Vol. 172, No. 10

JOURNAL OF BACTERIOLOGY, Oct. 1990, p. 5783-5788

0021-9193/90/105783-06$02.00/0 Copyright X 1990, American Society for Microbiology

Cloning and Expression of a Staphylococcus aureus Gene Encoding a Peptidoglycan Hydrolase Activity RADHESHYAM K. JAYASWAL,* YOON-IK LEE, AND BRIAN J. WILKINSON Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, Illinois 61761 Received 11 May 1990/Accepted 18 July 1990 A gene of Staphylococcus aureus PS47 encoding lytic activity was cloned and expressed in Escherichia coli. Deletion analysis of a recombinant plasmid carrying a 7.4-kilobase-pair fragment (kbp) of S. aureus DNA suggested that the gene was located within a 2.5-kbp EcoRI-XbaI fragment. Analysis of extracts of E. coli harboring recombinant plasmids on denaturing polyacrylamide gels containing purified cell walls of S. aureus showed a clearing zone by a polypeptide of apparent Mr 23,000. The release of dinitrophenylalanine but not reducing groups from purified cell wails by a cell extract of recombinant E. coli suggested that we had cloned an N-acetylmuramyl-L-alanine amidase.

Autolysins are enzymes capable of degrading bacterial cell walls. Roles have been proposed for these enzymes in wall growth, cell separation, wall turnover, and lysis initiated by cell wall-active antibiotics (26, 31). Recent evidence has also suggested a role for autolysins in bacterial pathogenicity (1). Four common types of autolysin are recognized: (i) a lysozymelike enzyme hydrolyzing N-acetylmuramyl-1,4-pN-acetylglucosamine bonds (muramidase); (ii) a ,-N-acetylglucosaminidase liberating the free reducing groups of N-acetylglucosamine (glucosaminidase); (iii) an N-acetylmuramyl-L-alanine amidase (amidase) hydrolyzing the bond between N-acetylmuramic acid and L-alanine, and (iv) peptidases hydrolyzing the stem or bridge peptides (26). In cases i and ii, both exo- and endoenzymes are known, but only the endoenzymes are believed to function as autolysins. The number, bond specificity, cellular location, and significance of Staphylococcus aureus autolysins is ambiguous. Tipper (30) reported that amidase was the major autolysin activity in crude cell walls (cell walls retaining autolytic activity), although glucosaminidase and glycine endopeptidase activities were also present. Huff et al. (11) reported purifying an amidase from culture supematants that had an Mr of 30,000, had a broad pH optimum, and required 20 mM MgCl2 for optimum activity. Singer et al. (27) purified amidase from the cytoplasmic fraction and reported an Mr of 800,000. Glucosaminidase has been purified from culture supernatants (28, 32, 34, 35), and Mrs of 51,000 (28) and 80,000 (32) have been reported. In addition, a bacteriophageelaborated lysin (termed virolysin) for S. aureus cell walls has been described by Ralston and McIvor (24) and Doughty and Mann (7). This enzyme was characterized as a peptidase yielding N-terminal alanine, glutamic acid, and glycine. There have been relatively few studies describing the cloning of autolysin genes. Autolysin genes have been cloned for the Streptococcus pneumoniae amidase (8, 9) and an unidentified Bacillus sp. autolysin (22). Biavasco et al. (2) have reported cloning a glucosaminidase gene from S. aureus encoding a polypeptide of M, 80,000. There is evidence that the glucosaminidase may play a role in the pathogenicity of S. aureus (2). However, the precise role(s) of other autolysins of S. aureus is not known. We have undertaken a molecular cloning approach to the study of S. aureus autol*

ysins. In this report, we describe the cloning of a gene encoding a low-molecular-weight lytic activity identified as an amidase from S. aureus. MATERIALS AND METHODS Bacterial strains and plasmids. S. aureus PS47 (21) was used as the source of chromosomal DNA. Two E. coli strains were used, HB101 and JM109, which bears a recA mutation (17). Micrococcus luteus cells were used as the substrate for testing lytic activity on plates. Plasmids pBR322, pUC13, pACYC184, and pTZ19R were used (4, 5, 18, 33). The E. coli strains were grown in Luria-Bertani (LB) medium, and M. luteus and S. aureus strains were grown in PYK (16) or nutrient broth (Difco Laboratories, Detroit, Mich.) medium at 37°C with shaking (200 rpm). When required, antibiotics were used at the following concentrations: ampicillin, 50 p,g/ml; tetracycline, 25 ,ug/ml; chloramphenicol, 20 ,ug/ml. Isolation and purification of S. aureus cell walls. Purified cell walls (PCW) were prepared from strain PS47 exponential-phase cultures (A580 of about 1.0) in PYK medium essentially as described by Wilkinson et al. (36). Organisms were broken by mixing with glass beads with a Bead-Beater (Biospec Products, Bartlesville, Okla.). PCW were treated with 10% trichloroacetic acid at 60°C for 90 min, followed by washing with water to yield peptidoglycan. Also, PCW were treated with 0.1 M NaOH for 10 min at 37°C to remove ester-linked D-alanine. These PCW were used in studies to determine the bond specificity of the cloned autolysin. In addition, sodium dodecyl sulfate (SDS)-treated cell walls (SDS-CW) were isolated from cells grown in nutrient broth for detection of autolytic activity in SDS-polyacrylamide gel electrophoresis (PAGE) gels (see below). Crude cell walls were isolated and treated with 4% (wt/vol) SDS for 90 min at 37°C, followed by 10 min at 100°C (22). Cell wall preparations were lyophilized. Isolation of DNA. S. aureus PS47 grown to mid-exponential phase in 200 ml of PYK was harvested by centrifugation, washed with 50 mM Tris hydrochloride (Tris-HCl) containing 50 mM EDTA (pH 7.8), and suspended in 25 ml of the same buffer containing lysostaphin (100 ,g/ml). After 1 h, pronase (0.5 mg/ml) and SDS (0.5%, wt/vol) were added, and the suspension was incubated for 10 min at 37°C. The resulting lysate was extracted three times with an equal volume of neutralized phenol-chloroform-isoamyl alcohol

Corresponding author. 5783



(25:24:1), followed by extraction with chloroform-isoamyl alcohol (24:1). Nucleic acid was precipitated by addition of 0.1 volume of 3.0 M sodium acetate and 2 volumes of 95% ethanol, recovered by centrifugation, and dissolved in TE buffer (1 mM Tris-HCl [pH 7.0], 0.1 mM EDTA). Plasmid DNA from E. coli was isolated by the alkaline SDS method (17), followed by CsCl density gradient centrifugation. Small-scale plasmid preparations were obtained by the boiling method described by Maniatis et al. (17). Construction of chromosomal gene bank of S. aureus in E. coli. Chromosomal DNA from S. aureus PS47 was partially digested with Sau3AI and then fractionated by centrifugation through a 12-ml 10 to 40% (wt/vol) sucrose gradient at 23,000 rpm (Beckman SW28 rotor) for 18 h at 25°C. Fragments in the range of 5 to 15 kbp were pooled and ligated into the BamHI-digested and alkaline phosphatase-treated pBR322 vector (17). Competent cells of E. coli HB101 were transformed (17) with the ligated mixture and selected for resistance to ampicillin. Clones resistant to ampicillin but sensitive to tetracycline were screened for lytic activity. Screening for lysis against M. luteus. About 1,700 colonies from the genomic bank were patched onto L agar medium containing ampicillin and grown at 37°C. After 16 h, colonies were exposed to chloroform vapor for 30 min and overlaid with soft agar containing 0.2% (wt/vol) autoclaved, lyophilized M. luteus cells (2, 22). After 8 to 12 h, clones were scored for lysis (clear zones) around the transformant colonies. Studies of the cloned lytic activity. E. coli clones grown overnight in LB broth (200 ml) were harvested by centrifugation (13,000 x g, 5 min, 4°C). Cells were suspended in 20 ml of 0.05 M KH2PO4-K2HPO4 buffer (pH 7.2) and broken with a French pressure cell (20). Unbroken cells and debris were removed by centrifugation (13,000 x g, 5 min, 4°C), and the cell extract was used directly for enzyme assays. To assay autolytic activity, S. aureus PS47 PCW were suspended (1 mg [dry weight] per ml) in 4 ml of 0.05 M KH2PO4-K2HPO4 buffer (pH 7.2); various amounts of cell extract (up to 900 ,ul) were added and incubated at 37°C. Turbidity was measured at intervals at 580 nm (23). In order to identify the specific bond cleaved, PCW were suspended at 10 mg/ml, and samples (0.4 ml) were removed during the incubation, diluted with 2.8 ml of water, and boiled for 10 min. Undigested PCW were removed by centrifugation (13,000 x g, 5 min, 20°C), and suitable samples of the supernatant were assayed for the appearance of amino groups with 1-fluoro-2,4-dinitrobenzene (FDNB) as described by Ghuysen et al. (10) and reducing groups by the method of Thompson and Shockman (29). The N-terminal amino acid released was determined by reacting supernatants from digested PCW with FDNB, followed by hydrolysis and chromatography as described by Ghuysen et al. (10). Preparation of S. aureus freeze-thaw autolysin activity. Freeze-thaw autolysin activity was obtained by the method of Huff et al. (11, 23) except that the extract was not

dialyzed.. Detection of lytic activity in SDS-PAGE gels. Enzymatic activity was detected in situ by using an SDS-PAGE gel containing 0.2% (wt/vol) PCW or SDS-CW from S. aureus PS47 as described by Leclerc and Asselin (14) and Potvin et al. (22). Cells were harvested by centrifugation (13,000 x g, 5 min, 4°C) and suspended in 25 mM Tris-HCl (pH 8.0) containing 2 mM dithiothreitol, 2% sucrose, 1% SDS, and 0.5 mM phenylmethylsulfonyl fluoride. Cells were disrupted by sonication on ice for 2 min (Sonifier Cell Disruptor 350; Branson Sonic Co.), and the extract was collected by


centrifugation (13,000 x g, 5 min, 4°C). The supernatant was heated for 2 min in boiling water prior to electrophoresis. After electrophoresis, the gels were incubated for 12 to 16 h at 37°C in 500 ml of 25 mM Tris-HCl (pH 8.0) containing 1% Triton X-100 to permit protein renaturation. Transparent bands of lysis in the translucent gel were rendered more visible by staining with 1% methylene blue in 0.01% KOH prior to photography. Protein analysis. E. coli strains harboring various-sized inserts were used for the identification of insert-encoded proteins. Cells grown for 8 to 12 h in LB medium containing the appropriate concentration of antibiotics were pelleted by centrifugation, suspended in lysis buffer (80 mM Tris-HCl [pH 6.8] containing 2% SDS, 100 mM dithiothreitol, 20% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride) and boiled for 5 min. Proteins were analyzed by SDS-PAGE with a 15% polyacrylamide gel as described by Jayaswal et al. (12). For in vivo labeling, 1-ml exponentially growing cultures in M9 medium (19) were centrifuged for 1 min, and the cells were suspended in 1 ml of M9 medium lacking S04 but containing 0.25 mM amino acids (minus methionine) and 50 ,uCi of [35S]methionine. After 15 min, the cells were pelleted by centrifugation and then lysed in 200 pd of lysis buffer. The proteins were resolved by SDS-PAGE (15% gel). The gel was dried and exposed to X-ray film. Protein concentration was determined by the method of Lowry et al. (15). Enzymes and chemicals. [35S]methionine and [32P]dCTP were purchased from ICN Biochemicals, Costa Mesa, Calif. Intestinal alkaline phosphatase was purchased from Boehringer Mannheim Biochemicals, Indianapolis, Ind. Lysozyme from chicken egg white, lysostaphin, bovine serum albumin, Triton X-100, antibiotics, dinitrophenyl (DNP)-alanine, DNP-serine, DNP-lysine, and DNP-glycine were purchased from Sigma Chemical Co., St. Louis, Mo. Restriction endonucleases, T4 DNA ligase, RNase, and the nick translation kit were purchased from Bethesda Research Laboratories, Inc., Bethesda, Md. All reagents were of analytical or ultrapure grade unless otherwise stated. Other procedures. DNA ligation, transformation, and plasmid isolation were performed as described by Maniatis et al. (17). All enzymes were used in accordance with the manufacturer's specifications. Recombinant DNA experiments were conducted under P1 containment conditions as specified by National Institutes of Health guidelines. RESULTS Isolation of an S. aureus gene coding for lytic activity. The experimental approach used for the cloning of the gene encoding lytic activity was to select clones of E. coli with the ability to lyse M. luteus cells after the E. coli were transformed with a genomic library of S. aureus. Because E. coli lacks lytic activity against M. luteus cells, it was a feasible host for this experimental approach. A partial Sau3A genomic library of S. aureus DNA consisting of fragments between 5 and 15 kb long was constructed by insertions within the BamHI site of plasmid pBR322. The ligation mixture was used to transform E. coli HB101, and the transformants were selected on LB-ampicillin plates. Approximately 1,700 recombinant clones were screened for their ability to form a clear zone around a transformant colony on LB agar overlaid with soft agar containing M. luteus cells. One of the clones displaying lytic activity (Fig. 1), designated pRJ2-19, was used for further analysis. Plasmid analysis showed that the recombinant clone contained a 7.4-kb insert. Retransformation of another strain of E. coli,

VOL. 172, 1990

whereas a strain bearing a plasmid containing the 3.8-kb EcoRI fragment (pRJ2-19E2) did not. This suggested that the gene encoding lytic activity was located within the 3-kb EcoRI fragment. Deletion of the EcoRI-XbaI (0.6 kb) fragment from pRJ2-19 had no effect on the expression of lytic activity in E. coli (Fig. 2, pRJ19H-X). However, any further deletion (>0.2 kb) from the XbaI site (pRJ19-A2, generated by exonuclease III deletion) resulted in the loss of lytic activity. These results led us to conclude that the lytic activity structural gene is located within the HindIII-XbaI fragment. Expression and characterization of the lytic activity in E. coli. S. aureus PS47 PCW (1 mg/ml) were suspended in 0.05 M KH2PO4-K2HPO4 buffer (pH 7.2), various amounts of cell extract from E. coli HB101 containing pBR322 and E. coli HB101 containing pRJ2-19 were incubated at 37°C, and the turbidity of the suspension was measured at intervals. No digestion of the PCW by the cell extract from the control strain without the cloned insert occurred. The cell extract of the cloned-insert-containing strain (1.0 mg of protein) showed rapid degradation of the PCW (80% loss of turbidity in 1 h). Lysis was more rapid in 0.05 M potassium buffer (pH 7.2) than 0.05 M Tris-HCl (pH 7.2) or boric acid-borax buffer (pH 7.5), and lytic activity was not stimulated by 25 or 50 mM MgCl2. Release of DNP-L-alanine, DNP-glycine, and reducing groups during digestion of PCW would indicate amidase, endopeptidase, and glucosaminidase activities, respectively. An incubation was set up with cell extract from strain HB101 containing pRJ2-19, and samples were taken for determination of amino groups and reducing groups. Cell extracts lacking PCW served as controls, and the values obtained from this incubation mixture were subtracted from those obtained from the PCW-containing mixture. No reducing groups were liberated, but amino groups were (Fig. 3), suggesting that we had cloned an amidase or an endopeptidase gene, but not a glucosaminidase gene. To identify the N-terminal amino acid released, tubes containing cell extract alone and cell extract plus PCW were set up, and the

FIG. 1. Plate test for lytic activity. Overnight cultures on LB agar medium were sprayed with chloroform and overlaid with 10 ml of soft agar containing 0.2% M. luteus cells. Plates were incubated at room temperature for 6 to 12 h, and lytic activity was shown by formation of a clear zone around colonies. (A) E. coli HB101 (pBR322); (B) E. coli HB101(pACYC184); (C) S. aureus PS47; (D) E. ccli HB101(pRJ2-19); (E) E. coli HB101(pRJ2-19E1).

JM109, with pRJ2-19 gave transformants that all expressed the lytic activity. This suggested that the insert encoded a lytic activity. E. coli recombinant clones showed over 100fold more lytic activity than S. aureus by the plate assay method (Fig. 1). This overproduction may be attributed to a gene dosage effect resulting from the cloning of the gene into the multicopy vector. Furthermore, chloroform may cause less release of lytic activity from S. aureus than E. ccli. Restriction analysis and subcloning of plasmid pRJ2-19. Various restriction enzymes were used to generate a physical map of the 7.4-kb insert (Fig. 2). No restriction sites were found for BamHl, Hindlll, Hpall, Kpnl, Sacl, Sall, Smnal, Sstl, or Xhol. Restriction endonuclease EcoRI digests of pRJ2-19 were ligated into the EcoRI site of the pACYC184 and pTZ19R cloning vectors. The ligation mixtures were used to transform competent F. coli HB101 or JM109, and transformants were screened for lytic activity. As shown in Fig. 2, strains bearing plasmids containing a 3-kb EcoRI fragment (pRJ2-19E1 and pRJ19E1) showed lytic activity,

7.4 Kbp










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a UJ

8 I



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' -C.)

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FIG. 2. Restriction endonuclease map of the 7.4-kb chromosomal DNA fragment inserted into the BamHI site of pBR322. The subclones were constructed into various plasmids as described in Materials and Methods. Each clone was tested for its ability to hydrolyze M. luteus whole cells. Symbols: +, lysis; -, no lysis. ---, pBR322 vector sequences.




700 4 600 2

cm z 500 Z


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Time (hr) FIG. 3. Digestion of S. aureus PCW by cell extract. PCW were suspended in KH2PO4-K2HPO4 buffer (pH 7.2) at a concentration of 10 mg/ml. To 4 ml of cell wall suspension, 600 ,ul of cell extract (200 ,ug of protein) from strain HB101 containing pRJ2-19 was added, and samples were removed at various intervals for the determination of turbidity at 580 nm (0) and the release of reducing groups (U) and free amino groups (A). No decrease in turbidity was seen with cell extract from HB101 (0).

supernatants were reacted with FDNB followed by hydrolysis. DNP-alanine was identified by thin-layer chromatography (Fig. 4). This indicated that we had cloned an N-acetylmuramyl-L-alanine amidase gene. In support of this, amino groups were released from peptidoglycan and NaOH-treated PCW and identified as DNP-alanine from NaOH-treated PCW. To determine the molecular size of the polypeptides

0~ 1













FIG. 4. Identification of DNP-amino acids by silica gel thin-layer chromatography. After dinitrophenylation and acid hydrolysis, DNP-amino acids were extracted by ether and analyzed by chromatography. The plate was first developed with solvent A (n-butanol-1% ammonia) at room temperature, and after drying it was developed with solvent B (chloroform-methanol-acetic acid, 85: 14:1) at 4°C. The figure shows the tracings of DNP-amino acids on the chromatogram. 1, DNP-lysine; 2, DNP-serine; 3, DNP-glycine; 4, DNP-alanine; 5, PCW; 6, cell extract; 7, cell extract plus PCW. Solid arrow, Direction of movement; open arrow, origin.

encoded by recombinant clones, electrophoretic analysis of proteins present in lysates was carried out by SDS-PAGE. Coomassie blue-stained gels did not show the synthesis of any fragment-encoded polypeptide by the recombinant clone (data not shown). Similar results were obtained when total E. coli proteins were labeled in vivo with [35S]methionine and analyzed by SDS-PAGE (data not shown). However, when the enzymographic techniques of Potvin et al. (22) were used to detect the lytic activity of renatured proteins previously separated by SDS-PAGE (containing 0.2% PCW or SDS-CW), a polypeptide of apparent Mr 23,000 showed a clearing zone (Fig. 5). The E. coli strain harboring the vector plasmid expressed no detectable lytic activity. DISCUSSION We have cloned a chromosomal fragment of S. aureus which is required for the synthesis of a lytic activity into a pBR322 vector in E. coli. The transfer of the recombinant plasmid into another E. coli strain resulted in production of lytic enzyme by all transformants. The lytic gene can be stably maintained and expressed in E. coli. In contrast, a decrease in expression of an S. aureus glucosaminidase gene in E. coli has been observed after a few subcultures (2). Deletion analysis of the plasmid showed that all the sequences required for the synthesis of lytic enzyme are located within a 2.5-kb segment of DNA. Subcloning of 3-kb EcoRI or 2.5-kb EcoRI-XbaI fragments in either orientation produced lytic activity in E. coli. This result suggests that the cloned gene contains its own promoter and is active in E. coli. However, unlike S. aureus, which releases lytic activity

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We are attempting to further localize the position of the amidase gene by DNA sequence analysis. Determination of the DNA sequence may give insights into the structure and function of the amidase gene. We are also attempting to construct S. aureus PS47 derivatives deficient in amidase production to determine its role in S. aureus pathogenicity. ACKNOWLEDGMENTS We thank Marcel Fernandez and Xin Wang for help in preparing the S. aureus library and Herman Brockman and Anthony Otsuka for critical reading of the manuscript. We thank John E. Gustafson for devising the staining procedure for the cell wall-containing gels. This work was supported by a grant-in-aid from the American Heart Association (Illinois affiliate) and an Organized Research grant from Illinois State University. FIG. 5. Zymogram of E. coli proteins separated by SDS-PAGE (15% polyacrylamide gel containing 0.2% SDS-CW as the substrate). After electrophoresis, proteins were renatured by treatment with 25 mM Tris-HCI (pH 8.0) containing 1% Triton X-100. The gel was stained with methylene blue solution (1% methylene blue in 0.01% KOH) prior to being photographed. Arrows show a clear zone due to active lytic enzymes. (A) S. aureus freeze-thaw extract; (B) E. coli HB102(pBR322); (C) E. coli HB1O1(pRJ2-19); (D) E. coli HB1O1(pACYC184); (E) E. coli HB1O1(pRJ2-19E1). Open arrow, Lysostaphin (used as a standard); solid arrows, lytic activity from

recombinant clones.

extracellularly, lytic activity in E. coli could only be detected after chloroform permeabilization or disruption of the cells, suggesting an intracellular or periplasmic location. This is analogous to the reported inability of some cloned S. aureus gene products to be released extracellularly by E. coli (2, 13, 25). SDS-PAGE was used to identify the insert encodedpolypeptide. Coomassie blue staining and in vivo labeling with [35S]methionine did not show synthesis of any insertspecific protein. This may be due to a low-level expression of the gene encoding lytic activity. This seems reasonable, as it is known that autolysins are potentially dangerous to the cells. Enzymographic studies showed that the recombinant E. coli produces lytic enzymes with apparent Mrs of 20,000 to 23,000 (Fig. 5). The molecular weights of these lytic activities are different from those of most lytic enzymes from staphylococci previously reported (2, 3, 11, 25, 27, 28, 32). A freeze-thaw extract of S. aureus contained several bands exhibiting lytic activity (Fig. 5, lane A). This does not necessarily and directly reflect the exact number of cell wall-lytic enzymes. It has been reported that posttranslational modifications such as glycosylation, nucleotidylation, and proteolytic processing can alter the molecular sizes of proteins without affecting their enzymatic activities (6, 14, 22). One of the active protein bands from S. aureus was of the same molecular weight as that produced by the recombinant E. coli. Immunoblot analysis with antibodies raised against purified recombinant lytic protein from E. coli will reveal whether the multiple lytic bands of S. aureus are related to each other. Substrate specificity studies showed that the lytic enzyme did not release reducing sugars but produced free amino groups. This suggests that either an amidase or an endopeptidase was cloned. Studies on the dinitrophenylation of N-terminal amino acids showed the release of DNP-alanine from the cell wall reaction mixture (Fig. 3). This result strongly suggests that we have cloned an amidase gene and not an endopeptidase gene. Amidase is one of the major autolysins of S. aureus (27, 30).

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30. 31.



34. 35. 36.

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Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

A gene of Staphylococcus aureus PS47 encoding lytic activity was cloned and expressed in Escherichia coli. Deletion analysis of a recombinant plasmid ...
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