4株霍乱弧菌非产毒株溶源性噬菌体pre-CTXΦ 的基因组结构分析

霍乱弧菌溶源性噬菌体CTXΦ携带霍乱毒素基因ctxAB，通过其结构基因gⅢ编码产生的PⅢ蛋白识别霍乱弧菌毒素共调菌毛(toxin co-regulated pilus, TCP)的主要结构亚单位TcpA，从而感染具有TCP的霍乱弧菌，使之成为产毒菌株。CTXΦ还有不携带ctxAB的前体pre-CTXΦ，根据CTXΦ基因组中调控基因rstR序列型不同，可分成不同的型别。在不同霍乱弧菌菌株的基因组中，已发现CTXΦ/pre-CTXΦ基因组及其亚型的多种组合排列方式。研究该噬菌体家族的基因组多样性，能够分析其进化及在霍乱弧菌产毒株形成中的作用。本研究发现了4株O1和O139群霍乱弧菌非产毒株具有pre-CTXΦ基因组及多样的rstR序列型，进一步对pre-CTXΦ在4株菌株中的基因组特征进行了分析。利用第3代基因测序法(短读长测序技术和单分子长读长测序技术)，获得了4株菌株的基因组序列。利用长读长测序和拼接分析，精确地获得了具有长片段重复序列结构的pre-CTXΦ基因组排列，明确了4株测序菌株中多样的pre-CTXΦ基因组排列。在非产毒株基因组菌株VC3193中发现了携带古典型pre-CTXΦ；还在菌株VC702的pre-CTXΦ基因组中首次发现了肺炎克雷白菌的转座子结构(Gen Bank序列号：SRIL00000000)。在这4株测序菌株中，受体TcpA以及pre-CTXΦ的PⅢ蛋白也具有明显差异的序列，有 TcpA和PⅢ新序列型，这提示了CTXΦ家族感染宿主菌的受体-配体相互识别的复杂对应关系。本研究丰富了对CTXΦ/pre-CTXΦ家族基因组及其整合排列的多样化认识，也为分析该溶源性噬菌体在不同遗传特征霍乱弧菌菌株间的水平转移和促使新产毒克隆形成方面提供了更多的证据。

Genomic structure and rearrangement of the lysogenic bacteriophage pre-CTXΦ in four non-toxigenic strains of Vibrio cholerae serogroups O1 and O139

The lysogenic phage CTXΦ of Vibrio cholerae can carry and transfer the cholera toxin gene ctxAB from toxigenic strains to non-toxigenic strains. CTXΦ also has a precursor, pre-CTXΦ，which does not carry ctxAB gene. In this study, we sequenced the genomes of four nontoxigenic serogroups O1 and O139 strains carrying pre-CTXΦ with the next generation sequencing. Both short-reading and long-reading sequencing were conducted to facilitate the assembly of long repeats. pre-CTXΦ prophage sequences were extracted from the genomes of these strains. The genes of CTXΦ receptor TcpA and the receptor-binding protein pIII in CTXΦ genome was subjected to sequence alignment, phylogenic tree construction and possible domains for protein-protein interactions. The tandem repeats of the different pre-CTXΦ alleles and their novel arrangement in the genomes were identified, and the precursor genome of classical type CTXΦ was also found. The arrangements of the pre-CTXΦ alleles in these strains were significantly different from the pandemic O1 El Tor strains. New sequences of the TcpA and the pIII were found in some strains. TcpA-pIII sequence correspondence analysis indicated the complicated interactions of the different sequence types of both proteins during the infection of pre-CTXΦ alleles. In addition, we have discovered a new type of pre-CTXΦ on small chromosome of VC702. The rstR-4 gene in VC702 has been disrupted by an insertion of a transposon which was firstly found in Klebsiella pneumoniae. Our study revealed new sequence types and arrangement of the pre-CTXΦ alleles and new evidence for better understanding the horizontal transfer of CTXΦ/pre-CTXΦ among the strains.