A plasmid from Staphylococcus sciuri DD 4747 had three open reading frames: a replication gene, an N-acetylmuramyl-l-alanine amidase-like gene, and a gene similar to the lysostaphin endopeptidase resistance gene (epr/lif). The epr-like gene was introduced into S. aureus RN4220; the recombinant strain was more resistant to lysostaphin endopeptidase and its cell wall peptidoglycan contained more serines and fewer glycines than the parental strain with the shuttle vector alone. Based on both its function and its similarity to femAB, this gene is a member of the femABX-like immunity gene family. Furthermore, this is the first example of a femABX-like immunity gene that is not linked to the gene for the bacteriolytic enzyme against which it specifies immunity.
FemABX-like proteins are peptidyl transferases involved in peptidoglycan cross-bridge formation in Gram-positive bacteria (reviewed in ). Some bacteria produce additional FemABX-like proteins that protect against interpeptide-specific endopeptidases by specifying amino acid substitutions during synthesis of the peptidoglycan cross-bridges. These proteins are called FemABX-like immunity factors . The best known of these immunity factors is the one that protects Staphylococcus simulans biovar staphylolyticus from the staphylolytic glycylglycine endopeptidase (lysostaphin) that it produces. The gene for lysostaphin endopeptidase resistance is called epr[3,4], for endopeptidase resistance, or alternatively lif, for lysostaphin immunity factor. Epr specifies the incorporation of serines in place of some glycines in the peptidoglycan cross-bridges of staphylococci . Epr forms heterodimers with FemA and with FemB; this results in the insertion of serine in place of glycine at positions 3 and 5 in the cross-bridges [6,7]. Other examples of FemABX-like immunity factor genes include epr in S. capitis, zif (zoocin A immunity factor) in Streptococcus equi subsp. zooepidemicus 4881 , and milF (millericin B immunity factor) in Streptococcus milleri. In all of these cases, the genes for the FemABX-like immunity factors have been closely linked to the genes for the endopeptidases against which they provide protection. In staphylococci these gene pairs have been found on plasmids [3,8] whereas in streptococci the gene pairs are chromosomal [9,10]. These genes are adjacent and, except for the millericin B operon , are transcribed in divergent directions.
In a previous study we identified three strains of S. sciuri that reacted with a probe for the lysostaphin endopeptidase gene (end), produced extracellular protein that reacted with a monoclonal antibody against lysostaphin endopeptidase, produced extracellular staphylolytic activity, and were more resistant to lysostaphin than the S. sciuri subsp. sciuri type strain . The present study was undertaken to determine if this lysostaphin endopeptidase resistance was due to an epr-like gene.
2Materials and methods
2.1Bacterial strains and plasmids
The bacterial strains and plasmids used in this study are summarized in Table 1. Unless otherwise indicated, all reagents were purchased from Sigma Chemical Co., St. Louis, MO, USA. E. coli strains were routinely grown in TYS, which consisted of 1% Bacto Tryptone (BD Diagnostic Systems, Sparks, MD, USA), 0.5% Bacto yeast extract (BD Diagnostic Systems) and 0.5% sodium chloride at 37°C with appropriate additions (ampicillin, 100 μg ml−1; kanamycin, 50 μg ml−1; chloramphenicol, 34 μg ml−1; 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside [X-gal], 50 μg ml−1; isopropyl β-d-thiogalactopyranoside [IPTG], 35 μM). Except for peptidoglycan analyses, staphylococcal strains were grown in Tryptic Soy Broth (BD Diagnostic Systems) at 37°C with appropriate additions (chloramphenicol, 17 μg ml−1; kanamycin, 50 μg ml−1). Liquid cultures were grown with aeration. Solid media contained 1.5% Bacto agar (BD Diagnostic Systems).
E. coli(AmpR) –S. aureus (ChlR) shuttle vector; 
4.4-kbp plasmid isolated from S. sciuri DD 4747; this study
Source of ω-Km2; , provided by S. Hollingshead, University of Alabama at Birmingham
pACK6::pBC SK (−)::ω-Km2 (plasmids were joined through their Xba I sites); this study
pACK6::pBC SK (−)::ω-Km2 (plasmids were joined through their Eco RI sites); this study
Cloned PCR product of eprsc; this study
2.2Determination of lysostaphin susceptibility
Susceptibility to lysostaphin was determined as previously described  using 1 U ml−1 lysostaphin (Sigma).
2.3Peptidoglycan purification and analysis
Cells were grown in lysostaphin production medium , and peptidoglycan purification and analysis were performed as previously described .
2.4DNA manipulations and analysis
Unless stated otherwise, standard procedures were as described by Sambrook et al. . Restriction endonucleases were from New England Biolabs, Inc., Beverly, MA, USA. Primers were from Invitrogen Corp., Carlsbad, CA, USA. Commercial products were used according to the manufacturers’ instructions.
Plasmid pACK6 was purified from S. sciuri DD 4747 by treatment with acetone, lysozyme, and sodium dodecyl sulfate (SDS) as previously described , digested with Xba I or Eco RI and ligated (Fast-Link DNA Ligation Kit, Epicentre Biotechnologies, Madison, WI, USA) to similarly digested and dephosphorylated (HK Thermolabile Phosphatase, Epicentre) pBC SK (−) (Stratagene, La Jolla, CA, USA). Because the chloramphenicol resistance determinant of pBC SK (−) is not active in staphylococci, gel-purified (QIAquick Gel Extraction Kit, Qiagen Inc., Valencia, CA, USA) ω-Km2 from pBR-ω-Km2 was inserted using the Bam HI sites to create pLSH (Xba I construct) and pJKAG (Eco RI construct).
Both strands of pACK6 in pLSH were sequenced by primer walking using BigDye? Terminator Cycle Sequencing v. 3.1 Ready Reaction (Applied Biosystems, Foster City, CA, USA), a GeneAmp? PCR System 9700 (Applied Biosystems), and an ABI Prism? 3100 Genetic Analyzer (Applied Biosystems). BioEdit  and BLAST  were used to analyze the sequence.
A 1.5-kb fragment of DNA containing eprsc was amplified from pLSH using thin-walled polypropylene PCR tubes in a RapidCycler (Idaho Technology Inc., Salt Lake City, UT, USA). Primers, 5′-GCAGGAGCTATTTTACAAACTTCA-3′ and 5′-GGGTTCAAAGTTTCTGTTCAAAAAGGA-3′, were annealed at 40°C for 30 s in buffer C (FailSafe PCR PreMix Selection Kit, Epicentre). The fragment was gel-purified, phosphorylated (End-It DNA End Repair Kit, Epicentre), and ligated into Eco RV-cut and dephosphorylated (Shrimp Alkaline Phosphatase, USB Corp., Cleveland, OH, USA) pBluescript II KS (+). The ligation products were electroporated (MicroPulser, Bio-Rad Laboratories, Hercules, CA, USA) into E. coli EC-100 cells, which were plated onto TYS containing ampicillin, IPTG, and X-gal. White colonies were tested for the presence of the correct-sized insert. One construct was selected and cut with Bam HI and Hind III for ligation into similarly cut and dephosphorylated pLI50. This ligation reaction mixture was used to electroporate E. coli EC-100 and ampicillin-resistant transformants were analyzed by restriction mapping; one was verified by sequencing and designated pLI50::eprsc. Plasmid pLI50::eprsc was then electroporated into S. aureus RN4220 as described by Schenk and Laddaga  and chloramphenicol-resistant transformants were selected.
2.5Renaturing activity gel analyses
Stationary-phase culture supernatants were concentrated 20-fold using Amicon Centriprep 10 filters (Millipore Corp., Bedford, MA, USA). Analytical polyacrylamide gel electrophoresis in the presence of SDS was performed by the method of Laemmli . Activity gel analyses to detect staphylolytic or micrococcolytic activities were performed as described previously  in gels (15% [w/v] acrylamide and 0.6% [w/v] N, N′-methylene-bisacrylamide) that contained heat-killed cells of S. aureus FDA 209P or Micrococcus luteus. Following electrophoresis the gels were washed in 200 ml deionized water for 2 h and then incubated for 48 to 72 h at 37°C in 0.05 M Tris–HCl (pH 7.2 at 25°C) containing 1% (v/v) Triton X-100 and 10 mM MgCl2. After incubation, unlysed cells were stained with 0.1% methylene blue for 10 min, destained with deionized water, and photographed with a Model 290 Electrophoresis Documentation and Analysis System (Kodak, Rochester, NY, USA).
3Results and discussion
In order to determine if the lysostaphin resistance of the three strains of S. sciuri (DD 4747, DD 5262, and DD 6282) was due to the presence of epr-like genes, we compared their peptidoglycan compositions to that of the more sensitive strain DD 4277. The only differences detected were in their cross-bridge compositions. As seen in Table 2, the three resistant strains had both increased resistance to lysostaphin and larger serine to glycine ratios than the more sensitive strain. These data suggested the presence of an epr-like gene in the three more resistant strains. Since all previous epr-like genes in staphylococci have been reported to be on plasmids, we determined if these three lysostaphin-resistant strains contained plasmids. All three resistant strains contained a 4.4-kb plasmid whereas the more sensitive strain did not. Strain DD 4747 was selected for further study.
Table 2. Lysostaphin resistance and peptidoglycan cross-bridge composition
aRate of lysis (change in OD620 in 10 min) of heat-treated cells of the lysostaphin assay organism (S. aureus FDA 209P) minus rate of lysis of test strain divided by the rate of lysis of the assay organism when standardized cell suspensions were reacted with lysostaphin (1 U ml−1).
bThe amount of each compound is expressed as a molar ratio relative to lysine; values shown are the averaged results from two separate analyses.
S. aureus FDA 209P
S. simulans biovar staphylolyticus
S. sciuri DD 6282
S. sciuri DD 5262
S. sciuri DD 4747
S. sciuri DD 4277T
The plasmid from strain DD 4747, designated pACK6, was sequenced (GenBank accession number AF093750). Sequence analysis revealed three open reading frames (Fig. 1). One coded for a protein that had homology to FemABX-like immunity factors, and was designated eprsc. It was closely related to epr from S. simulans biovar staphylolyticus and epr from S. capitis and was presumably responsible for insertion of serines in place of some glycines in the peptidoglycan cross-bridges. The second ORF, designated amisc, contained a region similar to the CHAP domain (pfam05257); this amidase domain is found in many proteins that are involved in bacterial cell wall metabolism, including several N-acetylmuramyl-l-alanine amidases revealed by BLAST. The third ORF was designated repsc because it coded for a putative protein that was similar to the Rep protein of several staphylococcal plasmids.
Additional relevant sequences on pACK6 (Fig. 1) included a sequence similar to the origin of replication on several staphylococcal plasmids. The Xba I site is within this region and when pACK6 and pBC SK (−) were joined through their Xba I sites to make pLSH, no transformants were obtained in S. aureus RN 4220. However, when pJKAG was made by joining pACK6 and pBC SK (−) through their Eco RI sites, which did not disrupt the putative origin of replication, transformants were obtained. Therefore we conclude that this region is required for replication. Another region on pACK6 was very similar to the putative tRNASer identified by Thumm and Götz  in pACK1 of S. simulans biovar staphylolyticus.
To prove that eprsc was in fact responsible for the increased serine content in the peptidoglycan cross-bridges, we attempted to cure pACK6 from strain DD 4747 by a number of different methods that previously had been successful in curing other plasmids from staphylococci . None of these methods was successful. This was consistent with a chromosomal gene for a lysostaphin-like endopeptidase providing a selective pressure for retention of the plasmid. Therefore as an alternative approach to prove that eprsc is functional, we amplified the gene by PCR, cloned the product into shuttle vector pLI50, and introduced it into S. aureus RN4220. As seen in Fig. 2, S. aureus containing pLI50::eprsc was more resistant to lysostaphin than the organism containing pLI50 alone. As shown in Table 3, eprsc caused the insertion of serines in place of some glycines in the peptidoglycan cross-bridges of S. aureus even though the construct did not contain the putative tRNASer. This confirmed that eprsc is a functional member of the femABX-like immunity gene family.
Table 3. Effect of eprsc on peptidoglycan cross-bridge composition
The amount of each compound is expressed as a molar ratio relative to lysine; values shown are the averaged results from two separate analyses.
S. aureus RN4220/pLI50
S. aureus RN4220/pLI50::eprsc
In order to determine if the amidase gene coded for an active bacteriolytic enzyme, pJKAG was introduced into S. aureus RN4220. No micrococcolytic activity bands at the predicted molecular mass (18 kDa) were seen in concentrated culture supernatants from S. sciuri DD 4747 or S. aureus RN4220/pJKAG (data not shown). However, as seen in Fig. 3, the recombinant S. aureus strain containing pJKAG produced an additional extracellular staphylolytic activity of the predicted molecular mass whereas the parental strain did not. This band corresponded to one of the staphylolytic bands produced by strain DD 4747. Because the amidase gene was located on pACK6 near eprsc, it might be the peptidoglycan hydrolase for which eprsc provides protection. However, the fact that the Eco RI site used to make pJKAG was in eprsc showed that an intact eprsc gene was not needed for resistance to the staphylolytic amidase and thus further supports our conclusion that eprsc is needed for resistance to a chromosomally encoded lysostaphin-like endopeptidase in strain DD 4747. In all previous cases the femABX-like immunity factor genes and the lytic enzyme genes for which they specify immunity are adjacent. Thus this is the first report of an organism in which the gene for the endopeptidase and its corresponding femABX-like immunity gene are not linked.
We thank Kelly Morrison of the University of Alabama at Birmingham Protein Analysis Laboratory for excellent technical assistance in the peptidoglycan analyses. We also thank Julie Kane and Aaron Glenn for their technical assistance. This work was supported in part by grant GM062197 from the National Institute of General Medical Sciences, NIH and by a grant from the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Program.