Biobeds, designed to minimize pesticide point source contamination, rely mainly on biodegradation processes. We studied the interactions of a biobed microbial community with the herbicide isoproturon (IPU) to explore the role of the pdmA gene, encoding the large subunit of an N-demethylase responsible for the initial demethylation of IPU, via quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR) and the effect of IPU on the diversity of the total bacterial community and its active fraction through amplicon sequencing of DNA and RNA, respectively. We further investigated the localization and dispersal mechanisms of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. strain SH (previously isolated from the soil used in the biobed) compared with the abundance of the pdmA gene and metagenomic fosmid library screening. pdmA abundance and expression increased concomitantly with IPU mineralization, verifying its major role in IPU transformation in the biobed system. DNA- and RNA-based 16S rRNA gene sequencing analysis showed no effects on bacterial diversity. The pdmAB-harboring plasmid pSH showed a consistently lower abundance than pdmA, suggesting the localization of pdmAB in replicons other than pSH. Metagenomic analysis identified four pdmAB-carrying fosmids. In three of these fosmids, the pdmAB genes were organized in a well-conserved operon carried by sphingomonad plasmids with low synteny with pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potentially crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon.

中文翻译：

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深入了解生物床系统中异丙隆降解基因 (pdmAB) 的功能和水平转移。

生物床旨在最大限度地减少农药点源污染，主要依靠生物降解过程。我们研究了生物床微生物群落与除草剂异丙隆 (IPU) 的相互作用，以探索pdmA基因的作用，该基因编码负责 IPU 初始去甲基化的N-去甲基化酶大亚基，通过定量 PCR (qPCR) 和反向分析转录-PCR (RT-qPCR) 以及分别通过 DNA 和 RNA 的扩增子测序观察 IPU 对总细菌群落及其活性部分多样性的影响。我们通过测量IPU 降解鞘氨醇单胞菌的质粒 pSH（含有pdmAB ）的丰度，进一步研究了pdmAB在生物床填充材料中的定位和分散机制。菌株SH（之前从生物床使用的土壤中分离出来）与pdmA基因的丰度进行比较并进行宏基因组fosmid文库筛选。pdmA丰度和表达量随着 IPU 矿化而增加，证实了其在生物床系统中 IPU 转化中的主要作用。基于 DNA 和 RNA 的 16S rRNA 基因测序分析显示对细菌多样性没有影响。携带pdmAB的质粒 pSH 显示出始终低于pdmA 的丰度，表明pdmAB定位于 pSH 以外的复制子中。宏基因组分析鉴定出四种携带pdmAB的 fosmids。在其中三个 fosmid 中，pdmAB基因组织在由与 pSH 低同线性的鞘单胞菌质粒携带的高度保守的操纵子中，而第四个 fosmid 包含位于红酸杆菌菌株基因组片段中的不完整pdmAB盒。进一步的分析表明 IS 6和 IS 256在pdmAB操纵子的转座和激活中具有潜在的关键作用。