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长距离 PCR 揭示了用于处理农药污染废水的农场生物净化系统复杂微生物群落中 IncP-1 质粒的遗传负荷。

Long-Range PCR Reveals the Genetic Cargo of IncP-1 Plasmids in the Complex Microbial Community of an On-Farm Biopurification System Treating Pesticide-Contaminated Wastewater.

机构信息

Division of Soil and Water Management, KU Leuven, Heverlee, Belgium.

Department of Biology, Section of Microbiology, University of Copenhagen, Copenhagen, Denmark.

出版信息

Appl Environ Microbiol. 2022 Feb 8;88(3):e0164821. doi: 10.1128/AEM.01648-21. Epub 2021 Dec 8.

Abstract

Promiscuous plasmids like IncP-1 plasmids play an important role in the bacterial adaptation to pollution by acquiring and distributing xenobiotic catabolic genes. However, most information comes from isolates and the role of plasmids in governing community-wide bacterial adaptation to xenobiotics and other adaptive forces is not fully understood. Current information on the contribution of IncP-1 plasmids in community adaptation is limited because methods are lacking that directly isolate and identify the plasmid borne adaptive functions in whole-community DNA. In this study, we optimized long-range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA. The DNA between the IncP-1 backbone genes and , a main insertion site of adaptive trait determinants, is amplified and its content analyzed by high-throughput sequencing. The method was applied to DNA of an on-farm biopurification system (BPS), treating pesticide contaminated wastewater, to examine whether horizontal gene exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. The cargo recovered from BPS community DNA encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, genes with catabolic traits composed only a minor fraction of the cargo, indicating that the IncP-1 region between and is not a major contributor to catabolic adaptation of the BPS microbiome. Instead, it contains a functionally diverse set of genes which either may assist biodegradation functions, be remnants of random gene recruitment, or confer other crucial functions for proliferation in the BPS environment. This study presents a long-range PCR for direct and cultivation-independent access to the identity of the cargo of a major insertion hot spot of adaptive genes in IncP-1 plasmids and hence a new mobilome tool for understanding the role of IncP-1 plasmids in complex communities. The method was applied to DNA of an on-farm biopurification system (BPS) treating pesticide-contaminated wastewater, aiming at new insights on whether horizontal exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. Unexpectedly, catabolic functions represented a small fraction of the cargo genes while multiple other gene functions were recovered. These results show that the cargo of the target insertion hot spot in IncP-1 plasmids in a community, not necessarily relates to the main obvious selective trait imposed on that community. Instead, these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

摘要

易变质粒,如 IncP-1 质粒,在细菌适应污染方面发挥着重要作用,它们通过获取和分配异生物质分解代谢基因来实现。然而,大多数信息来自于分离株,而质粒在调控整个群落对异生物质和其他适应性因素的适应方面的作用尚未完全了解。目前关于 IncP-1 质粒在群落适应中的贡献的信息是有限的,因为缺乏直接从整个群落 DNA 中分离和鉴定质粒携带的适应性功能的方法。在这项研究中,我们优化了长距离 PCR 方法,以直接访问和鉴定环境 DNA 中 IncP-1 质粒携带的货物。扩增 IncP-1 质粒骨架基因 和 之间的 DNA,这是适应性特征决定因素的主要插入位点,并通过高通量测序分析其内容。该方法应用于处理农药污染废水的农场生物净化系统(BPS)的 DNA 中,以检验由 IncP-1 质粒进行水平基因转移的代谢功能是否是 BPS 群落适应的主要驱动力。从 BPS 群落 DNA 中回收的货物编码了代谢功能,但也编码了抗性功能和各种其他(未知)功能。出乎意料的是,具有代谢功能的基因仅构成货物的一小部分,这表明 和 之间的 IncP-1 区域并不是 BPS 微生物组代谢适应的主要贡献者。相反,它包含了一组功能多样的基因,这些基因可能有助于生物降解功能,是随机基因招募的残余物,或者赋予了 BPS 环境中增殖的其他关键功能。本研究提出了一种长距离 PCR 方法,用于直接和无需培养即可访问 IncP-1 质粒中主要插入热点的适应性基因货物的身份,因此是一种用于理解 IncP-1 质粒在复杂群落中作用的新移动组工具。该方法应用于处理农药污染废水的农场生物净化系统(BPS)的 DNA 中,旨在深入了解由 IncP-1 质粒进行的代谢功能的水平交换是否是 BPS 群落适应的主要驱动力。出乎意料的是,代谢功能仅占货物基因的一小部分,而其他多个基因功能也被回收。这些结果表明,群落中 IncP-1 质粒的目标插入热点的货物不一定与施加于该群落的主要明显选择特征有关。相反,这些功能可能有助于适应未知的选择压力,或者代表随机基因招募的残余物。

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