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Isolation of Poly-3-Hydroxybutyrate Metabolism Genes from Complex Microbial Communities by Phenotypic Complementation of Bacterial Mutants

Chunxia Wang,^1, David J. Meek,² Priya Panchal,¹ Natalie Boruvka,¹ Frederick S. Archibald,^2,3 Brian T. Driscoll,² and Trevor C. Charles^1*

Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada,¹ Department of Natural Resource Sciences, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada,² Pulp and Paper Research Institute of Canada, 570 St-John's Blvd., Pointe-Claire, Quebec H9R 3J9, Canada³

Received 21 June 2005/ Accepted 10 October 2005

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate D-3-hydroxybutyrate due to absence of the enzyme D-3-hydroxybutyrate dehydrogenase activity. Clones that conferred D-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored D-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.

Present address: Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

This article has been cited by other articles:

• Wang, C., Sheng, X., Equi, R. C., Trainer, M. A., Charles, T. C., Sobral, B. W. S. (2007). Influence of the Poly-3-Hydroxybutyrate (PHB) Granule-Associated Proteins (PhaP1 and PhaP2) on PHB Accumulation and Symbiotic Nitrogen Fixation in Sinorhizobium meliloti Rm1021. J. Bacteriol. 189: 9050-9056 [Abstract] [Full Text]