Restriction-deficient Mutants of Staphylococcus aureus

Stobberingh, Ellen E.; Winkler, Kurt

doi:10.1099/00221287-99-2-359

Cited by 42 publications

(31 citation statements)

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“…S. aureus 879R4RF is reported to be R-M negative (29), although the reason for this phenotype is unknown and we could find no mutation in its sau1hsdR gene. It belongs to lineage CC51, as determined by a microarray analysis (18; data not shown), and has a typical sau1hsdS variant profile for this lineage.…”

Section: Methodsmentioning

confidence: 67%

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Sau1: a Novel Lineage-Specific Type I Restriction-Modification System That Blocks Horizontal Gene Transfer into Staphylococcus aureus and between S. aureus Isolates of Different Lineages

2006

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The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.Staphylococcus aureus is a commensal of the human nose and a common cause of both hospital-and community-acquired infection. It is becoming increasingly virulent and resistant to antibiotics due to the horizontal transfer of mobile genetic elements (MGE) encoding virulence and resistance genes (17). S. aureus can be classified into approximately 10 dominant lineages, each with unique surface protein profiles and each capable of causing disease (18). In addition, approximately 15% of any S. aureus genome consists of MGE, such as bacteriophage, transposons, plasmids, and pathogenicity islands (17, 18). These elements may be transferred horizontally between isolates at high frequency, both in the laboratory and in vivo (19,21). However, this is not reflected in the epidemiological spread of resistance and virulence genes among naturally occurring S. aureus strains. For example, the transfer of vanA to S. aureus from vancomycin-resistant enterococci has been exceedingly slow (only four cases) given the high incidence of patients harboring both vancomycin-resistant enterococci and S. aureus and treated with vancomycin (30). Another example is that over more than 40 years, only some S. aureus lineages have acquired SCCmec elements (12, 25) despite the widespread use of methicillin-type antibiotics. Another example is that virulence genes in S. aureus carried on MGE include the Panto...

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Section: Methodsmentioning

confidence: 67%

“…Mutagenised S. aureus isolates with enhanced susceptibility to bacteriophage lysis were identified many years ago, and it was proposed that R-M systems were responsible for this phenotype (11,29). The first S. aureus R-M system to be characterized in detail was Sau3A, a type II system that specifically digests DNA at GATC sites (28).…”

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confidence: 99%

Sau1: a Novel Lineage-Specific Type I Restriction-Modification System That Blocks Horizontal Gene Transfer into Staphylococcus aureus and between S. aureus Isolates of Different Lineages

2006

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“…Smith and Condemine (36) stated that "technology needs to be developed to simplify map construction by using maps that already exist for related organisms." This project outlines one such technology that was applied to Staphylococcus aureus.The species is divided into five phage groups on the basis of typing patterns produced on infection with staphylococcal phages (23,32,33,42,45,48). A detailed genetic and physical map has been established in the phage group III S. aureus NCTC 8325 by using methods of classical genetics and pulsedfield gel electrophoresis (PFGE) analysis (27,28,31).…”

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confidence: 99%

Construction of a chromosome map for the phage group II Staphylococcus aureus Ps55

Bannantine

Pattee

1996

J Bacteriol

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The genome size and a partial physical and genetic map have been defined for the phage group II Staphylococcus aureus Ps55. The genome size was estimated to be 2,771 kb by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI, CspI, and SgrAI. The Ps55 chromosome map was constructed by transduction of auxotrophic and cryptic transposon insertions, with known genetic and physical locations in S. aureus NCTC 8325, into the Ps55 background. PFGE and DNA hybridization analysis were used to detect the location of the transposon in Ps55. Ps55 restriction fragments were then ordered on the basis of genetic conservation between the two strains. Cloned DNA probes containing the lactose operon (lac) and genes encoding staphylococcal protein A (spa), gamma hemolysin (hlg), and coagulase (coa) were also located on the map by PFGE and hybridization analysis. This methodology enabled a direct comparison of chromosomal organization between NCTC 8325 and Ps55 strains. The chromosome size, gene order, and some of the restriction sites are conserved between the two phage group strains.A physical map of a chromosome consists of an ordered set of restriction fragments that places genomic sequence in the order it exists inside the cell. The resolution of chromosome structure can vary from a macrorestriction map consisting of only a few large, restriction fragments (10, 14, 17, 37) to thousands of small clones of ordered chromosomal DNA (4, 13) to a complete nucleotide sequence (11). The advantages of a physical map over a genetic map are severalfold. Physical maps are faster to construct and require considerably less laborintensive methods. Methods in genetic mapping, such as conjugation, transformation, transduction, and mutagenesis, can be bypassed in physical map construction. Many prokaryotic physical maps have been constructed as a starting point to characterize the chromosome because methods of genetic analysis were underdeveloped (1,2,5,14,15,17,43,47). Other physical maps were constructed to confirm previously established genetic maps (10,28,46).With the establishment of long-range genomic mapping techniques (20,36), there has been a recent surge in the field of prokaryotic genomic mapping. With these maps constructed, a preliminary assessment of chromosomal diversity among the prokaryotes may become possible. This assessment of chromosomal diversity has already been examined for species in the genus Neisseria (8), where a newly constructed Neisseria meningitidis map was compared with a previously constructed map of a gonococcal strain. Smith and Condemine (36) stated that "technology needs to be developed to simplify map construction by using maps that already exist for related organisms." This project outlines one such technology that was applied to Staphylococcus aureus.The species is divided into five phage groups on the basis of typing patterns produced on infection with staphylococcal phages (23,32,33,42,45,48). A detailed genetic and physical map has been established in the phage group III S. aure...

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“…Phage w42 was originally isolated from S. aureus strain PS42D, which is the propagating strain for the typing phage w42D (Coleman et al, 1989). In this study, w42 was propagated on the phage-free S. aureus host strain 80CR3 (Stobberingh & Winkler, 1977). S. aureus transformations were carried out with S. aureus strains 80CR3 and RN4220 (Table 1).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2005

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The serotype F phage w42 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 b-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 w42 resistance to lysis by exogenous phages was unknown. The authors report here that phage w42 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 w42 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 w42 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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Restriction-deficient Mutants of Staphylococcus aureus

Cited by 42 publications

References 10 publications

Sau1: a Novel Lineage-Specific Type I Restriction-Modification System That Blocks Horizontal Gene Transfer into Staphylococcus aureus and between S. aureus Isolates of Different Lineages

Sau1: a Novel Lineage-Specific Type I Restriction-Modification System That Blocks Horizontal Gene Transfer into Staphylococcus aureus and between S. aureus Isolates of Different Lineages

Construction of a chromosome map for the phage group II Staphylococcus aureus Ps55

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

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