Isolation of poly-3-hydroxybutyrate metabolism genes from complex microbial communities by phenotypic complementation of bacterial mutants
Isolation of poly-3-hydroxybutyrate metabolism genes from complex microbial communities by phenotypic complementation of bacterial mutants
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DOI：
10.1128/aem.72.1.384-391.2006
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发表时间：
2006-01-01
影响因子：
4.4
通讯作者：
Charles, TC
中科院分区：
文献类型：
--
作者：
Wang, CX;Meek, DJ;Charles, TC
本研究的目的是在群落水平上启动细菌聚 3-羟基丁酸酯 (PHB) 代谢的遗传学研究。我们从广泛宿主范围的 IncP cosmid pRK7813 中的活性污泥和土壤微生物群落构建了宏基因组文库。通过苜蓿中华根瘤菌 bdhA 突变体的功能异源互补，从这些文库中分离出几个独特的克隆，由于缺乏 D-3-羟基丁酸脱氢酶活性，该突变体无法在 PHB 循环中间体 D-3-羟基丁酸上生长。还分离了赋予大肠杆菌 D-3-羟基丁酸利用能力的克隆。尽管许多苜蓿中华根瘤菌bdhA突变体互补克隆恢复了突变体宿主的D-3-羟基丁酸脱氢酶活性，但对于一些克隆来说，这种活性是检测不到的。大肠杆菌选择中分离出的几乎所有克隆也是如此。对苜蓿苜蓿互补中分离的克隆进行了进一步分析。通过转座子诱变来定位互补基因，随后对其中三个基因进行 DNA 序列分析，揭示了编码序列存在很大差异，但属于已知短链脱氢酶/还原酶编码基因的多样性范围内。在一些情况下，推导的BdhA蛋白对之间的氨基酸序列同一性<35%，在该水平下通过基于核酸杂交的方法进行检测可能不会成功。
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.