Characterization of the stability and dynamics of Tn6330 in an Escherichia coli strain by nanopore long reads
Characterization of the stability and dynamics of Tn6330 in an Escherichia coli strain by nanopore long reads
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通过纳米孔长读长表征大肠杆菌菌株中 Tn6330 的稳定性和动态
DOI：
10.1093/jac/dkz117
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发表时间：
2019-07-01
影响因子：
5.2
通讯作者：
Chen, Sheng
中科院分区：
文献类型：
--
作者：
Li, Ruichao;Chen, Kaichao;Chen, Sheng
目的研究Tn6330在单个细菌种群中的稳定性和稳定性。方法通过聚合酶链式反应、接合反应、S1-PFGE、稳定性实验、单分子长读测序和生物信息学等方法对携带Tn6330基因的细菌进行鉴定。结果在一株临床分离的大肠杆菌中同时检测到Tn6330。该质粒含有IncFIB复制子和类噬菌体复制子，以及两个整合子，整合子含有多种可移动元件和抗性基因，包括mcr-1、flor、bla(Tm-1b)和Strab。Tn6330转座子和质粒型Tn6330转座子均可重组为环状中间体，在mcr-1基因的传递中发挥作用。Tn6330在有或无粘菌素选择压力的情况下，经过30代12h的培养后，在质粒和染色体中都非常稳定。在细菌种群的真实单DNA分子中，Tn6330的衰变结构虽然出现的频率很低，但仍是首次被检测到，其中Tn6330被降解为单一的ISApl1元件。结论长读测序技术是研究细菌遗传元件进化和稳定性的一种很好的工具。细菌质粒和染色体中编码mcr-1的元件的超稳定性使其不太可能通过减少粘菌素的使用而迅速根除，导致Tn6330频繁死亡的因素值得进一步研究。
Background The mcr-1 gene has been widely reported in both bacterial chromosomes and plasmids, while its stability in these genetic materials is not well understood.Objectives Our aim was to characterize the stability and dynamics of Tn6330 elements in both a plasmid and the chromosome in a single bacterial population.Methods Plasmid-borne and chromosomal Tn6330 were characterized by PCR, conjugation, S1-PFGE, stability assay, single-molecule long-read sequencing and bioinformatics analysis.Results Tn6330 was simultaneously detected in both a plasmid and the chromosome of a clinical Escherichia coli strain. The plasmid was found to comprise the IncFIB replicon and a phage-like replicon, as well as two integrons that harboured various mobile elements and resistance genes including mcr-1, floR, bla(TEM-1b) and strAB. Both plasmid-borne and chromosomal Tn6330 transposons could be re-organized into a circular intermediate that played a role in transmission of the mcr-1 gene. Tn6330 was found to be very stable in both the plasmid and chromosome after 30 passages of 12h with or without colistin selective pressure. The decayed structure of Tn6330 in the genuine single DNA molecules of bacterial populations, although occurring at a very low frequency, could be detected for the first time, in which Tn6330 was degraded into a single ISApl1 element.Conclusions Long-read sequencing technology is a good tool to study the evolution and stability of genetic elements in bacteria. The ultrastability of an mcr-1-encoding element in a bacterial plasmid and chromosome renders it unlikely to be eradicated quickly by the reduced use of colistin, and factors leading to the frequent demise of Tn6330 warrant further studies.