Dynamics of cell wall-binding proteins at a single molecule level: B. subtilis autolysins show different kinds of motion.

The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move through the cell wall to find sites of enzymatic activity. Cell wall synthesis for cell elongation involves the integration of new peptidoglycan strands by integral membrane proteins, as well as the degradation of existing strands by so-called autolysins, soluble proteins that are secreted through the cell membrane. Autolysins comprise different classes of proteases and glucanases and mostly contain cell-wall binding domains in addition to their catalytic domain. We have studied dynamics of Bacillus subtilis autolysins LytC, a major endopeptidase required for lateral cell wall growth, and LytF, a peptidase acting at the newly formed division site in order to achieve separation of daughter cells. We show that both proteins, fused to moxVenus are present as three distinct populations of different diffusion constants. The fastest population is compatible with free diffusion in a crowded liquid environment, that is similar to that of cytosolic enzymes, likely reflecting autolysins diffusing through the periplasm. The medium mobile fraction can be explained by constrained motion through a polymeric substance, indicating mobility of autolysins through the wall similar to that of DNA-binding proteins within the nucleoid. The slow-mobile fraction are most likely autolysins bound to their specific substrate sites. We show that LytF is more static during exponential phase, while LytC appears to be more active during the transition to stationary phase. Both autolysins became more static in backgrounds lacking redundant other autolysins, suggesting stochastic competition for binding sites. On the other hand, lack of inhibitor IseA or autolysin CwlS lead to an altered preference for polar localization of LytF within the cell wall, revealing that inhibitors and autolysins also affect each other's pattern of localization, in addition to their activity.

Design of a novel affinity probe using the cell wall-binding domain of a Listeria monocytogenes autolysin for pathogen detection.

IMPORTANCE: Human listeriosis is caused by consuming foods contaminated with the bacterial pathogen Listeria monocytogenes, leading to the development of a severe and life-threatening foodborne illness. Detection of L. monocytogenes present in food and food processing environments is crucial for preventing Listeria infection. The L. monocytogenes peptidoglycan hydrolase IspC anchors non-covalently to the bacterial surface through its C-terminal cell wall-binding domain (CWBD), CWBDIspC. This study explored the surface binding property of CWBDIspC to design, construct, characterize, and assess an affinity molecular probe for detecting L. monocytogenes. CWBDIspC recognized a cell wall ligand lipoteichoic acid that remains evenly displayed and mostly unoccupied on the bacterial surface for interaction with the exogenously added CWBDIspC. CWBDIspC, when fused to the enhanced green fluorescent protein reporter or covalently conjugated onto magnetic beads, exhibited the functionality as an antibody alternative for rapid detection and efficient separation of the pathogen.

1H, 13C, and 15N resonance assignments of a conserved putative cell wall binding domain from Enterococcus faecalis.

Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence.

Potential Role of the Host-Derived Cell-Wall Binding Domain of Endolysin CD16/50L as a Molecular Anchor in Preservation of Uninfected Clostridioides difficile for New Rounds of Phage Infection.

Endolysin is a phage-encoded cell-wall hydrolase which degrades the peptidoglycan layer of the bacterial cell wall. The enzyme is often expressed at the late stage of the phage lytic cycle and is required for progeny escape. Endolysins of bacteriophage that infect Gram-positive bacteria often comprises two domains: a peptidoglycan hydrolase and a cell-wall binding domain (CBD). Although the catalytic domain of endolysin is relatively well-studied, the precise role of CBD is ambiguous and remains controversial. Here, we focus on the function of endolysin CBD from a recently isolated Clostridioides difficile phage. We found that the CBD is not required for lytic activity, which is strongly prevented by the surface layer of C. difficile. Intriguingly, hidden Markov model analysis suggested that the endolysin CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but possesses a higher binding affinity to bacterial cell-wall polysaccharides. Moreover, the CBD forms a homodimer, formation of which is necessary for interaction with the surface saccharides. Importantly, endolysin diffusion and sequential cytolytic assays showed that CBD of endolysin is required for the enzyme to be anchored to post-lytic cell-wall remnants, suggesting its physiological roles in limiting diffusion of the enzyme, preserving neighboring host cells, and thereby enabling the phage progeny to initiate new rounds of infection. Taken together, this study provides an insight into regulation of endolysin through CBD and can potentially be applied for endolysin treatment against C. difficile infection. IMPORTANCE Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment. To utilize endolysin for the therapeutic propose, understanding of the fundamental role of endolysin becomes important. Here, we investigate the function of cell-wall binding domain (CBD) of an endolysin from a C. difficile phage. The domain is homologous to a cell-wall associating module of bacterial cell-wall proteins, likely acquired during phage-host coevolution. The interaction of CBD to bacterial cell walls reduces enzyme diffusion and thereby limits cell lysis of the neighboring bacteria. Our findings indicate that the endolysin is trapped to the cell-wall residuals through CBD and might serve as an advantage for phage replication. Thus, employing a CBD-less endolysin might be a feasible strategy for using endolysin for the treatment of C. difficile infection.

Analysis of Murein Hydrolases and Proteases Through Zymography.

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.

Competence-Stimulating-Peptide-Dependent Localized Cell Death and Extracellular DNA Production in Streptococcus mutans Biofilms.

Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide called competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm.IMPORTANCE Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased, as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years it has been noted that the contribution of phenotypic heterogeneity within biofilms requires analysis at the single-cell or subpopulation level to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

False positives in using the zymogram assay for identification of peptidoglycan hydrolases.

Zymogram assays have been used extensively to identify novel peptidoglycan hydrolases. In this study it is reported that the zymogram is susceptible to false positive results when highly positively charged proteins are assayed. As an example, we report on the case of the ChiZ membrane protein from the Mycobacterium tuberculosis divisome, which previously was described as a peptidoglycan hydrolase. Even though the full length ChiZ protein was able to produce positive assay results, other direct methods for measuring peptidoglycan hydrolysis do not provide convincing evidence that ChiZ has peptidoglycan hydrolysis activity. We show that the false positive result is produced by the highly positively charged N-terminal region of ChiZ. Thus, we developed a zymogram control that can be used to identify false positives results. This control assay lacks the refolding step in the normal zymogram assay. For lysozyme the control assay shows no activity, while the N-terminal region of ChiZ shows a false positive result. Given the limitations of the zymogram assay to reliably identify peptidoglycan hydrolases, we recommend using the zymogram control assay together with other methods to evaluate possible peptidoglycan hydrolysis activity.

SpyB, a Small Heme-Binding Protein, Affects the Composition of the Cell Wall in Streptococcus pyogenes.

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to ß-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.

Monoclonal antibodies recognizing the surface autolysin IspC of Listeria monocytogenes serotype 4b: epitope localization, kinetic characterization, and cross-reaction studies.

Listeria monocytogenes serotype 4b is responsible for a high percentage of fatal cases of food-borne infection. In a previous study, we created 15 monoclonal antibodies (MAbs) against a ≈ 77 kDa antigen that is associated with the cell surface of live L. monocytogenes serotype 4b cells. Here we report an extensive characterization of these MAbs to further their development as diagnostic reagents. The ≈ 77 kDa target antigen was identified by mass spectrometry and N-terminal sequencing to be IspC, a novel surface associated autolysin. Epitope localization experiments revealed that each of the 15 MAbs recognized the C-terminal cell-wall binding domain of IspC. The presence of IspC was shown to be highly conserved within L. monocytogenes serotype 4b, as evidenced by a strong reaction between anti-IspC MAbs and all 4b isolates. To determine the range of cross-reactivity with other L. monocytogenes serotypes ELISA was used to test each MAb against multiple isolates from each of the L. monocytogenes serotypes. Of the 15 MAbs, five: M2774, M2775, M2780, M2790 and M2797, showed specificity for L. monocytogenes serotype 4b and only cross reacted with serotype 4ab isolates. The kinetics of the interaction between each of the MAbs and IspC was measured using surface plasmon resonance. The MAbs M2773, M2792, M2775, M2797 and M2781 each had very low dissociation constants (4.5 × 10(-9) to 1.2 × 10(-8) M). While several of these antibodies have properties which could be useful in diagnostic tests, the combined high fidelity and affinity of M2775 for the IspC protein and serotype 4b isolates, makes it a particularly promising candidate for use in the development of a specific L. monocytogenes serotype 4b diagnostic test.

The nine peptidoglycan hydrolases genes in Lactobacillus helveticus are ubiquitous and early transcribed.

Peptidoglycan hydrolases (PGHs) are bacterial enzymes that can hydrolyze the peptidoglycan in bacterial cell wall leading to autolysis. By releasing intracellular enzymes, autolysis of Lactobacillus helveticus has important applications in cheese ripening as its extent varied from strain to strain. Nine genes coding PGHs were previously annotated in the genome of the high autolytic strain L. helveticus DPC 4571. This study was conducted to evaluate the clone diversity of the nine PGHs genes within a collection of 24 L. helveticus strains, highly diverse in terms of origin, biotope and autolytic activity. Pulsed field gel electrophoresis was applied to assess the genomic diversity of the 24 strains. The presence or absence of nine PGHs genes was verified for all L. helveticus strains. Nucleotide and deduced amino acid sequence were compared for six relevant strains. Finally, gene expression was monitored by reverse transcription during growth and by zymogram for 12 strains. The nine PGHs genes are ubiquitous and transcripted early during growth. Zymograms were similar in terms of molecular size of the bands, but exhibited strain to strain variations in the number of bands revealing from 2 to 5 lytic bands per strain.

A bedside assay to detect streptococcus pneumoniae in children with empyema.

BACKGROUND: Empyema is a complication of pneumonia, commonly caused by Streptococcus pneumoniae. AIMS: To validate the utility of an immunochromatographic test for the detection of S. pneumoniae antigen in the pleural fluid of children with empyema. METHODS: Empyema patients had blood and pleural fluid cultured, and polymerase chain reaction (PCR) to detect the S. pneumoniae autolysin gene, lytA, in pleural fluid. Pleural fluid was tested using the Binax NOW S. pneumoniae antigen detection assay and compared with lytA PCR results and/or culture in blood or pleural fluid. RESULTS: S. pneumoniae was detected by PCR in pleural fluid of 68 of 137 (49.6%) patients, by culture in 11 of 135 (8.1%) pleural specimens and 16 of 120 (13.3%) blood specimens. Pleural fluid Binax NOW testing from 130 patients demonstrated a sensitivity of 83.8% and specificity of 93.5% (positive predictive value of 93.4% and negative predictive value of 84.1%). CONCLUSIONS: In pediatric empyema, high predictive values of pleural fluid Binax NOW S. pneumoniae antigen test suggest that this test may help rationalize antibiotic choice in these patients.

Insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytC.

The first structure of a pneumococcal autolysin, that of the LytC lysozyme, has been solved in ternary complex with choline and a pneumococcal peptidoglycan (PG) fragment. The active site of the hydrolase module is not fully exposed but is oriented toward the choline-binding module, which accounts for its unique in vivo features in PG hydrolysis, its activation and its regulatory mechanisms. Because of the unusual hook-shaped conformation of the multimodular protein, it is only able to hydrolyze non-cross-linked PG chains, an assertion validated by additional experiments. These results explain the activation of LytC by choline-binding protein D (CbpD) in fratricide, a competence-programmed mechanism of predation of noncompetent sister cells. The results provide the first structural insights to our knowledge into the critical and central function that LytC plays in pneumococcal virulence and explain a long-standing puzzle of how murein hydrolases can be controlled to avoid self-lysis during bacterial growth and division.

Development of a novel fluorescent substrate for Autolysin E, a bacterial type II amidase.

The bifunctional Autolysin E from Staphylococcus epidermidis, contains a Zn(2+)-dependent N-acetylmuramoyl-L-alanine amidase AmiE (EC 3.5.1.28). This enzyme hydrolyzes the amide bond between the carbohydrate chain and the peptide stem of bacterial peptidoglycan. Since peptidoglycan is the mayor component of bacterial cell walls, type II amidases like Autolysin E play an essential role in the bacterial life cycle. Therefore bacterial amidases are appropriate drug targets in the development of antibiotics. The drug discovery process relies on sensitive enzyme assay systems to test possible lead candidates for enzyme inhibition. However, specific determination of bacterial amidase activity is complicated because a simple and accurate enzyme assay is currently unavailable. In this study we developed a sensitive fluorescent substrate for the type II amidase Autolysin E from S. epidermidis, which is suitable for quantifying amidase activity in a high throughput compatible fashion. Using derivatives of the substrate Mca-Ala-D-isoGln-Lys(Dnp)-D-Ala-Arg-OH, we were further able to characterize the amidase substrate specificity of Autolysin E.

Molecular properties of the putative autolysin Atl(WM) encoded by Staphylococcus warneri M: mutational and biochemical analyses of the amidase and glucosaminidase domains.

The putative autolysin Atl(WM) of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side amidase (ami(atlwm)-R1-R2) and a C-terminal side glucosaminidase (R3-glu(atlwm)). Zymographic analysis of the protein overproduced in Escherichia coli showed that both enzymes were active toward 17 Gram-positive bacteria, including staphylococci, lactobacilli, lactococci, enterococci, and micrococci. The purified enzyme core ami(atlwm) (or glu(atlwm)) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) degrees C, respectively. ami(atlwm) was inactivated by EDTA, and was stimulated by such salts as CoCl(2), MnCl(2), CaCl(2), or ZnCl(2). Six mutations within ami(atlwm), (H362A, E421A, H467A, H479, D481A, and Y491D) drastically reduced cell-lytic activity. Comparative analysis with other related amidases suggested that the three residues H362, H467, and D481 likely act as ligands (and/or active sites). The lytic activity of glu(atlwm) markedly declined in four mutants (E1238A, E1238Q, T1239A, and Y1332A). For determination of the putative cell-recognition regions, four domains (R1-R2, R1, R2, and R3) were purified; all the proteins substantially bound to S. warneri M cells from exponential to stationary growth phases, and R1-R2 aggregated the cells. Protein sequencing and immunoblot analysis suggested that the extacellular Atl(WM) might be primarily processed at two specific sites (one between pro and ami(atlwm), and the other between R2 and R3) to yield the mature amidase and glucosaminidase.

Structural analysis of bacteriophage-encoded peptidoglycan hydrolase domain KMV36C: crystallization and preliminary X-ray diffraction.

The C-terminus of gp36 of bacteriophage varphiKMV (KMV36C) functions as a particle-associated muramidase, presumably as part of the injection needle of the phiKMV genome during infection. Crystals of KMV36C were obtained by hanging-drop vapour diffusion and diffracted to a resolution of 1.6 A. The crystals belong to the cubic space group P432, with unit-cell parameters a = b = c = 102.52 A. KMV36C shows 30% sequence identity to T4 lysozyme (PDB code 1l56).

Evaluation of cell wall binding domain of Staphylococcus aureus autolysin as affinity reagent for bacteria and its application to bacterial detection.

We evaluated the cell wall binding (CWB) domain of Staphylococcus aureus autolysin as an affinity reagent for bacteria. A fusion of CWB domain and green fluorescent protein (CWB-GFP) bound to S. aureus with a dissociation constant of 15 nM. CWB-GFP bound to a wide range of gram-positive bacteria, but not to most gram-negative bacteria. We suspected that the outer membrane of gram-negative bacteria inhibits the access of CWB-GFP to peptidoglycan layer. Indeed, CWB-GFP bound to gram-negative bacteria when they were treated with benzalkonium chloride. Because CWB-GFP bound to the bacterial peptidoglycan layer, it appeared to be an effective affinity reagent for bacteria and CWB fusion with reporter proteins could be applied to detect bacteria. We also constructed a fusion of CWB and luciferase, which can be used for the rapid detection of bacteria.

A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays.

Current spectrophotometers measure murein hydrolase activity simultaneously under many conditions and in small intervals. A correct interpretation of these large data sets requires clear and standardized criteria. Furthermore, there is a need for a uniform unit definition to express enzymatic activity, because application of variable definitions seriously hampered comparison between different studies. The method presented here is based on maximizing R(2)-values of incremental data sets. Combined with an appropriate unit definition, it provides a statistically sound background and warrants reproducible and reliable results. Activity calculations are further simplified by an online available Excel spreadsheet. This method is especially suited for experiments where individual curves differ extensively from each other (e.g. low versus high activity conditions) and can be expanded to other similar high-throughput bioassays.

Identification of IspC, an 86-kilodalton protein target of humoral immune response to infection with Listeria monocytogenes serotype 4b, as a novel surface autolysin.

We identified and biochemically characterized a novel surface-localized autolysin from Listeria monocytogenes serotype 4b, an 86-kDa protein consisting of 774 amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial infection. Recombinant IspC, expressed in Escherichia coli, was purified and used to raise specific rabbit polyclonal antibodies for protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during infection. The protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. The recombinant IspC was capable of hydrolyzing not only the cell walls of the gram-positive bacterium Micrococcus lysodeikticus and the gram-negative bacterium E. coli but also that of the IspC-producing strain of L. monocytogenes serotype 4b, indicating that it was an autolysin. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range of between 3 and 9, with a pH optimum of 7.5 to 9. Analysis of various truncated forms of IspC for cell wall-hydrolyzing or -binding activity has defined two separate functional domains: the N-terminal catalytic domain (amino acids [aa] 1 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW modules responsible for anchoring the protein to the cell wall. In contrast to the full-length IspC, the N-terminal catalytic domain showed hydrolytic activity at acidic pHs, with a pH optimum of between 4 and 6 and negligible activity at alkaline pHs. This suggests that the cell wall binding domain may be of importance in modulating the activity of the N-terminal hydrolase domain. Elucidation of the biochemical properties of IspC may have provided new insights into its biological function(s) and its role in pathogenesis.

Comparative proteomic analysis of Staphylococcus aureus strains with differences in resistance to the cell wall-targeting antibiotic vancomycin.

Three isogenic strains derived from a clinical vancomycin-intermediate Staphylococcus aureus isolate were examined by comparative protein abundance analysis. Subcellular fractionation was followed by protein separation in 2-DE gels and spot identification by MALDI-TOFTOF-MS and LC-MS/MS. Sixty-five significant protein abundance changes were determined. Numerous enzymes participating in the purine biosynthesis pathway were dramatically increased in abundance in strain VP32, which featured the highest minimal inhibitory concentration for vancomycin, compared to strains P100 and HIP5827. Peptidoglycan hydrolase LytM (LytM) and the SceD protein, a putative transglycosylase, were increased in abundance in the cell envelope fraction of strain VP32, whereas the enzyme D-Ala-D-Ala ligase was decreased in its cytosol fraction. Furthermore, penicillin-binding protein 2 (PBP2) had substantially higher activity in strain VP32 compared to that in strain HIP5827. LytM, PBP2 and D-Ala-D-Ala ligase catalyze reactions in the biosynthesis or the metabolism of cell wall peptidoglycan. It is plausible that expression and activity changes of these enzymes in strain VP32 are responsible for an altered cell wall turnover rate, which has been observed, and an altered peptidoglycan structure, which has yet to be elucidated for this highly vancomycin-resistant strain.

Effects of oxacillin and tetracycline on autolysis, autolysin processing and atl transcription in Staphylococcus aureus.

Autolysins are peptidoglycan hydrolases involved in cell growth and cell lysis. Atl is an important autolysin of Staphylococcus aureus and is essential for penicillin-induced autolysis. The objective of our study was to examine the effect of oxacillin, chloramphenicol and tetracycline on autolysis, peptidoglycan hydrolase profiles and transcription of atl encoding the major S. aureus autolysin on cells grown in the presence of minimum inhibitory concentrations of the antibiotics. Growth of methicillin-susceptible strains in the presence of oxacillin led to increased autolysis, a loss of low molecular weight and a gain of high molecular weight peptidoglycan hydrolase bands suggesting altered proteolytic processing of peptidoglycan hydrolases, and a decrease in atl transcription. In contrast, growth in the presence of tetracycline led to a decrease in autolysis, an increase in atl transcription, and a drastic decrease in the protein concentration of freeze-thaw extracts obtained for peptidoglycan hydrolase analysis. Growth of methicillin-resistant strains in the presence of oxacillin had only moderate effects on autolysis and peptidoglycan hydrolase profiles.