all of which were individually inactivated. Only inactivation of ugdBCAL2946 resulted in increased sensitivity to polymyxin B. The sensitivity to polymyxin B could be overcome when either ugdBCAL2946 or ugdBCAM0855 but not ugdBCAM2034 was expressed from plasmids. The growth of a conditional ugdBCAL2946 mutant, created in the {Delta}ugdBCAM0855 background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugdBCAL2946 or ugdBCAM0855 expressed in trans, but not ugdBCAM2034. Biochemical analysis of the purified, recombinant forms of UgdBCAL2946 and UgdBCAM0855 revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD+. Purified UgdBCAM2034 showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugdBCAL2946 was 5·4- and 135-fold greater than ugdBCAM0855 and ugdBCAM2034, respectively. Together, these data indicate that the combined activity of UgdBCAL2946 and UgdBCAM0855 is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugdBCAL2946, is required for polymyxin B resistance.

3 E-mail: miguel.valvano{at}schulich.uwo.ca




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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]


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