Dempsey Rita M, Carroll David, Kong Huimin, Higgins Lauren, Keane Conor T, Coleman David C
Department of Microbiology, University of Dublin, Trinity College, Dublin, Ireland.
New England Biolabs, Beverly, MA, USA.
Microbiology (Reading). 2005 Apr;151(Pt 4):1301-1311. doi: 10.1099/mic.0.27646-0.
The serotype F phage Phi42 of Staphylococcus aureus is a triple-converting bacteriophage that encodes the staphylokinase gene (sak) and the enterotoxin A gene (entA). Lysogeny results in loss of expression of the chromosomal beta-haemolysin gene (hlb) (negative conversion), the expression of staphylokinase and enterotoxin A (positive conversion), and the acquisition of resistance to lysis by all 23 phages of the International Basic Set (IBS) of S. aureus typing phages. Until this study, the basis of Phi42 resistance to lysis by exogenous phages was unknown. The authors report here that phage Phi42 encodes a restriction-modification (R-M) system, termed Sau42I, adjacent to and in the same orientation to the phage integrase gene int. The genes encoding Sau42I were cloned and sequenced, and found to consist of two overlapping reading frames, ORF S (specificity) and ORF RM (restriction-modification), in the same orientation. The ORFs share a high degree of DNA and amino acid sequence homology with the previously characterized BcgI R-M system of Bacillus coagulans. Expression of the cloned Sau42I ORF S and ORF RM in S. aureus 80CR3 transformants from a plasmid vector conferred resistance to lysis by all 23 IBS phages. Similarly, transformants of S. aureus RN4220 harbouring recombinant plasmids containing both ORFs were resistant to lysis by the IBS typing phages. However, transformants harbouring plasmids encoding either ORF S or ORF RM were susceptible to lysis by the IBS phages, and they had the same phage-susceptibility pattern as the respective parental isolates. In vitro analysis of crude and partially purified extracts of S. aureus transformants harbouring both the Phi42 ORF S and ORF RM genes indicated that Sau42I has endonuclease activity and requires co-factors Mg(2+) and S-adenosylmethionine in order to function, and activity is optimized at pH 8, although the precise recognition sequence has yet to be determined. The findings of this study confirm that Phi42 is a quadruple-converting phage, believed to be the first described for S. aureus, and show that it encodes a novel R-M system termed Sau42I.
金黄色葡萄球菌的血清型F噬菌体Phi42是一种三重转化噬菌体，它编码葡萄球菌激酶基因（sak）和肠毒素A基因（entA）。溶原性导致染色体β-溶血素基因（hlb）表达缺失（负向转化）、葡萄球菌激酶和肠毒素A表达（正向转化），并获得对金黄色葡萄球菌分型噬菌体国际基本集（IBS）的所有23种噬菌体裂解的抗性。在本研究之前，Phi42对外源噬菌体裂解的抗性基础尚不清楚。作者在此报告，噬菌体Phi42编码一种限制修饰（R-M）系统，称为Sau42I，与噬菌体整合酶基因int相邻且方向相同。编码Sau42I的基因被克隆并测序，发现由两个重叠的阅读框组成，即ORF S（特异性）和ORF RM（限制修饰），方向相同。这些开放阅读框与先前鉴定的凝结芽孢杆菌的BcgI R-M系统具有高度的DNA和氨基酸序列同源性。从质粒载体在金黄色葡萄球菌80CR3转化体中克隆的Sau42I ORF S和ORF RM的表达赋予了对所有23种IBS噬菌体裂解的抗性。同样，携带包含两个开放阅读框的重组质粒的金黄色葡萄球菌RN4220转化体对IBS分型噬菌体的裂解具有抗性。然而，携带编码ORF S或ORF RM的质粒的转化体对IBS噬菌体的裂解敏感，并且它们具有与各自亲本分离株相同的噬菌体敏感性模式。对同时携带Phi42 ORF S和ORF RM基因的金黄色葡萄球菌转化体的粗提物和部分纯化提取物的体外分析表明，Sau42I具有内切核酸酶活性，并且需要辅因子Mg(2+)和S-腺苷甲硫氨酸才能发挥作用，尽管精确的识别序列尚未确定，但在pH 8时活性最佳。本研究结果证实Phi42是一种四重转化噬菌体，据信是首次描述的针对金黄色葡萄球菌的噬菌体，并表明它编码一种称为Sau42I的新型R-M系统。
