Molecular genetic analysis of the evolution of cytotoxin-converting phages and the horizontal transfer of toxin genes.

细胞毒素转化噬菌体进化和毒素基因水平转移的分子遗传学分析。

基本信息

批准号：
09670277
负责人：
HAYASHI Tetsuya
金额：
$ 2.05万
依托单位：
Shinshu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

φCTX is a cytotoxin-converting phage isolated from Pseudomons aeruginosa. In this study, we determined the complete nucleotide sequence of φCTX phage genome. The precise genome size was 35,538 bp. Forty seven ORFs were identified on the φCTX genome, including two previously identified genes, ctx and int. Among them, fifteen gene products were identified in the phage particle. The most striking feature of the φCTX genome was an extensive homology with the coliphage P2 and P2-related phages; more than half of the ORFs had marked homology to P2 genes. The gene arrangement on the genome was also highly conserved for the two phages, though the G+C content and codon usage of most φCTX genes were similar to those of the host P. aeruginosa chromosome. In addition, φCTX was found to share several common features with P2, including the morphology, non-inducibility, use of LPS core oligosaccharide as receptor and CaィイD22+ィエD2-dependent receptor binding. These findings indicate that φCTX is a P2-l … More ike phage well adapted to P. aeruginosa, and provide a clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of φCTX, P2 and other P2-relatives revealed the presence of several hot spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.Pseudomonas aeruginosa produces three types of bacteriocins; R-type, F-type, and S-type pyocins. The S-type pyocin is a simple colicin-like protein, whereas the R-type resembles a contractile tail structure of bacteriophage, and the F-type a non-contractile tail of bacteriophage. Since φCTX is a member of R pyocin-related phages which are genetically and serologically related to R pyocin, above-mentioned results suggested the relatedness of R pyocin to the P2 phage family. Thus, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analyzed in order to elucidate the relationship between the pyocins and bacteriophages. Further, in order to specify the R2 pyocin locus, we determined the nucleotide sequence of the corresponding region of the strain PML14 which produced only F2 pyocin. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with the P2 phage family and the F-type from the λ phage family. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that the both pyocins are not simple defective phages but are phage tails that have been evolutionarily specialized as bacteriocins. Less

φCTX 是从铜绿假单胞菌中分离出来的细胞毒素转化噬菌体。在本研究中，我们确定了 φCTX 噬菌体基因组的完整核苷酸序列，在 φCTX 基因组上鉴定了 47 个 ORF，其中包括两个先前鉴定的基因。其中，噬菌体颗粒中鉴定出十五种基因产物。 φCTX基因组与大肠杆菌噬菌体P2和P2相关噬菌体具有同源性；超过一半的ORF与P2基因具有明显的同源性，尽管G+C含量较高，但两种噬菌体在广泛基因组上的基因排列也高度保守。大多数 φCTX 基因的密码子使用与宿主铜绿假单胞菌染色体的密码子使用相似。此外，还发现 φCTX 与 P2 具有一些共同特征，包括形态、非诱导性、使用 LPS 核心寡糖作为受体以及 CaD22+D2 依赖性受体结合这些发现表明 φCTX 是一种非常适合铜绿假单胞菌的 P2-1 噬菌体，并提供了属间传播的明确证据。此外，对 φCTX、P2 和其他 P2 相关基因组结构的比较分析揭示了几个热点的存在。插入的外源DNA，包括细胞毒素基因，似乎与噬菌体感染水平转移的各种基因的获得密切相关。铜绿假单胞菌产生三种类型的细菌素：R型、F型和S型。 S型脓毒素是一种简单的大肠菌素样蛋白质，而R型类似于噬菌体的收缩尾部结构，而F型则类似。由于 φCTX 是 R pyocin 相关噬菌体的成员，其在遗传和血清学上与 R pyocin 相关，因此上述结果表明 R pyocin 与 P2 噬菌体家族的相关性。 R2 化脓菌素基因，以及位于铜绿假单胞菌染色体上 R2 基因簇下游的 F2 化脓菌素基因为了阐明脓毒菌素和噬菌体之间的关系，对PAO1进行了分析。此外，为了确定R2脓毒菌素基因座，我们确定了仅产生F2脓毒菌素的菌株PML14的相应区域的核苷酸序列。 R 型脓毒菌素与 P2 噬菌体家族具有共同祖先，而 F 型脓菌素则来自 λ 噬菌体家族。这一观点得到了支持。与噬菌体类似的裂解基因盒的鉴定然而，R2 和 F2 脓毒素基因簇的基因组织表明，这两种脓毒素不是简单的缺陷噬菌体，而是在进化上专门化为细菌素的噬菌体尾部。