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Autolysins in bacteria are peptidoglycan hydrolases with roles in growth, turnover, and cell lysis. LytM was identified as the only autolysin in a previously reported autolysis-deficient (lyt−) strain of Staphylococcus aureus. Purified LytM has been studied in great detail for its lytic properties and its production is elevated in vancomycin resistant S. aureus. However, the postulated roles of LytM in S. aureus are largely speculative. Studies utilizing a reporter strain where the lytM promoter was cloned in front of a promoterless lacZ gene and fused in S. aureus strain SH1000 suggest that the expression of lytM is highest during the early exponential phase. Additionally, lytM expression was down-regulated in agr− mutants. Expression of lytM was not affected by the presence of cell wall inhibitors in the growth medium. To further determine the significance of LytM in staphylococcal autolysis, the gene encoding LytM was deleted by site directed mutagenesis. Deletion of lytM, however, did not alter the rate of staphylococcal cell autolysis. Surprisingly, when the lytM mutation was combined with the lyt− mutant, the lytic activity band of the lyt− strain was still apparent in the lytM:lyt− double mutant. Purified full-length His-tagged LytM did not demonstrate any lytic activity against S. aureus cells. Surprisingly, cultures of S. aureus lytM deletion mutant lysed at a significantly faster rate compared to the wild-type S. aureus in the presence of oxacillin. Findings of this study raise questions about LytM as an autolysin and the significance of this protein should thus be investigated beyond its role as an autolysin.
Staphylococcus aureus is an aggressive pathogen that is responsible for a wide array of diseases ranging from pyogenic skin infections and food poisoning to complicated life-threatening diseases such as bacteremia and endocarditis (Plata et al., 2009). The emergence of multidrug-resistance in S. aureus is generating enormous public health concern and an urgent need for alternative therapeutic targets for infections caused by this bacterium.
Peptidoglycan hydrolases are enzymes that hydrolyse the peptidoglycan of the bacterial cell wall. These enzymes in S. aureus include N-acetyl muramidase, N-acetyl glucosaminidase, N-acetylmuramyl-L-alanine amidase and endopeptidase (Ingavale et al., 2003; Ramadurai et al., 1999). Cellular levels and activities of autolysins are believed to be intricately regulated and these enzymes are proposed to play key roles in bacterial cell wall metabolism, daughter-cell separation, antibiotic mediated cell lysis and pathogenicity (Ingavale et al., 2003; Ramadurai et al., 1999).
LytM was identified and proposed to be the only autolysin present in a previously reported autolysis-defective lyt− mutant strain of S. aureus (Mani et al., 1993; Ramadurai & Jayaswal, 1997). LytM is suggested to be a lysostaphin-type peptidase that is found mostly in bacteria and bacteriophages and are believed to be glycyl-glycine endopeptidases (Bochtler et al., 2004; Ramadurai & Jayaswal, 1997; Sugai et al., 1997). Glycyl-glycine peptide bonds are involved in cross-linking peptidoglycan in many Staphylococcus species including S. aureus (Schleifer & Kandler, 1972). These lysostaphin-type peptidases have similar active sites and share a core folding motif but they have highly divergent folds (Bochtler et al., 2004). The presence of endopeptidases in gram-positive bacteria such as Bacillus subtilis and many gram-negative bacteria that lack glycyl-glycine peptidoglycan cross links suggests additional roles for these enzymes beyond peptidoglycan hydrolases (Bochtler et al., 2004).
LytM has been studied extensively for its lytic properties in recent years. The protein has been crystallized and its active site domains has been mapped (Firczuk et al., 2005; Odintsov et al., 2004). In addition, LytM production has been shown to be elevated in vancomycin-resistant S. aureus (Pieper et al., 2006; Renzoni et al., 2006).
In this study, the expression pattern of lytM during stages of bacterial growth and the significance of LytM as an autolysin were investigated. LytM was determined to be an early exponential phase protein and the expression of lytM was determined to be down-regulated by Agr. This study however, raises questions about the physiological role of this protein as an autolysin and suggests that the significance of this protein should be investigated beyond its role as an autolysin.
The bacterial strains and plasmid constructs used in this study are shown in Table 1. S. aureus and Escherichia coli cells were routinely grown aerobically at 37°C in tryptic soy broth/agar (TSB/TSA; Beckton Dickinson) and Luria-Bertani broth/agar (LB/LBA; Fisher), respectively. Broth cultures were grown in a shaking incubator (220 r.p.m.) unless stated otherwise. When needed, ampicillin (50 μg ml−1), tetracycline (10 μg ml−1), erythromycin (10 μg ml−1), and chloramphenicol (10 μg ml−1) were added to the bacterial growth medium.
Plasmid DNA was isolated using the Qiaprep kit (Qiagen Inc.); chromosomal DNA was isolated using the DNAzol kit (Molecular Research Center) from lysostaphin (Sigma) treated S. aureus cells as per the manufacturer’s instructions. All restriction and modification enzymes were purchased from Promega. DNA manipulations were carried out using standard procedures. PCR was performed with the PTC-200 Peltier Thermal Cycler (MJ Research). Oligonucleotide primers (Table 2) were obtained from Sigma Genosys.
For this study, the lytM nucleotide sequence was obtained from the http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=610 database that suggests an additional 18 nucleotides at the 5′-end to be part of the lytM gene compared to what has been suggested by others (Ramadurai & Jayaswal, 1997; Ramadurai et al., 1999). To create a lytM deletion mutant, a set of two primers, P1 and P2, were used to amplify a 1083 bp DNA fragment using genomic DNA extracted from S. aureus strain SH1000 as template. This amplicon represented 192 nt of the 5′-end and additional DNA upstream of the lytM gene. Primers P3 and P4 were used to amplify an 834 bp DNA fragment that represented 68 nt of the 3′-end of the lytM gene and additional downstream region. These two fragments were cloned individually in to plasmid pGEMT (Promega) and subsequently ligated together in plasmid pTZ18R (Mead et al., 1986) resulting in the construct pTZ-lytM that simultaneously generated a unique BamH1 restriction site between the ligated fragments. A 2.2 kb tetracycline resistance cassette was subsequently inserted at this BamH1 site resulting in the construct pTZ-lytM-tetM which was used as a suicidal construct to transform S. aureus RN4220 cells by electroporation (Schenk & Laddaga, 1992). Selection of the transformants on tetracycline plates led to the integration of the entire construct into the chromosome. Phage-80α was propagated on these transformants and used to resolve the mutation in the lytM gene in the S. aureus strains by performing transductional outcrosses as described (Singh et al., 2001b; Singh et al., 2007; Singh et al., 2008). Mutation in the lytM gene was subsequently transduced into the S. aureus lyt− strain (Mani et al., 1993; Ramadurai & Jayaswal, 1997) to potentially create an autolysin free lyt−:lytM double mutant.
For genetic complementation of the lytM mutant, an approximately 2.2 kb DNA fragment was PCR amplified using primers P5 and P6 and S. aureus SH1000 genomic DNA as template. This amplicon represents a fragment starting 890 nt upstream and ending 364 nt downstream of the lytM gene that was cloned in to the BamHI and HindIII sites of shuttle plasmid pCU1 (Augustin et al., 1992) and subsequently transferred to a lytM mutant of S. aureus SH1000.
Mid-exponential phase cultures (OD600 = 0.6) were diluted 50-fold in a nephelo culture flask (Wheaton) containing 50 ml fresh TSB with a flask-to-medium volume ratio of 6:1 and growth was followed by measurement of optical density spectrophotometrically at 600 nm. In another experiment, cultures pre-grown to an OD600 = 0.5 were added with oxacillin at a final concentration of 15 μg ml−1 and subsequent growth was measured spectrophotometrically.
Primers P7 and P8 were used to amplify a 1223 bp DNA fragment using genomic DNA from S. aureus SH1000 as template. This amplicon represents the upstream and 23 nt of the 5′-end of the of the lytM gene. The amplicon was cloned in the correct orientation upstream of a promoterless lacZ gene of vector pAZ106 (Chan et al., 1998) and was introduced into the chromosome of S. aureus RN4220 by electroporation with selection on erythromycin. Phage 80α lysate of the resulting transformant was used to transduce the lytM promoter:lacZ fusion into strain S. aureus SH1000 and its derivative agr mutant (Shenkman et al., 2001). A single copy insertion of the fusion in the chromosome was confirmed by Southern blot analysis. The activity of β-galactosidase in the reporter strain was assayed using O-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate as described previously (Singh et al., 2001a; Singh et al., 2001b).
The lytM open reading frame was PCR amplified using primer pairs P9 and P10 and S. aureus SH1000 genomic DNA as the template. The amplified lytM gene was cloned in frame at the BamHI and HindIII sites of the overexpression vector pRSETa (Invitrogen) to produce pRSETa-lytM which was then transferred into E. coli BLR(DE3) pLysS (Novagen). The resulting transformants were grown in LB containing ampicillin (50 μg ml−1), chloramphenicol (30 μg ml−1), and tetracycline (12 μg ml−1) to OD600 of 0.4 and induced for the synthesis of His-tagged LytM by the addition of 2.5 mM of isopropyl-β-thiogalactopyranoside (IPTG) for 2.5 h. The induced culture was harvested and resuspended in 50 mM Tris-HCl buffer (pH 7.5), sonicated, and centrifuged. The supernatant fluid was applied to a nickel-charged agarose affinity column and eluted with 400 mM imidazole using the Xpress Purification system (Invitrogen). Fractions containing the overexpressed His-tagged LytM were pooled, dialyzed, and concentrated against 50 mM potassium phosphate buffer, pH 7.2.
Autolysis assays were performed as previously described (Singh et al., 2008). Briefly, wild-type and the lytM mutant cultures of S. aureus were grown to an OD600 of 0.7 at 37°C in PYK medium (0.5% Bacto peptone, 0.5% yeast extract, 0.3% K2HPO4, pH 7.2). After one wash with cold water (8,500×g, 4°C, 15 min), cells were suspended in 0.05 M Tris-HCl buffer, pH 7.2, containing 0.05% Triton X-100 to a OD600 of 1.0. Cell suspension was incubated in flasks at 37°C with shaking (125 r.p.m.) and autolysis was determined by measuring decline in the turbidity spectrophotometrically at 600 nm every 30 min. Autolysis was also analyzed using a zymographic procedure as described previously (Singh et al., 2008). The total autolysins were extracted after bead beating bacterial cells in 0.25 M phosphate buffer (pH 7.2) using a BioSpec Mini-Beadbeater after growth in PYK to an OD600 = 0.7. Purified His6-LytM, extracts from E. coli cells overexpressing His6-LytM, and S. aureus bead-beated cell free extract were analyzed for the presence of autolysins in a zymographic method using autoclaved S. aureus 8325-4 cells as described previously (Singh et al., 2008).
To construct a mutation, lytM upstream and downstream flanking regions were PCR amplified and sandwiched with a tetracycline resistance cassette in plasmid pTZ18R. This construct was used to replace the wild-type lytM gene in S. aureus chromosome by double homologous recombination. This mutant represents a deletion of 706 nt of the 966 nt lytM gene. In PCR assays, primers P9 and P10 amplified a ~1.0 kb lytM region when the genomic DNA from the wild-type S. aureus was used as the template (Fig. 1, lane 1) as compared to a ~2.5 kb amplicon when genomic DNA from the lytM mutant strain was used as template (Fig. 1, lane 2). The mutation in lytM gene was also confirmed by Southern blot analysis (data not shown).
Deletion of LytM was investigated for any impact on the growth of S. aureus in TSB or in modified TSB to impose stresses such as acidic stress (pH 5.5), alkaline stress (pH 9.0), or salt stress (TSB added with additional 1.5 M NaCl). No growth defect was observed whether the lytM mutants used were in S. aureus strain SH1000 or 8325-4 (data not shown). Surprisingly, the presence of oxacillin led to increased lysis of mid-log phase lytM mutant cells compared to a culture of wild-type S. aureus 8325-4 cells under identical conditions (Fig. 2). To verify if this was indeed the lack of a functional LytM that is responsible for oxacillin induced lysis, the mutant was complemented with the lytM gene under its own promoter in trans on plasmid pCU1. As evident in Fig. 2, the level of resistance to oxacillin induced lysis was restored in the complemented strain.
Expression of lytM was monitored using lytM promoter - lacZ fusion in S. aureus SH1000. An overnight culture of the reporter strain was diluted (1:100) in a 300 ml flask containing 40 ml of fresh TSB and incubated with shaking at 37°C. The bacterial cells were harvested at 120, 210, 300, 440 and 560 min and the level of β-galactosidase activity was determined. The level of β-galactosidase was reflective of the lytM promoter activity. The highest lytM expression was determined in cells from early to mid exponential phase and this activity declined during the late-exponential phase and was lowest during the stationary phase of growth (Fig. 3A). A higher expression of lytM was also observed in S. aureus cells from early-to-mid exponential phase of growth in a real time RT-PCR assay (data not shown). This observation is consistent with a prior report showing increased lytM transcript levels in early exponential phase S. aureus cells (Ramadurai & Jayaswal, 1997). It was also reported by Ramadurai et al. (1999) that the transcription of lytM was suppressed in the agr mutant cells of S. aureus. In this study also, we observed a noticeable decrease in the expression of lytM in an agr mutant of S. aureus SH1000 compared to the wild-type SH1000 (Fig. 3B). The lytM gene, however, was not identified as a gene regulated by Agr in transcriptional profiling studies that compared gene expression in agr mutant relative to their wild-type parent (Cassat et al., 2006; Dunman et al., 2001). It is possible that in these studies the level of lytM regulation was below the cut-off set for the Agr regulated genes.
Considering the role of LytM as a peptidoglycan hydrolase and its abundance in cells resistant to vancomycin (Mongodin et al., 2003; Pieper et al., 2006), lytM expression was also determined in cells stressed with various cell wall inhibitors. The cells were allowed to grow to a density of 0.6 and at this point the cell wall inhibitors were added at final concentrations of 5 μg ml−1. The cells were allowed to grow for 60 min with these antibiotics and the level of β-galactosidase was subsequently determined. There was no real growth inhibition in cultures growing in the presence of vancomycin and bacitracin in 60 min, but with the other antibiotics there was about 20–30% growth inhibition relative to lytM reporter culture without the addition of any antibiotic. There was no appreciable change, however, in the level of β-galactosidase in these antibiotic stressed cells suggesting that the expression of lytM is not affected when S. aureus cells are challenged with cell wall-active antibiotics (data not shown). This observation is consistent with the prior report that did not identify lytM as a gene with altered expression in S. aureus cells challenged with cell wall-active antibiotics (Utaida et al., 2003).
The autolysis subsequent to mutation in the lytM gene in S. aureus was initially investigated in strain SH1000. However, no difference in the autolysis of the lytM mutant cells of S. aureus strain SH1000 was observed compared to the autolysis of the wild-type SH1000. We consistently observe slower rate of autolysis of the SH1000 cells compared to S. aureus 8325-4 cells. To see if there was any impact of LytM deletion on S. aureus autolysis, the lytM mutation was transferred to the S. aureus strain 8325-4 and the lyt− transposon mutant of strain 8325-4. There was no appreciable difference in the autolysis of the lytM mutant cells of strain 8325-4 relative to wild-type 8325-4 (Fig. 4). Additionally, no autolysis was observed in case of lyt− and lyt−:lytM double mutant during the course of experiment (5 h) when autolysis was measured periodically (Fig. 4). The turbidity of lyt− and lyt−:lytM cell suspension remained unchanged even after 24 h (data not shown).
In zymographic investigations, several lytic-activity bands were seen in samples from the wild-type S. aureus strain 8325-4 (Fig. 5, lane 1). The pattern of autolytic bands was almost identical in samples from the lytM mutant of S. aureus strain 8325-4 (Fig. 5, lane 3). In these experiments, the S. aureus lyt− lytM double mutant was expected to be autolysin free based on the previous report that suggested the LytM protein to be responsible for the residual autolytic activity in the lyt− S. aureus (Ramadurai & Jayaswal, 1997). Surprisingly, in the zymographic investigations, the pronounced 36 kDa lytic activity band in lyt− S. aureus (Fig. 5, lane 2) postulated to be due to LytM, was present in the lyt−:lytM double mutant (Fig. 5, lane 4). This observation suggests that LytM is not responsible for the residual activity of the lyt− strain of S. aureus.
To address the presence of 36 kDa lytic activity band in the lyt−:lytM double mutant, the lytM gene was cloned in vector pRSETA and overexpressed in E. coli. The protein band that appeared to be induced after the addition of IPTG was a 36 kDa protein (Fig. 6A, arrow comparing lanes 2 and 3). The size expected for the full length His-tagged LytM was 40 kDa. The protein that was repeatedly purified following metal chromatography was also of the size of 40 kDa (Fig. 6A, lane 1). It has been reported that the LytM signal peptide undergoes cleavage even in E. coli cells (Odintsov et al., 2004; Ramadurai & Jayaswal, 1997). This leads to the loss of the signal peptide and the approximately 4 kDa His-tag present on the N-terminus of the recombinant His-tagged LytM. It is speculated that the majority of the overexpressed LytM undergoes cleavage of the signal peptide and only a small fraction of LytM remains intact with the His-tag which could be purified. In zymographic experiments, Ramadurai & Jayaswal (1997) reported three autolysin bands of 36, 22, and 19 kDa in extracts of E. coli cells that overproduced LytM and proposed that the lower lytic-activity bands were LytM degraded products. However, in our zymographic experiments, no autolytic band was visualized even after prolonged incubation of the zymographic gel in the lane corresponding to purified His-tagged LytM (Fig. 6B, lane 4). There were smaller autolytic activity bands between 14–19 kDa in the lanes corresponding to the whole cell extract of the E. coli cells expressing His-tagged LytM (Fig. 6B, lane 3) but a 36 kDa lytic activity band was not visualized. The 14 kDa protein band that was apparent in E. coli cells that contained only plasmid pRSETA (Fig. 6B, lane 2) may be attributed to high level expression of T7 lysozyme in BL21(DE3) pLysS cells.
LytM was originally identified and proposed to be responsible for the residual autolytic activity in an autolysis-defective lyt− mutant strain of S. aureus (Ramadurai & Jayaswal, 1997). It has subsequently been shown that the expression of lytM is negatively regulated by RAT, a regulator of autolysis of the S. aureus cells (Ingavale et al., 2003). In proteomic and transcriptomic analysis, the level of LytM has been shown to be elevated 2–3 fold in derivative S. aureus strains with increased vancomycin resistance compared to its level in the parent S. aureus strain with lower level of vancomycin resistance (Mongodin et al., 2003; Pieper et al., 2006). It has also been shown by electrophoretic mobility shift and DNase protection assays that the expression of lytM in S. aureus is regulated by the essential two-component regulatory system WalK/WalR (YycG/YycF) (Dubrac & Msadek, 2004; Dubrac et al., 2007). The response regulator WalR activates the expression of nine genes involved in staphylococcal cell wall degradation. Conditions that depleted WalR in S. aureus cells led to significant reduction in the levels of cell wall hydrolytic enzymes including a 36 kDa hydrolytic enzyme that was speculated by the authors to be LytM (Dubrac et al., 2007).
The results of this study, however, suggest that LytM which is an early to mid exponential phase protein is not responsible for the 36 kDa lytic activity band present in the lyt− mutant strain of S. aureus. This conclusion is based on the fact that there was no decrease in the intensity of 36 kDa lytic band subsequent to the deletion of lytM gene from S. aureus cells. In addition, the lytic activity present in the lyt− mutant strain of S. aureus could not be abolished after the deletion of lytM gene in this autolysis resistant strain. Our findings are further supported by the observations with LytM protein and its lytic activity during the course of its crystal structure determination (Odintsov et al., 2004). The authors demonstrated LytM to be a Zn++-dependent two domain metalloprotease (Odintsov et al., 2004). The N-terminal domain of LytM (45–98) makes very limited contact with the LytM C-domain (Odintsov et al., 2004). The LytM C-domain (99–316) comprises two ordered regions located up- and down-stream of a disordered (147–182) region. The authors detected no lytic activity in assays using pentaglycine as substrate with the full length LytM or a truncated LytM that lacked the N-terminal and the upstream ordered region (Odintsov et al., 2004). However, truncated LytM (185–316) or a trypsin product of LytM (180–316) that only contained the downstream ordered region demonstrated activity in these assays (Odintsov et al., 2004). The crystal structure of this active fragment of LytM185–316 has since been determined (Firczuk et al., 2005).
The abundance of LytM in the form of a 36 kDa protein in vancomycin resistant S. aureus (Pieper et al., 2006) suggests some role for this protein in resistance against vancomycin and probably other cell wall inhibitors. This speculation is supported by observation in this study where the lack of a functional LytM led to induced lysis of staphylococcal cells in the presence of oxacillin. However, the expression of lytM was not impacted by exposure to cell wall inhibitors either in this study or in a prior study (Utaida et al., 2003).
Several S. aureus mutants are described in the literature with dramatically reduced rates of autolysis. Similar to lyt− mutant, a mutation in the atl gene in S. aureus abolished most of the lytic bands except for a 36 kDa autolysin band and few minor bands of smaller sizes (Foster, 1995). It is still to be ascertained what gene or genes have been inactivated in lyt− S. aureus strain subsequent to transposon insertion that led to reduced autolysis of the mutant cells. On the other hand, the atl gene is well characterized, encodes a 137 kDa protein, and it has been proposed that most autolysins in S. aureus are the processed products of ATL protein (Foster, 1995; Sugai et al., 1997). In another study, suppression of the expression of a putative S. aureus glycoprotease led to dramatically reduced autolysis of S. aureus cells. However, there was no change in the expression levels of any of the known autolysin regulators or autolysins including LytM in these autolysis-resistant cells with reduced level of the glycoprotease (Zheng et al., 2007). Expression level of lytM and and other major autolytic enzymes was also not suppressed in transcriptomic analysis of an autolysis-deficient methicillin-resistant strain of S. aureus (Renzoni et al., 2006).
In summary, the findings of this study suggest that LytM is an insignificant player in terms of autolysins in S. aureus and is not responsible for the 36 kDa lytic protein many investigators have proposed to be due to this protein. There are several genes like lytN and aaa (Gill et al., 2005; Heilmann et al., 2005) that are postulated to be peptidoglycan hydrolases and encode proteins of approximately 36 kDa that might be responsible for the pronounced lytic activity band of this size that is typically visualized in zymographic analysis of staphylococcal autolysins. Based on the findings of this study, it is thus proposed that the LytM protein be investigated in S. aureus beyond its role as an autolysin.
The authors thank R.K Jayaswal (Illinois State University) for providing some of the strains used in this work. This work was supported in part by a Warner/Fermaturo & ATSU Board of Trustees Research Funds and grant 1R15AI090680-01 from the National Institutes of Health to V.K.S, a grant from KCOM Biomedical Sciences Graduate Program to K.S. and ASDOH summer internship to M.R.C.