Exploring the metabolic characteristics of indigenous PAH degraders is critical to understanding the PAH bioremediation mechanism in the natural environment. While stable-isotopic probing (SIP) is a viable method to identify functional microorganisms in complex environments, the metabolic characteristics of uncultured degraders are still elusive. Here, we investigated the naphthalene (NAP) biodegradation of petroleum polluted soils by combining SIP, amplicon sequencing and metagenome binning. Based on the SIP and amplicon sequencing results, an uncultured Gammaproteobacterium sp. was identified as the key NAP degrader. Additionally, the assembled genome of this uncultured degrader was successfully obtained from the ¹³C-DNA metagenomes by matching its 16S rRNA gene with the SIP identified OTU sequence. Meanwhile, a number of NAP degrading genes encoding naphthalene/PAH dioxygenases were identified in this genome, further confirming the direct involvement of this indigenous degrader in the NAP degradation. The degrader contained genes related to the metabolisms of several carbon sources, energy substances and vitamins, illuminating potential reasons for why microorganisms cannot be cultivated and finally realize their cultivation. Our findings provide novel information on the mechanisms of in situ PAH biodegradation and add to our current knowledge on the cultivation of non-culturable microorganisms by combining both SIP and metagenome binning.

中文翻译：

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揭示在石油污染土壤中负责原位 PAH 生物降解的未培养 Gammaproteobacterium 的代谢特征

探索原生多环芳烃降解物的代谢特征对于了解自然环境中多环芳烃的生物修复机制至关重要。虽然稳定同位素探测 (SIP) 是一种在复杂环境中识别功能微生物的可行方法，但未培养的降解物的代谢特征仍然难以捉摸。在这里，我们通过结合 SIP、扩增子测序和宏基因组分箱研究了石油污染土壤的萘 (NAP) 生物降解。根据 SIP 和扩增子测序结果，未培养的 Gammaproteobacterium sp。被确定为关键的 NAP 降解剂。此外，这种未培养降解物的组装基因组是从¹³C-DNA 宏基因组通过将其 16S rRNA 基因与 SIP 识别的 OTU 序列进行匹配。同时，在该基因组中鉴定出许多编码萘/多环芳烃双加氧酶的 NAP 降解基因，进一步证实了该原生降解剂直接参与了 NAP 降解。该降解物含有与几种碳源、能量物质和维生素代谢相关的基因，阐明了微生物无法培养并最终实现培养的潜在原因。我们的研究结果提供了关于原位PAH 生物降解机制的新信息，并增加了我们目前通过结合 SIP 和宏基因组分箱来培养不可培养微生物的知识。