志贺毒素2型噬菌体FMin27的stx2基因突变株构建及其感染特性

Construction of a stx2 deletion mutant of Shiga toxin 2 phage FMin27and its infectious properties

摘要:

【目的】通过构建一株stx2基因突变的志贺毒素2型噬菌体，来观察它对不同血清型大肠杆菌的感染特性。【方法】利用l噬菌体的Red重组系统，将氯霉素抗性基因（cat）插入到大肠杆菌O157:H7 Min27株的stx2基因中，获得O157:H7 Min27的突变菌株Min27(Δstx∷cat)。用丝裂霉素对该突变菌株进行诱导，结合抗性标记和PCR方法筛选出一株突变的志贺毒素2型噬菌体, 命名为FMin27(Δstx∷cat)。采用双层琼脂平板法和氯霉素抗性筛选的方法，观察FMin27(Δstx∷cat) 感染21株不同血清型大肠杆菌后的裂解和溶原转换情况。【结果】2株血清型分别为Ｏ60和Ｏ138的大肠杆菌可以被FMin27(Δstx∷cat)溶原感染，表现出对氯霉素的抗性，但没有形成噬菌斑，而大肠杆菌MG1655株既可以被溶原又可以被裂解。溶原的菌株经丝裂霉素诱导后，能释放出感染性的FMin27(Δstx∷cat)颗粒，并对大肠杆菌MC1061进行裂解形成噬菌斑。【结论】FMin27(Δstx∷cat)可以感染和溶原特定的大肠杆菌，且溶原菌株能释放出原感染性的噬菌体，显示出FMin27噬菌体具有携带外源基因在若干不同血清型的大肠杆菌间水平转移的能力，为进一步研究Stx噬菌体的感染机理和志贺毒素表达调控奠定了基础。

关键词:大肠杆菌O157:H7;Stx2噬菌体;FMin27(Δstx∷cat);感染特性

Abstract:

[Objective] To construct an stx2 gene mutant phage FMin27(Δstx∷cat) and to observe its infectiousness of various serotypes Escherichia coli strains. [Methods] With the help of Red recombinant system, the stx2 gene of the E. coli O157:H7 Min27 strain isolated from intestinal feces of piglet with diarrhea at a swine farm of Shanghai, was replaced by the chloramphenicol acetyltrasferase (cat) gene from plasmid pLacI. Phage FMin27(Δstx∷cat) was isolated after in-duction of E. coli Min27(Δstx∷cat) strain with mitomycin C. Twenty-one E. coli strains with various serotypes were infected with FMin27(Δstx∷cat), and plaque formation and lysogenic conversion of them were investigated. [Results] Of the 21 E. coli isolates, 2 with the serotypes of O60 and O138 integrated the FMin27(Δstx∷cat) in their chromosomes and expressed resistance to chloramphenicol. With the exception of one laboratory E. coli strain MG1655, none of the tested E. coli strains supported the formation of plaques and lysogenization when used as indicators for FMin27(Δstx∷cat). Fol-lowing induction with mitomycin C, these lysogenic strains released infectious particles of FMin27(Δstx∷cat) that formed plaques on a lawn of E.coli laboratory strain MC1061. [Conclusion] These results demonstrated that FMin27(Δstx∷cat) was able to infect and lysogenize particular E. coli strains and that the lysogens could produce infec-tious phage progeny. It could be inferred that Stx bacteriophages were able to spread exogenous genes among E. coli strains. The work provided a basis for further study on mechanisms of Stx phages infection and control of Stx expression.

Key words:Escherichia coli O157:H7; Stx2 bacteriophage; FMin27(Δstx∷cat); infectious property