Sau42I, a BcgI-like restriction–modification system encoded by the Staphylococcus aureus quadruple-converting phage π42

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Abstract
The serotype F phage π42 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 β-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 π42 resistance to lysis by exogenous phages was unknown. The authors report here that phage π42 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 π42 ORF S and ORF RM genes indicated that Sau42I has endonuclease activity and requires co-factors Mg2+ 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 π42 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.

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