1 Department of Microbiology, University of Dublin, Trinity College, Dublin, Ireland
2 New England Biolabs, Beverly, MA, USA
3 Department of Clinical Microbiology, St James's Hospital, Dublin, Ireland
4 Microbiology Research Unit, Department of Oral Medicine and Oral Pathology, University of Dublin, Trinity College, Dublin 2, Ireland
Correspondence
David C. Coleman
dcoleman{at}dental.tcd.ie
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is X94423.
Present address: Dublin Institute of Technology, Dublin, Ireland.
Present address: University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Present address: BioHelix, Beverly, MA, USA.
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INTRODUCTION |
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The specific mechanisms by which S. aureus manages to exclude exogenous bacteriophages, and thereby become non-typable, have not been investigated in detail. It is generally considered that all S. aureus phages adsorb to their host cell surface, but may be prevented from further activity after DNA has entered the cell (Parker, 1983; Novick, 1990
). Restrictionmodification (RM) systems are the best-known mechanisms for defence by bacteria against invasion by foreign DNA. One of the advantages of RM systems over mutations conferring phage resistance is that one RM system can simultaneously protect against a variety of phages, thus giving the host an advantage over competing organisms. In general, bacterial RM systems consist of a restriction endonuclease (responsible for recognition of, and cleavage within, a specific DNA sequence) and a DNA methyltransferase (which modifies unmethylated or hemimethylated DNA within the same sequence) (Wilson & Murray, 1991
; Cheng, 1995
; Malone et al., 1995
; Timinskas et al., 1995
). Restriction endonucleases usually require Mg2+ or other divalent cations in order to cleave DNA. Some also require, or are stimulated by, ATP or S-adenosylmethionine (AdoMet). Modification methyltransferases require AdoMet as a co-factor, which serves as the methyl donor (Wilson & Murray, 1991
; Cheng, 1995
; Malone et al., 1995
; Timinskas et al., 1995
). In some RM systems, the restriction endonuclease can operate independently of the modification machinery, while other systems require both the restriction and modification components in order to function (Wilson & Murray, 1991
).
On the basis of a number of criteria, including enzyme subunit composition, cofactor requirements, DNA specificity and reaction products, RM systems have been separated into three classes designated I, II and III (Yuan, 1981). However, new systems are being discovered that do not fit into this original classification. A rapidly growing new class of RM systems includes the BcgI-like enzymes (Kong et al., 1993
, 1994
; Kong & Smith, 1997
; Kong, 1998
), which to date includes BaeI (Sears et al., 1996
), BplI (Vitkute et al., 1997
), CjeI and CjePI (Vitor & Morgan, 1995
), Bsp24I (Degtyarev et al., 1993
), and finally the one reported in this present study, Sau42I, which is the only one other than BcgI to have its gene sequences characterized in detail. An unusual feature of this family is that the enzymes cleave on both sides of the recognition sequence. They have been classified as type IIb restriction endonucleases (Roberts et al., 2003
).
We have found two overlapping unidirectional ORFs adjacent to the int gene region of the S. aureus phage 42 that have extensive nucleotide and amino acid sequence homology to the RM system BcgI from Bacillus coagulans. The purpose of this study was to clone, analyse and express these two ORFs in S. aureus, and to investigate if they encode a functional RM system that is responsible for the phage non-typable phenotype expressed by S. aureus
42 lysogens.
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METHODS |
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Bacteriophage DNA preparation and purification.
To obtain high concentrations of phage 42 particles for DNA analysis and cloning, S. aureus 80CR3 was grown for 1216 h in 5 ml phage broth, according to Blair & Williams (1961)
, containing 0·01 M CaCl2 at room temperature, and then transferred to an orbital shaker at 150 r.p.m. for 2 h at 37 °C. Bacteriophage particles were added to 1 ml cells at a ratio of approximately 1 : 10 in a sterile glass 5 ml Kahn tube, and incubation continued for 20 min for phage adsorption at room temperature. The mixture of phage and cells was then transferred to a 2 l Erlenmeyer flask containing 200 ml phage broth with 0·01 M CaCl2, and incubated in an orbital shaker at 150 r.p.m. at 37 °C for 68 h, or until complete lysis had taken place. This was detected by observing a drop in the optical density of the culture, or the appearance of fine thread-like material and cellular debris resulting from cell lysis. Phage particles were collected, and their DNA was extracted as described below.
Phage 42 DNA was purified from 500 ml lysis cultures using a modification of a method for purification of bacteriophage lambda DNA (Sambrook et al., 1989
). The lysed cultures were treated with DNase (10 µg ml1) and RNase (25 µg ml1) for 24 h at room temperature. Sodium chloride (1 M final concentration) was then added to the lysate, dissolved by swirling, and placed on ice for 1 h. The culture was then centrifuged for 5 min at 6000 g. The supernatant was transferred to clean centrifuge buckets, and solid PEG MW 8000 (PEG 8000, Sigma-Aldrich) was added to a final concentration of 10 % (w/v), and incubated at 4 °C for 212 h after dissolving. The solution was centrifuged at 6000 g for 10 min, and the supernatant was carefully decanted. The resulting pellet containing the phage particles plus PEG was then resuspended gently in 12 ml SM buffer (per litre: 5·8 g NaCl; 2 g MgSO4.7H2O; 50 ml 1 M Tris/HCl, pH 7·5; 5 ml 2 %, w/v, gelatin) using a sterile Pasteur pipette, and 0·5 ml was transferred to a sterile Eppendorf tube. The solution was then extracted two or three times with an equal volume of liquid phenol equilibrated with 10 mM Tris/HCl buffer, pH 8, and once with a phenol/chloroform mixture (1 : 1, v/v) before the DNA was finally precipitated with 2 vols absolute ethanol. The DNA was recovered by centrifugation, and washed once with 70 % (v/v) ethanol, dried briefly and resuspended in TE buffer (Tris/HCl, 10 mM; EDTA, 1 mM; pH 8). The DNA was stored in aliquots at 20 °C. Restriction endonuclease digests of
42 DNA were carried out with enzymes purchased from Promega, according to the manufacturer's instructions.
Sau42I enzyme extraction, purification and assay.
Sau42I endonuclease activity was initially tested from small-scale crude cell extracts from untransformed cells, and cells transformed with the Sau42I genes. Subsequently, extracts were obtained from active fractions of large-scale crude preparations. Small-scale crude cell extracts of S. aureus cells were obtained as follows. TSB (900 ml) was inoculated with 100 ml of an overnight culture grown in TSB. In the case of S. aureus transformants harbouring the pShCm shuttle plasmid or its derivatives, the medium was supplemented with chloramphenicol. After incubation for 4 h at 37 °C, the cells were harvested by centrifugation, and resuspended in 1 ml 0·05 M Tris/HCl, 0·015 M trisodium citrate, pH 7·4. Approximately 5 g cells (wet weight) was obtained by this procedure. Lysostaphin (Sigma-Aldrich) was added to a final concentration of 5 U ml1. After incubation for 15 min at 37 °C, 4 µl 0·01 M Tris/HCl, 0·01 M -mercaptoethanol, pH 7·4, was added to the cells. The cell suspension was sonicated at 0 °C for 5x1 min with a Branson Sonifier, then centrifuged at 4 °C for 30 min at 35 000 r.p.m. in an 8x25 ml angle rotor (Beckman Instruments). The supernatant was designated crude extract, and was immediately tested for enzymic activity as follows: 10 µl crude extract was added to 1 µg phage
or other test DNA (New England Biolabs) in 65 µl cleavage buffer (10 mM Tris/HCl, pH 8; 10 mM MgCl2; 100 mM NaCl; 1 mM DTT; 20 mM AdoMet). Three further serial dilutions of 25 µl of the previous dilution of crude extract in 50 µl cleavage buffer were immediately carried out, followed by incubation at 37 °C for 10 min. DNA cleavage was assayed by gel electrophoresis of a 20 µl sample on a 1 % (w/v) agarose gel.
Large-scale cell preparations were used in an attempt to purify the endonuclease component of Sau42I (i.e. the RM component). Frozen cells (100 g) from fermentation cultures grown at 37 °C overnight were thawed to room temperature. The cells were resuspended in 100 ml lysis buffer, and 500 U lysostaphin was added. The cells were incubated for 20 min at 37 °C on a shaking incubator. After 20x1 min pulses on a Branson Sonifier (equipped with a 5 mm tip) at 4 °C, the cell debris was removed by centrifugation as described above, and the resulting supernatant was immediately poured onto a 15x5 cm heparin sulphate column (Pharmacia LKB) equilibrated with buffer A (10 mM Tris/HCl, pH 7·4; 100 mM EDTA; 10 mM -mercaptoethanol) containing 200 mM NaCl. The column was subsequently washed with 200 ml of the same buffer, then eluted with a 200 ml linear gradient of 0·051·0 M NaCl in buffer A (see above). To detect the active fractions (i.e. those with DNA cleavage activity), 5 µl of each fraction was incubated with phage
DNA for 5 min at 37 °C, and samples were tested by gel electrophoresis. Maximum activity was observed in the samples obtained at approximately 700 mM NaCl. Fractions showing some activity were pooled, and then run on a 15x2·5 mM source Q column (Pharmacia LKB). The active fractions from this step were detected as described above, pooled, and dialysed against 50 % (v/v) glycerol in buffer for 16 h at 4 °C to remove salts.
Western immunoblotting.
Polyclonal antibodies raised against the BcgI large and small subunits (Kong, 1998) were tested against cell extracts of S. aureus transformants as follows. Crude cell extracts and column-purified fractions of transformed and untransformed S. aureus were electrophoresed on SDS-PAGE gels, and transferred to a nitrocellulose filter by electroblotting. The nitrocellulose filter was then incubated for 1 h with rabbit primary antibody raised against BcgI. The filter was then washed before the addition of anti-rabbit antiserum linked to alkaline phosphatase (New England Biolabs) (Kong, 1998
). The filter was then developed with the addition of 23 ml alkaline phosphatase substrate (NBT/BCIP; New England Biolabs), and the protein bands were viewed.
Transformation of bacteria.
Competent E. coli cells for plasmid transformation were prepared, and transformed according to the method outlined by Sambrook et al. (1989). S. aureus electroporation-competent cells were prepared as follows. A 1 ml aliquot of cells from an overnight culture was inoculated into 100 ml TSB, and grown for a further 1·52 h to an OD600 of 0·5. The cells were harvested at 6000 g for 10 min, and the cells were resuspended in 50 ml ice-cold (0·5 M) sucrose solution. The cells were harvested as before, resuspended in 1 ml sucrose solution, and transferred to a sterile 1·5 ml Eppendorf tube on ice for 20 min. Following incubation, the cells were harvested by centrifugation, and resuspended in 1 ml 0·5 M ice-cold sucrose solution. Aliquots of competent cells (200 µl) were transferred to 0·5 mm electroporation cuvettes (Bio-Rad) with 0·110 µg DNA, and electroporated at the following settings: 2·5 mV and 100
. Immediately after electroporation, 500 µl TSB was added to the cells, and they were incubated at 37 °C for 1 h before being plated on appropriate selective agar medium. Transformant colonies were usually observed after 2448 h.
Construction of recombinant plasmids, and DNA sequencing.
Plasmid pRD-RM10 (Fig. 1c), which encodes the complete putative S. aureus
42 RM system, was constructed by digesting pDC107 (Carroll et al., 1995
) with NsiIPstI, and ligating the resulting 1·8 kb fragment containing sequences with homology to the specificity subunit (S) of BcgI to Pst-digested pRD21. Plasmid pRD21 (Fig. 1c
) was constructed as follows. A 6·5 kb PstISalI fragment from
42 DNA (Fig. 1b
; Coleman et al., 1989
), which contains some homology to the restriction methylation RM gene of BcgI, was cloned into the PstISalI site of pBluescript KS to yield plasmid pRDSK20. This plasmid was digested with ClaI, which produced two fragments: a 6·5 kb fragment, which contained the plasmid vector and the region of interest, i.e. the endonuclease modification gene; and a fragment of 2·5 kb containing sequences upstream from the remaining portion of the PstISalI insert fragment of
42. This digested plasmid DNA was then religated, and transformed into E. coli. Plasmid pRD21 was selected from the transformants for further study.
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Bioinformatics.
Database searches and sequence comparison were performed using the CLUSTAL W and BLAST algorithms (Higgins & Sharp, 1988; Altschul et al., 1990
), and the National Center for Biotechnology Information (NCBI) GenBank/EMBL databases.
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RESULTS |
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Subcloning and sequencing 42 RM genes
Fig. 1(a, b) shows the structural organization of the linear
42 genome. ORF A, now renamed ORF S (see below), is located within a 5 kb HindIII fragment, and was originally cloned in plasmid pDC107 (Fig. 1c
; Carroll et al., 1995
). This plasmid was found to also contain a partial ORF with sequence homology to the 3' end of the BcgI RM subunit. S. aureus ORF A was therefore renamed ORF S (specificity subunit) in analogy with the BcgI system, and the partial ORF was designated ORF RM (restriction and methyltransferase subunit) (Fig. 1c
). The adjoining fragment on the phage genome was cloned from purified
42 DNA on a 4 kb PstIClaI fragment (Fig. 1b
) into vector plasmid pBluescript II KS+. The insert in the resulting plasmid, pRD21 (Fig. 1c
), was sequenced, and revealed a 1·5 kb partial ORF in the same orientation as ORF RM with a high level of homology to the 5' end of the BcgI RM subunit. The ORF S and ORF RM genes were combined on plasmid pRD-RM10 by ligation of the 1·8 kb PstINsiI fragment of pDC107 to PstI-digested pRD21 (Fig. 1c
). The complete DNA sequence of the two ORFs was submitted to the GenBank/EMBL nucleotide sequence database, and assigned the accession no. X94423.
Sau42I, a putative novel RM system
Based on the findings described above, we believe that we have discovered a new RM system encoded by S. aureus bacteriophage 42, and we have therefore named this putative system Sau42I.
The genetic organization of Sau42I is similar to that of the BcgI RM system, i.e. the genes are encoded by two ORFs of the same orientation, but in different reading frames which overlap by several bases (Fig. 1c). The G+C content of the overall sequence is 32 mol%, which is similar to the content of other S. aureus phage genes, which averages 30 mol%; the G+C content of S. aureus bacterial genes averages 35 mol% (Novick, 1990
).
The DNA sequence of ORF S, the putative specificity subunit, contains two contiguous potential ATG start codons in the same reading frame. Either of two translation products of 337 or 336 aa residues could therefore be produced with a similar molecular mass of approximately 38 kDa. Analysis of the amino acid sequence revealed that the second half of the protein sequence shows similarity to the first half, suggesting dimer symmetry in the protein. The amino acid homology between ORF S and the BcgI specificity subunit is 36 % identity and 50 % similarity. This is significantly lower than the level of homology between the RM subunits. An amino acid alignment of Sau42I specificity subunit ORF S and the BcgI S subunit is shown in Fig. 2. A search of the GenBank database with the predicted protein sequence indicated the closest homology to BcgI, and partial homology to a putative type IIs restriction enzyme from the Helicobacter pylori genome (accession nos AE001559 and AE000647, respectively).
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DISCUSSION |
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In order to investigate whether Sau42I is functional in S. aureus, a 42 DNA fragment encoding both genes was cloned in shuttle plasmid pRDCm11 (Fig. 1c
), and transformed into S. aureus 80CR3. Transformants harbouring pRDCm11 were found to exhibit resistance to lysis by all 23 phages of the IBS typing set, as did S. aureus 42CR3-L (Table 1
), a
42 lysogenic derivative of 80CR3. In contrast, 80CR3 was susceptible to lysis by all 23 IBS phages. Similarly, S. aureus RN4220 transformants harbouring pRDCm11 were resistant to lysis by all the IBS phages, whereas the parental strain was susceptible to lysis by 17/23 IBS phages (Fig. 5
). Interestingly, transformants harbouring pRDCmT1 (Fig. 1c
), which encoded ORF RM and a truncated derivative of ORF S lacking sequences for the C-terminal 20 aa, were also resistant to lysis by all 23 IBS typing phages. Presumably the functional domains encoded by ORF S are still intact in plasmid pRDCmT1. Transformants harbouring pRDCmT2 encoding ORF RM and a truncated ORF S lacking the C-terminal 174 aa were susceptible to lysis by the IBS typing phages. Similarly, transformants harbouring pRDCmT3 encoding ORF S and a truncated ORF RM encoding the C-terminal 112 aa only were susceptible to lysis by the IBS typing phages. These findings demonstrated that the
42 ORF S and ORF RM genes are responsible for the broad-range phage lysis resistance exhibited by
42 lysogens, and that both genes are required for this to occur.
The observed homology between the ORF S and ORF RM determinants of the Sau42I and BcgI systems, and the resistance to lysis by exogenous phages of the IBS typing set exhibited by S. aureus transformants harbouring cloned 42 ORF S and ORF RM genes, strongly suggested that Sau42I is a functional RM system. Timinskas et al. (1995)
classified the BcgI-encoded methyltransferase protein as belonging to class N12, based on the presence and order in the primary sequence of characteristic conserved amino acid motifs. All methyltransferases identified to date contain conserved amino acid motifs, despite the fact that there is little overall sequence homology. Fig. 3
shows an amino acid alignment of the Sau42I and BcgI methyltransferase subunits, and the four conserved motifs (CM) identified by Timinskas et al. (1995)
are present in both sequences. The submotif FXGXG (where X can be any residue), located within CM I, is common to virtually all enzymes that utilize AdoMet for the source of methionine, apart from N12 methyltransferases such as BcgI and Sau42I, where instead of invariant F, other amino acid residues are found (Fig. 3
). The submotif is believed to be the AdoMet binding domain (Wilson & Murray, 1991
; Timinskas et al., 1995
). The second motif present in both the Sau42I and BcgI amino acid sequences is CM II, comprising a sequence of 10 aa residues (Fig. 3
). CM II motifs are present in all methyltransferases, and very often the sequence PPY is conserved (Timinskas et al., 1995
). The nature of the residues preceding PPY is characteristic for different classes of methyltransferase (D in D12 and D21 classes, S in S21 and S12, and N in N12) and it correlates with the base methylation specificity of the enzymes (Klimasauskas et al., 1989
; Timinskas et al., 1995
). This element appears to be unique to the amino methyltransferases (Zhang et al., 1993
). In the Sau42I with BcgI methyltransferase sequences the submotif NPPY is present within CM II, and thus both of these enzymes belong to class N12 (Fig. 3
). Timinskas et al. (1995)
reported that class N methyltransferases include a third CM, termed CM Is, preceding CM I. This sequence is usually 17 aa in length in almost all class N methyltransferases, and is present in both the Sau42I and BcgI (Fig. 3
). In class N methyltransferases, CM I and CM II are thought to be involved in AdoMet binding and methyl group transfer (Timinskas et al., 1995
). Close location of CM Is to CM I in the primary amino acid sequence is indicative of possible interactions (Fig. 3
; Timinskas et al., 1995
). Timinskas et al. (1995)
identified a fourth CM, termed CM III, downstream from CM II, consisting of a region of 19 aa residues in almost all class N methyltransferases. CM III motifs are also present in both the Sau42I and BcgI methyltransferase sequences (Fig. 3
; Timinskas et al., 1995
). A study by Malone et al. (1995)
suggested that the CM III motif is a catalytic region.
In order to investigate whether the ORF RM determinant encoded detectable endonuclease activity in S. aureus transformants, crude and partially purified cell lysates of S. aureus 80CR3 and RN4220 transformants harbouring plasmid pRDCm11 were investigated for endonuclease activity on bacterial, plasmid and phage DNA. Endonuclease activity was detected; however, despite repeated attempts, complete digestion of substrate DNA was never achieved. This was probably due to concurrent methylation and cleavage of substrate DNA by the endonuclease and methylation subunit encoded by ORF RM, resulting in a proportion of the substrate DNA being protected from cleavage. Thus the exact cleavage site of Sau42I has not yet been elucidated. In the BcgI RM system, both the S and RM subunits are required to bind, cleave and methylate DNA (Kong et al., 1994). Both the endonuclease and methylase domains are located in the RM subunit, with the target recognition domain located in the S subunit (Kong, 1998
). Further studies are in progress to dissect the functional organization of the Sau42I system, and to identify the target recognition sequence.
The finding of an RM system encoded by 42 distinguishes this phage as a quadruple-converting phage, believed to be the first reported for S. aureus. Lysogenization of S. aureus by
42 or similar phages enhances the virulence potential of lysogens by giving them the ability to express enterotoxin A and staphylokinase. The ability of
42 to confer protection on its host against lysis by a wide variety of exogenous phages, while not a virulence factor in itself, would confer a selective advantage over phage-susceptible cells in the presnce of lytic phages, providing lysogens with a potentially significant survival advantage. It is possible that phages similar to
42 may be partly responsible for the high prevalence of phage non-typable strains reported among clinical isolates of S. aureus.
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ACKNOWLEDGEMENTS |
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Received 22 September 2004;
revised 16 December 2004;
accepted 22 December 2004.
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