College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China.
College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China.
J Hazard Mater. 2023 Sep 5;457:131777. doi: 10.1016/j.jhazmat.2023.131777. Epub 2023 Jun 3.
Nicotine and nornicotine are all toxic alkaloids involved in the formation of carcinogenic tobacco-specific nitrosamines. Microbes play an important role in removing these toxic alkaloids and their derivatives from tobacco-polluted environments. By now, microbial degradation of nicotine has been well studied. However, limited information is available on the microbial catabolism of nornicotine. In the present study, a nornicotine-degrading consortium was enriched from a river sediment sample and characterized by metagenomic sequencing using a combination of Illumina and Nanopore technologies. The metagenomic sequencing analysis demonstrated that Achromobacter, Azospirillum, Mycolicibacterium, Terrimonas, and Mycobacterium were the dominant genera in the nornicotine-degrading consortium. A total of 7 morphologically distinct bacterial strains were isolated from the nornicotine-degrading consortium. These 7 bacterial strains were characterized by whole genome sequencing and examined for their ability to degrade nornicotine. Based on a combination of 16 S rRNA gene similarity comparisons, 16 S rRNA gene-based phylogenetic analysis, and ANI analysis, the accurate taxonomies of these 7 isolated strains were identified. These 7 strains were identified as Mycolicibacterium sp. strain SMGY-1XX, Shinella yambaruensis strain SMGY-2XX, Sphingobacterium soli strain SMGY-3XX, Runella sp. strain SMGY-4XX, Chitinophagaceae sp. strain SMGY-5XX, Terrimonas sp. strain SMGY-6XX, Achromobacter sp. strain SMGY-8XX. Among these 7 strains, Mycolicibacterium sp. strain SMGY-1XX, which has not been reported previously to have the ability to degrade nornicotine or nicotine, was found to be capable of degrading nornicotine, nicotine as well as myosmine. The degradation intermediates of nornicotine and myosmine by Mycolicibacterium sp. strain SMGY-1XX were determined and the nornicotine degradation pathway in strain SMGY-1XX was proposed. Three novel intermediates, myosmine, pseudooxy-nornicotine, and γ-aminobutyrate, were identified during the nornicotine degradation process. Further, the most likely candidate genes responsible for nornicotine degradation in Mycolicibacterium sp. strain SMGY-1XX were identified by integrating genomic analysis, transcriptomic analysis, and proteomic analysis. The findings in this study will help to expand our understanding on the microbial catabolism of nornicotine and nicotine and provide new insights into the nornicotine degradation mechanism by consortia and pure culture, laying a foundation for the application of strain SMGY-1XX for the removal, biotransformation, or detoxification of nornicotine.
尼古丁和去甲烟碱都是参与形成致癌烟草特异性亚硝胺的有毒生物碱。微生物在从受烟草污染的环境中去除这些有毒生物碱及其衍生物方面发挥着重要作用。到目前为止，尼古丁的微生物降解已经得到了很好的研究。然而，关于去甲烟碱的微生物代谢的信息有限。在本研究中，从河流沉积物样本中富集了一个去甲烟碱降解生物群落，并通过 Illumina 和 Nanopore 技术相结合的宏基因组测序进行了表征。宏基因组测序分析表明，无色杆菌、固氮螺菌、分枝杆菌、地杆菌和分枝杆菌是去甲烟碱降解生物群落中的主要属。从去甲烟碱降解生物群落中分离出 7 株形态不同的细菌。通过全基因组测序对这 7 株细菌进行了表征，并考察了它们降解去甲烟碱的能力。根据 16S rRNA 基因相似性比较、16S rRNA 基因系统发育分析和 ANI 分析的综合结果，确定了这 7 株分离株的准确分类。这 7 株菌被鉴定为分枝杆菌 SMGY-1XX、山原假单胞菌 SMGY-2XX、鞘氨醇单胞菌 SMGY-3XX、游动球菌 SMGY-4XX、噬几丁质菌科 SMGY-5XX、地杆菌 SMGY-6XX 和无色杆菌 SMGY-8XX。在这 7 株菌中，分枝杆菌 SMGY-1XX 以前没有报道过具有降解去甲烟碱或尼古丁的能力，但它被发现能够降解去甲烟碱、尼古丁和肌氨酸。确定了分枝杆菌 SMGY-1XX 降解去甲烟碱和肌氨酸的中间产物，并提出了 SMGY-1XX 菌株中去甲烟碱的降解途径。在去甲烟碱降解过程中鉴定出 3 种新的中间产物，即肌氨酸、假氧去甲烟碱和γ-氨基丁酸。此外，通过整合基因组分析、转录组分析和蛋白质组分析，确定了分枝杆菌 SMGY-1XX 中负责去甲烟碱降解的最可能候选基因。本研究的结果将有助于扩大我们对去甲烟碱和尼古丁微生物代谢的理解，并为生物群落和纯培养物中去甲烟碱降解机制提供新的见解，为 SMGY-1XX 菌株在去除、生物转化或去毒去甲烟碱方面的应用奠定基础。
