Novel Plasmid-Borne Fimbriae-Associated Gene Cluster Participates in Biofilm Formation in Escherichia coli
Novel Plasmid-Borne Fimbriae-Associated Gene Cluster Participates in Biofilm Formation in Escherichia coli
复制标题
新型质粒携带菌毛相关基因簇参与大肠杆菌生物膜形成
DOI：
10.1089/mdr.2020.0512
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发表时间：
2021-06-01
影响因子：
2.6
通讯作者：
Liu, Ya-Hong
中科院分区：
文献类型：
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作者：
He, Yu-Zhang;Xu, Ying;Liu, Ya-Hong
这项研究报道了接合质粒的基因簇参与大肠杆菌生物膜的形成。我们使用新型 EZ-Tn5 转座子技术来生成转座子文库，并使用任意引物 PCR 来检测生物膜形成缺陷突变体中的插入位点。为了验证候选生物膜形成基因的功能，将这些基因克隆到质粒 pBluescript II SK (+) 中并转化到大肠杆菌 DH5 α 中。然后使用结晶紫染色和显微镜检查表型生物膜形成来评估转化体的生物膜产生。总共筛选了 3,000 个大肠杆菌 DH5 α-p253 转座子突变体，其中 28 个发现生物膜形成缺陷。进一步的表征表明，24/28 个突变在染色体插入中被检测到，而其余 4 个突变则证明生物膜形成的功能基因隐藏在质粒中。有趣的是，质粒测序显示这四个转座子突变都被插入到菌毛相关基因簇（fim-cluster）中。该 fim 簇是一个跨越 7,949 bp 序列的混合片段，具有末端反向重复序列和两个编码区。总之，我们对基于EZ-Tn5的转座子方法构建的文库进行了高效筛选，并鉴定了负责大肠杆菌生物膜产生的基因簇，特别是质粒中包含的基因。需要进一步的研究来了解这种新型质粒介导的生物膜形成基因在临床大肠杆菌分离株中的传播及其临床影响。
This study reported the involvement of a gene cluster from a conjugative plasmid in the biofilm formation of Escherichia coli. We used a novel EZ-Tn5 transposon technique to generate a transposon library and used arbitrarily primed PCR to detect the insertion sites in biofilm formation-deficient mutants. To validate the function of candidate biofilm formation genes, the genes were cloned into plasmid pBluescript II SK (+) and transformed into E. coil DH5 alpha. Biofilm production from the transformants was then assessed by phenotypic biofilm formation using Crystal Violet staining and microscopy. A total of 3,000 transposon mutants of E. coli DH5 alpha-p253 were screened, of which 28 were found to be deficient in biofilm formation. Further characterization revealed that 24/28 mutations were detected with their insertions in chromosome, while the remaining 4 mutations were evidenced that the functional genes for biofilm formation were harbored in the plasmid. Interestingly, the plasmid sequencing showed that these four transposon mutations were all inserted into a fimbriae-associated gene cluster (fim-cluster). This fim-cluster is a hybrid segment spanning a 7,949 bp sequence, with a terminal inverted repeat sequence and two coding regions. In summary, we performed a high-efficiency screening to a library constructed with the EZ-Tn5-based transposon approach and identified the gene clusters responsible for the biofilm production of E. coli, especially the genes harbored in the plasmid. Further studies are needed to understand the spread of this novel plasmid-mediated biofilm formation gene in clinical E. coli isolates and the clinical impacts.