Staphylococcus aureus Created by Tn917-lacZ Mutagenesis

JOURNAL OF BACTERIOLOGY, Mar. 1993, p. 1493-1499

Vol. 175, No. 5

0021-9193/93/0501493-07$02.00/0 Copyright © 1993, American Society for Microbiology

Isolation and Characterization of Autolysis-Defective Mutants of Staphylococcus aureus Created by Tn917-lacZ Mutagenesis NAGRAJ MANI, PHILIP TOBIN, AND RADHESHYAM K. JAYASWAL* Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, Illinois 61761-6901 Received 7 August 1992/Accepted 14 December 1992

Two autolysis-defective mutants (LytV1 and LytV2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants was negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage +11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring f3-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands.

Peptidoglycan hydrolases are a class of enzymes that are ubiquitous among bacteria (15). Some of these enzymes, responsible for cell lysis, are known as autolysins (40). Roles have been proposed for these enzymes in wall growth, cell separation, wall turnover, lysis initiated by cell wall-active antibiotics, competence for genetic transformation, formation of flagella, sporulation, and bacterial pathogenicity (2, 18, 33, 40). However, the involvement of Staphylococcus aureus autolysins in all of the above processes is speculative, as they have not been thoroughly investigated at the molecular or genetic level. Peptidoglycan hydrolases in S. aureus were first reported by Welsch and Salmon (51), and subsequent studies reported a variety of staphylococcal extracellular and intracellular lytic enzymes (19, 38, 43). Regulation of autolysin activity is believed to occur most commonly at the posttranslational level, including specific activation of the enzyme by substrate modification, topological restriction of enzyme distribution in the cell wall, and control at the site of export (18). Autolytic activity is stimulated by lysozyme (49), pancreatic RNase, cytochrome c, poly-L-lysine, cationic peptides (4, 17, 50), detergents (31), dodecylglycerol (44), proteolytic enzymes (45), and monovalent cations (8, 24), and it is inhibited by lipoteichoic acids (9). No information, however, is available on the regulation of the lytic gene(s) at the transcriptional level. Little information is available on the molecular mechanism of induction or repression of autolysins. A global regulatory system, agr, that controls the postexponential production of exoproteins in S. aureus has been identified (32). However, we found that an agr mutation had no effect on the synthesis and secretion of autolysins (20). An autolysis-deficient mutant of S. aureus was isolated *

and characterized with respect to cell wall turnover and sensitivity to penicillin G by Chatterjee et al. (7). The autolysin-defective bacterium showed penicillin tolerance, which seems to be of clinical relevance. However, the relationship between the production of autolysins and tolerance to antibiotics is still a matter of speculation. This mutant, unfortunately, is not available for further study. In this article, we describe the isolation and preliminary characterization of two S. aureus mutants defective in the production of major autolysins, as determined by zymogram. Transposon mutagenesis was used to isolate mutants because it has several advantages over chemical mutagenesis. We used transposon Tn917-lacZ, which has been shown to transpose to random sites in S. aureus (52). The gene fusion techniques originally developed for gram-negative bacteria (6) have also been used for gram-positive bacteria. Tn917-lacZ contains a promoterless lacZ gene within the transposon which, if inserted in the proper orientation downstream of the promoter of the mutated gene, creates a transcriptional lacZ fusion. Thus, the promoter activity of the mutated gene can be determined by measuring 3-galactosidase activity. Here, we also report the effect of growth conditions on mutant lyt gene expression. MATERIALS AND METHODS

Bacterial strains and cultures. S. aureus ISP2018 (a derivative of strain RN450 containing plasmid pTV32), used for transposon mutagenesis, was obtained from Peter Pattee, Iowa State University, Ames. S. aureus RN450 (8325-4; no prophage) was used for the construction of the genomic library and isogenic Lyt- strains. Phage 411 (26) was used in the transduction experiments. The S. aureus strains were grown in PYK medium (21), tryptic soy broth (Difco Laboratories, Detroit, Mich.) CY medium (27), or defined medium (42) at 30'C with shaking (200 rpm). When required,

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chloramphenicol at 20 pug/ml and erythromycin at 20 ,ug/ml were used. Transposon mutagenesis. Transposon Tn9l 7-lacZ is present in a plasmid, pTV32, which has a chloramphenicol resistance marker outside the transposon and a temperaturesensitive replicon, so that transposition can be forced to occur at 430C. To induce random transposition of the transposon, strain ISP2018 carrying plasmid pTV32 was grown on defined medium at 430C in the presence of erythromycin (Emr conferred by Tn917). Erythromycin-resistant colonies were screened for the autolysis-defective (Lyt-) phenotype by overlaying plates with soft agar containing 0.2% heatkilled, lyophilized S. aureus cells as the substrate, as described previously (21). After 8 to 12 h, mutants were scored for their inability to lyse by the presence or absence (Lyt-) of clear zones around the colonies. Lysis of whole cells. Cultures (50 ml) growing exponentially (optical density at 580 nm [OD580] of approximately 0.7) in PYK medium containing 1 M NaCl were collected by centrifugation (13,800 x g, 4°C, 10 min). Cells were washed twice with 50 ml of ice-cold water and resuspended in 50 ml of 0.05 M Tris-HCl (pH 7.2) containing 0.05% (vol/vol) Triton X-100 (Sigma Chemical Co., St. Louis, Mo.). The cells were incubated at 30°C with shaking, and the OD580 was measured at 30-min intervals for 3 to 5 h. Preparation of autolysins. (i) LiCl extracts. Autolytic enzymes were extracted from S. aureus with LiCl essentially as described by Qoronfleh and Wilkinson (30). Mid-exponential-phase cultures growing in PYK medium (500 ml) were collected, washed with ice-cold water, and resuspended in 5 ml of 3 M LiCl. The suspension was stirred for 10 min at 4°C. The cells were removed by centrifugation, and the supernatant was used to determine enzyme activity (autolysins) after dialysis overnight against 0.01 M KPO4 buffer (pH 7.2). (ii) Freeze-thaw extracts. The method used for the preparation of freeze-thaw extracts was essentially that of Huff et al. (19). Mid-exponential-phase cultures growing in PYK medium (500 ml) were collected by centrifugation, washed with ice-cold water as described above, and resuspended in about 10 ml of 0.05 M KPO4 buffer (pH 7.2). The cell suspension was placed in a freezer at -60°C. After 6 to 8 h, the cell suspension was thawed at room temperature, vortexed, and replaced in the freezer. This procedure was repeated twice, and the cells were finally removed by centrifugation (30,000 x g for 20 min at 4°C). The supernatant was used for the determination of lytic activity. (iii) Intracellular autolysins. Soluble autolytic activity present in the cytoplasm was isolated from the exponentially growing cultures after mechanical disintegration of the cells with a Bead-Beater (Biospec Products, Bartlesville, Okla.). The cells were removed by centrifugation (30,000 x g, 10 min, 4°C), and the supernatant was used for the determination of activity. (iv) Extracellular autolysins. Cultures were grown to the stationary phase in CY medium. The cells were removed by centrifugation, and the culture supernatant was concentrated 100-fold by ultrafiltration with an Amicon YM-10 membrane. This concentrated supernatant was used for exoprotein and autolysin analyses. (v) Isolation of cell walls. Purified cell walls and crude cell walls (CCW) were prepared from exponential-phase cultures of S. aureus strains grown in PYK medium as described by Jayaswal et al. (21). Enzyme assays. (i) Autolytic activity. To determine autolytic activity, S. aureus CCW were resuspended (1 mg [dry weight]/ml) in 1 ml of 0.05 M KH2PO4-K2HPO4 buffer (pH

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7.2); various amounts of enzyme extracts were added and incubated at 370C. Turbidity was measured at 30-min intervals at 580 nm (21). Protein concentration was determined by the method of Bradford (5).

(ii) 13-Galactosidase activity. P-Galactosidase was assayed

at 30'C by the method of Miller (25) with o-nitrophenyl-pD-galactopyranoside (ONPG; Sigma Chemical Co.) as the substrate. The cells were permeabilized by chloroform treatment. The reaction was stopped by the addition of 0.5 ml of 1 M sodium carbonate, and the OD420 was determined after the removal of cell debris. P-Galactosidase specific activity was determined according to the formula: Miller units of P-galactosidase = (OD420) (1,000)/(OD600) (1) (V), where T is the reaction time and V is the volume of cells assayed (25). Detection of lytic activity in SDS-PAGE gels. The lytic activity of enzyme extracts was determined on a 15% polyacrylamide-sodium dodecyl sulfate (SDS) gel containing 0.2% (wt/vol) crude cell walls from S. aureus as described by Leclerc and Asselin (23). The enzyme extracts were mixed with loading buffer and heated for 3 min in a boiling-water bath prior to polyacrylamide gel electrophoresis (PAGE). The gels were incubated for 12 to 16 h at 370C in 25 mM Tris-HCl (pH 8.0) containing 1% Triton X-100 to permit protein renaturation. Bands with lytic activity were observed as clear zones in the opaque gel. Gels were stained with 1% methylene blue in 0.01% KOH prior to photography. For protein analysis, SDS-15% PAGE gels were used, and bands were visualized after staining with Coomassie blue dye. Construction of isogenic Lyt- strains. Strain RN450 was transduced with phage +11, which was propagated on the Lyt- mutants as described by Novick (27). Strain RN450 was infected with phage (>11 (transducing phage) in a ratio of 1:1. The mixture was plated on tryptic soy agar (TSA) plates containing 0.05 M CaCl2 and incubated at 37°C. After 4 h, the plates were overlaid with 4 ml of soft agar (0.7% TSA containing 0.1 M sodium citrate and 20 ,ug of erythromycin per ml) and incubated for an additional 12 h. Erythromycinresistant transductants were purified and used for further characterization. Southern blot analysis. For Southern blot analyses (34), chromosomal DNA was isolated as described earlier (21) and digested with KjnI and SalI. After electrophoresis on a 0.7% agarose gel, the fractionated DNA fragments were transferred to a nylon membrane and probed with a Sail fragment (Tn917-lacZ) from plasmid pTV32 labeled with [32P]dCTP. Other procedures. The phage propagation and transduction and other genetic techniques used were as described by Novick (27). DNA digestion, Southern blotting, and random labeling of DNA probes were performed as described by Sambrook et al. (34).

RESULTS Isolation of autolysis-defective mutants. Among 6,000 erythromycin-resistant colonies screened for the autolysisdefective phenotype, two mutants were found in which the insertion of the transposon resulted in the loss of a clearing zone around the colonies. These mutants were designated Lyt-l and Lyt-2 and were used for further studies. Growth rate and cell division. Peptidoglycan hydrolases have been proposed to play an important role in cell morphogenesis. To determine whether the Lyt-1 and Lyt-2 mutants were impaired in morphogenesis, their growth characteristics were measured and microscopic studies were performed. As shown in Fig. 1, the generation times of the

AUTOLYSIS-DEFECTIVE MUTANTS OF S. AUREUS

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Time (h) mutants were comparable to that of the parent strain RN450 in PYK and defined media. However, autolysis did not occur in the mutants after the end of exponential growth. Cell division and cell separation seemed to be normal when observed under the microscope. It has been reported earlier (7) that autolysis-deficient mutants of S. aureus form clumps, but we have not seen any clumping of our mutants. The tolerance of the mutants to NaCl was identical to that of the wild type. Lysis of whole cells. When S. aureus RN450 and erythromycin-resistant (Lyt+) strains grown in PYK medium containing 1 M NaCl were resuspended in 0.05 M Tris-HCI buffer (pH 7.2) containing 0.05% Triton X-100, lysis was observed. However, under the same conditions, the autolysis-deficient mutants did not show any significant lysis (Fig. 2). Cell wall-bound lytic activity. To measure cell wall-bound lytic activities, cells in the mid-exponential growth phase were collected, and enzymes were extracted by the freezethaw and LiCl methods as described in Materials and Methods. Lytic activities were measured with CCW of S. aureus. As shown in Fig. 3, reaction mixtures containing enzyme

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FIG. 2. Autolysis of whole cells of S. aureus. Mid-exponentialphase cultures were resuspended in 0.05 M Tris-HCl (pH 7.2) containing 0.05% Triton X-100 and incubated at 30°C. The changes in A580 were determined as described in Materials and Methods. Symbols: [1, RN450; *, Lyt-l; *, Lyt-2; 0, Lyt+.

FIG. 3. Lytic activity in freeze-thaw extracts of S. aureus. Freeze-thaw extracts were mixed with CCW, and changes in A580 were measured at various times. A reaction mixture lacking enzyme extract (control) showed a rate of change in OD580 similar to that of the Lyt- mutants. Because of too many overlapping data points, the control curve has been omitted. Symbols: , RN450; U, ISP2018; *, Lyt-1; *, Lyt-2.

extracts from the Lyt- mutants showed the same rate of change in OD580 as the reaction mixture lacking enzyme extract (data not shown). Under the same conditions, enzyme extracts from the parent RN450 and Lyt' strains lysed CCW (as shown by a decrease in OD580) significantly faster than did the control. These results indicate that the Lytmutants were deficient in cell wall-bound lytic activities, as they were unable to degrade CCW preparations. Intracellular lytic activities. It was reported earlier that most of the autolytic activity of S. aureus is located in the cytoplasm (37). To rule out the possibility that the Lytphenotype of the mutants is due to a defect in enzyme translocation from the cytoplasm to the cell wall, the intracellular concentration of lytic enzyme was determined. Cells in mid-exponential-phase cultures were collected, homogenized with a Bead-Beater, and centrifuged, and the supernatant was used for lytic activity measurement. Lytic activity was undetectable in the cell extracts of Lyt- mutants but was detectable in those of the parent strain (data not shown). The data suggest that the mutants also lack intracellular lytic activity. However, a single lytic enzyme band was detected in the intracellular extracts of Lyt- mutants (Fig. 6, lanes 3 and 4). Analysis of the protein profiles of the intracellular extracts from the mutants and parent strains by SDS-PAGE did not show any obvious difference (Fig. 4, lanes 3 through 6). Extracellular lytic activities. Some S. aureus peptidoglycan hydrolases are secreted into the growth medium (43). To determine the effect of the Lyt mutation on extracellular lytic activity, the spent medium was concentrated by Amicon filtration (YM10), and lytic activity was determined with SDS-PAGE gels containing CCW of S. aureus. The medium from the Lyt- strains showed negligible activity compared with that from the Lyt+ strain. This result does not appear to be due to the inability of Lyt- mutants to secrete proteins, because these mutants secreted most of the proteins secreted by the parent strain (Fig. 4, lanes 1 and 2). Three protein bands were missing in the Lyt- strain compared with the wild type when stained with Coomassie blue dye.

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FIG. 4. Protein analysis by SDS-15% PAGE. Enzyme extracts prepared from various S. aureus strains were separated by SDS15% PAGE, and proteins were visualized after staining with Coomassie blue dye. Equivalent amounts of protein were loaded in lanes 1 and 2 and in lanes 3 through 6 so that they can be compared. Lanes: 1, extracellular extract of Lyt+; 2, extracellular extract of Lyt-; 3, intracellular extract of Lyt-2; 4, intracellular extract of Lyt-1; 5, intracellular extract of ISP2018; 6, intracellular extract of RN450; 7, size marker proteins (sizes shown in kilodaltons). The arrows show the missing protein bands in the extracellular extract of Lyt-1.

Whether these bands correspond to any lytic enzyme bands remains to be determined. Detection of lytic activity in SDS-PAGE gels. The zymographic method used to detect peptidoglycan hydrolase activity is more sensitive than the in vitro assay for CCW degradation. Therefore, to determine whether any residual lytic activity was present in the Lyt- mutants, enzyme extracts prepared by different methods were analyzed for enzymatic activity in situ by using an SDS-PAGE gel containing 0.2% crude cell walls from S. aureus. LiCl extracts of the parent strain showed multiple lytic enzyme bands (Fig. 5B, lanes 1 to 4), but only a single lytic band was detected in the extracts of mutants (Fig. 5B, lanes 5 and 6). The parent A kDa

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364 FIG. 5. Zymogram of S. aureus extracts prepared by the (A) freeze-thaw method and (B) LiCl method and separated by SDSPAGE with 0.2% SDS-treated CCW. Equivalent amounts of protein were loaded in each lane. After electrophoresis, proteins were renatured by treatment with 25 mM Tris-HCl (pH 8.0) containing 1% Triton X-100. The gel was stained with methylene blue before being photographed. Lanes: 1, NCTC 8325; 2, RN450; 3, ISP2018; 4, Lyt+; 5, Lyt-1; 6, Lyt-2. The arrows show the lytic enzyme bands of Lyt mutants. Prestained size markers are macroglobulin (180 kDa), 0-galactosidase (116 kDa), fructose-6-phosphate kinase (84 kDa), fumarase (48 kDa), and lactic dehydrogenase (36 kDa).

FIG. 6. Intracellular and extracellular lytic enzyme profile of S. aureus strains on an SDS-15% PAGE gel containing 0.2% SDStreated CCW. Equivalent amounts of protein were loaded in lanes 1 to 4 (intracellular extracts) and lanes 5 and 6 (extracellular extracts) so that they can be compared. After electrophoresis, proteins were renatured and stained with methylene blue as described in Materials and Methods. Lanes: 1, RN450; 2, ISP2018; 3, Lyt-1; 4, Lyt-2; 5, Lyt-1; 6, Lyt'. Size markers (in kilodaltons) are shown on the left.

strains (Fig. 5A, lanes 1 to 4) showed multiple bands of lytic activity, but mutant strains showed a single band of lytic activity in freeze-thaw extracts (Fig. 5A, lanes 5 and 6). The amount of protein analyzed for the mutants was about one-third of that analyzed for the parent strain. The reason for this poor extraction of proteins from the mutants is not known. Although the intracellular extracts prepared from the mutants did not show any detectable change in optical density when incubated with a CCW preparation of S. aureus, a single lytic enzyme band was observed in the zymogram of these extracts (Fig. 6, lanes 3 and 4). The parent strain (cell extract), on the other hand, showed multiple lytic enzyme bands (Fig. 6, lanes 1 and 2). Likewise, exoproteins prepared from the mutants did not show any lytic activity when assayed in vitro, but a single lytic enzyme band was detected in the exoproteins of the Lytmutants (Fig. 6, lane 5). These results show that the in situ gel assay is more sensitive than the in vitro liquid assay. Insertional inactivation of lysis genes. The mutant DNA was analyzed by Southern blotting to show the presence of a single copy of the transposon in the bacterial chromosome. DNA from the mutants, digested with KpnI and EcoRI and probed with the Sail fragment of Tn917-lacZ, showed 9.5and 10.3-kbp hybridizing fragments. This indicated the insertion of a single copy of the transposon into the bacterial

chromosome. To prove that the Lyt- phenotype is due to transposon insertion, we mobilized the erythromycin marker from the mutants into another strain by using phage 411. Analyses of the erythromycin-resistant transductants showed no autolysis or autolytic activity in in situ or in vitro assays, indicating that the Lyt- phenotype was linked to the transposon. Sensitivity of CCW of mutant strains to wild-type autolysins. To determine whether the mutations had any effect on the susceptibility of cell walls to autolysins, CCW from the Lyt- mutants were incubated with freeze-thawed autolysin extracts from the wild-type strain. The rates of degradation of the CCW of the mutant and wild-type strains were similar (data not shown), suggesting that there was no significant alteration in the mutants' cell wall structure and composition. Sensitivity of Lyt- mutants to penicillin. The effects of penicillin G on the growth and viability of Lyt-1 and wild-type S. aureus strains were compared. The MIC of penicillin G for both strains was identical (0.25 ,ug/ml). As shown in Fig. 7, addition of penicillin (1 ,ug/ml) to growing cultures caused the growth of both the mutant and the

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Time(h) FIG. 7. Effect of penicillin G on the growth of S. aureus. Penicillin (1 pug/ml) was added to exponentially growing cultures, and the A580 was recorded at various times. Symbols: U, ISP2018; i, ISP2018 plus penicillin;+, LytV1; N, Lyt-i plus penicillin.

wild-type strain to cease. Lysis of cultures was not observed until 8 h; subsequently, the wild-type strain lysed but the Lyt- strain did not. We determined the viability of the mutants and the wild-type strain at various times during penicillin treatment. The number of CFU in the exponential and early stationary phases was same for the three strains, but in the late stationary phase, the CFU of the wild-type strain declined compared with those of the mutant strains (data not shown). lyt-IacZ gene fusion studies. Both mutants have the transposon Tn917-lacZ inserted in the appropriate orientation for gene expression studies (as indicated by the blue color on plates containing X-Gal [5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside]). We measured P-galactosidase activity during the growth cycle by the calorimetric assay (25) and found that the activity increased twofold during the exponential phase and declined during the decline phase of bacterial growth. We also measured the f3-galactosidase activity of Lyt- mutants during whole-cell autolysis. When actively growing cells of a Lyt- mutant were resuspended in autolysis buffer (50 mM Tris-HCl [pH 7.2] containing 0.05% Triton X-100), no change in OD580 was observed, but an 80% decline in 3-galactosidase activity was found (data not shown). These results suggest that modification or relocation rather than induction of existing enzymes may be occurring during autolysis. Growth conditions can influence the production of autolysins in S. aureus (36). Therefore, the activity of the mutated gene was determined under various growth conditions. Addition of NaCl to the growth medium has been shown to stimulate autolysis (16). As shown in Fig. 8A, addition of NaCl (0.5 to 2.5 M) induced P-galactosidase activity, and maximum induction (threefold) was seen at 1.5 M NaCl. Similarly, induction was seen with sucrose (data not shown). However, KCl had no effect on gene expression. The pH of the growth medium also affected gene activity. Acidic pH favored gene induction, and maximum activity was observed at pH 6.0 (Fig. 8B). The final pH of the growth medium at the time of 13-galactosidase measurement was about 7.0 in all cases. Autolysins extracted from cells grown at various pHs showed remarkable differences in

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P-galactosidase activity. (A and B) Cells were grown in PYK medium at 300C for 12 h and washed with Z buffer (25) before 3-galactosidase activity measurement. (C) Cells were grown in PYK medium for 12 h at various temperatures, and 13-galactosidase activity was determined.

specific activity and molecular forms (39). Gene expression was also modulated by temperature. The 13-galactosidase activity of cultures grown at 30, 37, and 420C was measured. The activity was highest at the lowest temperature tested (30'C) (Fig. 8C). Anaerobic conditions had no observable effect on P-galactosidase activity. Thus, the gene activity seems to be regulated by NaCl, sucrose, pH, and temperature.

DISCUSSION We have isolated two autolysis-deficient mutants of S. aureus by transposon mutagenesis. Both mutants showed normal growth and cell division but lacked autolysis. The autolysis-deficient phenotype in the plate assay was not due to the inability of the mutants to secrete lytic enzymes. Lytic activities, determined by a decrease in OD58 of CCW, were insignificant in enzyme extracts prepared by the freeze-thaw and LiCl methods. Also, the concentrations of enzymes inside and outside the cells were negligible. These results suggested that the Lyt- phenotype was due to a defect in enzyme synthesis rather than a defect in secretion. Southern blot analysis of Lyt-1 and Lyt-2 DNAs mutant digested with Kpnl and EcoRI showed similar hybridization patterns, which indicated that a single copy of the transposon was inserted in both mutants, probably at the same site on the chromosome. Construction of isogenic Lyt- mutants by phage +11 transduction further supports our hypothesis that the insertion of the transposon is responsible for the Lyt- phenotype of the mutants. Several forms of autolysins have been found by SDSPAGE in lyophilized cells of Micrococcus luteus (21, 23, 29, 38). It has been proposed that posttranslational modifications such as glycosylation, nucleotidylation (10), and proteolytic processing are responsible for the generation of different forms of autolytic enzymes. Recently, proteolytic processing of autolytic enzymes was shown to occur in

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Bacillus megaterium as a means of regulation by activation from latent precursors (13). Cloned autolysins of grampositive bacteria expressed in Eschenichia coli also gave multiple bands (12, 23, 29), which suggests the occurrence of proteolytic processing of autolysins. However, the number of genes responsible for the production of these different forms of lytic enzymes is not known. Autolysis-deficient mutants of S. aureus (7), Streptococcus pneumonieae (35, 41), and Bacillus subtilis (11, 46) have been reported. Recently, lytic enzyme bands have been correlated with specific autolysis mutations in B. subtilis (12). It has been reported that the Lyt- phenotype in S. aureus was due to the lack of glucosaminidase activity (28). We have shown, by zymographic analyses of enzyme extracts from mutants, that a single mutation can abolish most forms of lytic activity (Fig. 5 and 6). A survey of the literature indicates that this is the first report on the isolation and characterization of such mutants. The data presented in this article suggest that the transposon insertion occurred in a gene which probably regulates the expression or processing of autolysins in S. aureus. We are unable to say from the present data whether the mutation is in a structural gene or in a gene whose product could be involved in autolysin processing or whether we have mutated a gene which regulates autolysin gene expression. The major loss of autolytic activity without any effect on the cell growth and development of the mutated strains suggests a redundant or nonessential role for the missing autolysins. We speculate that the single lytic enzyme band present in the mutants may be vital for cell morphogenesis. Autolysins have been known for quite some time, and various factors affecting lytic activity have been reported for a variety of bacteria (18). However, no information is available on the regulation of autolysin genes at the transcriptional level. Since both the Lyt- mutants had the transposon (Tn917-lacZ) inserted in the proper orientation, the effect of environmental factors on the expression of the mutated gene was determined by measuring the activity of the P-galactosidase reporter gene. Addition of NaCl to the growth medium increased the 3-galactosidase activity, suggesting that Na' ions, by some unknown mechanism, induce the mutated gene (Fig. 8A). This induction did not occur when K+ ions were substituted for Na' ions. Bierbaum and Sahl (4) have proposed that activation was probably due in some cases to the displacement of autolysins bound to teichoic acid in the walls. The precise mechanism by which NaCl stimulated autolysis remains a mystery. The modulation of the mutated gene activity by the pH, temperature, sucrose concentration, and ionic strength of the medium indicated that the autolysins are regulated by environmental stimuli. We also compared the gene fusion data on the effects of these factors with those for the wild-type S. aureus strain, and the effects were found to be similar (39). Gene cloning and nucleotide sequencing techniques have greatly facilitated structure-function analyses of genes. The genes for Streptococcus pneumoniae amidase (14), Streptococcus faecalis autolysin (1), an amidase of B. subtilis (12, 22), and an unidentified Bacillus sp. autolysin (29) have been cloned. So far, two peptidoglycan hydrolase genes from S. aureus have been cloned and analyzed (3, 21, 47, 48). To obtain a better understanding of the mechanism of autolysis and to characterize the gene product, we are currently cloning the mutated gene.

J. BACTrERIOL.

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