一株耐镉细菌Exiguobacterium acetylicum TC13的分离鉴定及全基因组分析

Isolation, identification, and whole-genome analysis of a cadmium-tolerant bacterium Exiguobacterium acetylicum TC13

摘要:

【背景】 一些具有金属抗性的微生物被发现能降低土壤中金属的有效性，减少金属在植物中的转运和积累，同时具有促进植物生长的作用，表现出较低的成本效益和环境生态友好性，在农业、工业和环境领域具有广阔的应用前景。【目的】 分离筛选并获得耐Cd(II)优质菌株，并对其进行生物学鉴定，分析菌株形貌及耐镉的生物学特性，进一步从基因组层面剖析菌株的分子遗传特征，初步探究其潜在功能及耐重金属Cd(II)相关基因，并探究Cd(II)胁迫下菌株对小麦籽粒萌发特性的影响。【方法】 收集四川省德阳市磷矿、煤矿产区10余份土样，利用筛选培养基分离获得耐Cd(II)菌株，采用扫描电镜(scanning electron microscopy, SEM)、透射电镜(transmission electron microscopy, TEM)观察其形貌特征；测定培养液OD₆₀₀验证菌株耐Cd(II)特性。基于Illumina NovaSeq和Oxford Nanopore ONT测序平台，采用全基因组测序技术进行菌株鉴定及全基因组功能分析。利用生物信息学软件对原始数据组装、功能注释，挖掘耐Cd(II)适应机制相关基因。观察不同处理下的发芽生根率及芽根长势，研究Cd(II)胁迫下菌株对小麦籽粒的萌发特性。【结果】 分离获得一株优质耐Cd(II)菌株，经16S rrna基因测序和全基因测序鉴定为Exiguobacterium acetylicum，命名为TC13。菌株TC13全基因组序列组装得到1个染色体和1个环状质粒，大小分别为3 192 165 bp和167 401 bp；G+C含量分别为46.62%和44.63%；分别包含3 231个和154个DNA编码序列，染色体同时包含54个ncRNA。功能基因数据库比对结果发现菌种中多个基因涉及重金属转运、外排及应激响应等生物学过程，这可能与其耐Cd(II)特性有关。菌株对镉胁迫下小麦籽粒萌发率及长势特性具有显著提升作用。【结论】 本研究通过全基因组测序探究了E. acetylicum TC13的基因结构，丰富了同属种菌株遗传学信息，解析了Cd(II)耐受的相关基因，证实了菌株TC13对Cd(II)胁迫下小麦籽粒生长的促进作用，这表明菌株TC13在土壤修复及促生中具有潜在的应用前景。

Abstract:

[Background] Some metal-tolerant microorganisms have been found to reduce the availability of metals in soil, reduce the transportation and accumulation of metals in plants, and promote plant growth. With low cost-effectiveness and environmental friendliness, these microorganisms demonstrate promising prospects for applications in agriculture, industry, and the environment. [Objective] To isolate and identify high-quality strains tolerant to Cd(II), examine the morphological characteristics and Cd(II) tolerance of the strains, analyze the molecular genetic characteristics of the strains at the genomic level, mine their potential functions and Cd(II) tolerance-related genes, and observe the effects of the strains on wheat seed germination under cadmium stress. [Methods] Over ten soil samples were collected from phosphate and coal mining areas in Deyang City, Sichuan Province. A Cd(II)-tolerant strain was isolated in selective culture media, and its morphological characteristics were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The Cd(II) tolerance of the strain was examined based on the OD₆₀₀. Whole-genome sequencing was performed using Illumina NovaSeq and Oxford Nanopore ONT platforms for identification and genetic function analysis of the strain. Bioinformatics tools were used for genetic assembly, functional annotation, and mining of genes related to Cd(II) adaptation. The wheat seed germination rate, the bud and root growth of wheat seedlings under different treatments were determined to reveal the germination characteristics of wheat seeds under Cd(II) stress. [Results] One Cd(II)-tolerant strain was isolated, identified as Exiguobacterium acetylicum and named TC13. The whole-genome sequence assembly of TC13 resulted in one chromosome and one circular plasmid with the lengths of 3 192 165 bp and 167 401 bp and the G+C content of 46.62% and 44.63%, respectively. The chromosome and circular plasmid carried 3 231 and 154 DNA coding sequences, and the chromosome contained 54 ncRNAs. Functional gene database alignment results revealed that multiple genes were involved in biological processes such as heavy metal transport, efflux, and stress response in the strain, which may be associated with its cadmium tolerance. In addition, the strain increased the germination rate of wheat seeds and improved the growth of wheat seedlings under Cd(II) stress.[Conclusion] This study explored the genome structure of E. acetylicum TC13 by whole-genome sequencing, enriching the genetic information of strains of the same species, revealing the Cd(II) tolerance-related genes and confirmed the strain TC13 could promot the growth of wheat grain under Cd(II) stress. The strain TC13 has potential application prospects in soil remediation and plant growth promotion.