Open AccessIsolation of Poly-3-Hydroxybutyrate Metabolism Genes from Complex Microbial Communities by Phenotypic Complementation of Bacterial Mutants
Author(s) -
Chunxia Wang,
David J. J. Meek,
Priya Panchal,
Natalie Boruvka,
Frederick Archibald,
Brian T. Driscoll,
Trevor C. Charles
Publication year - 2006
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.72.1.384-391.2006
Subject(s) - complementation , sinorhizobium meliloti , biology , cosmid , transposon mutagenesis , mutant , gene , genetics , escherichia coli , plasmid , protein fragment complementation assay , biochemistry , transposable element
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 aSinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediated -3-hydroxybutyrate due to absence of the enzymed -3-hydroxybutyrate dehydrogenase activity. Clones that conferredd -3-hydroxybutyrate utilization onEscherichia coli were also isolated. Although many of theS. meliloti bdhA mutant complementing clones restoredd -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 theE. coli selection. Further analysis was carried out on clones isolated in theS. 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.