Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1

Sugai, Motoyuki; Fujiwara, Takanori; Akiyama, Tomoko; Ohara, Masaru; Komatsuzawa, Hitoshi; Inoue, Satoshi; Suginaka, Hidekazu

doi:10.1128/jb.179.4.1193-1202.1997

Cited by 97 publications

(106 citation statements)

References 66 publications

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“…Protein homology searches revealed the presence of a 38-amino-acid motif, T y r -X -Hi s-X '-Va I-X ,20 -GI y -XSp6-Hi s , and the p resence of conserved histidine residues. Inhibition of lysostiiphin and ALE-1 enzyme activities by DEPC or iodoacetic acid has been reported previously (Park et al, 199.5;Sugai et al, 1997). It has been suggested that the conserved histidine residues may be important for the lytic activity of these enzymes and serve as ligands to zinc.…”

Section: Zinc Ligands In Lytmmentioning

confidence: 81%

“…An attempt was made to determine whether the specific activity of LytM could be modified by various physical and chemical factors, as tested previously by Sugai et al (1990Sugai et al ( , 1997 for the lytic enzymes of staphylococci. Among the compounds tested, ampicillin, Ba2+, Cu2+, DTT, EDTA, Fe2+, Mg2', Mn", PMSF and Zn2+ appeared to have some inhibitory effect on lytic activity.…”

Section: Qualitative Effects Of Physical and Chemical Factors On The mentioning

confidence: 99%

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Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus

Ramadurai¹,

Lockwood²,

Lockwood³

et al. 1999

Microbiology

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IL 61 790-41 20, USA The authors previously reported the cloning of a lytic-enzyme-encoding gene, lytM, from an autolysis-defective mutant of Staphylococcus aureus. In the present work, the lytM gene was overexpressed in Escherichia coli and the product was purified to homogeneity by affinity chromatography and HPLC. Biochemical analysis of LytM-cleaved peptidoglycan fragments indicated that LytM is a glycylglycine endopeptidase. lmmunoelectron microscopic studies with anti-LytM rabbit IgG showed that LytM is expressed during the early exponential phase and is overexpressed in an autolysis-defective mutant compared with the parent strain. Also, a uniform distribution of gold particles on the surface of actively growing bacterial cells indicates that LytM plays a role in cell growth. Northern blot analyses of /ytM expression in two global regulatory mutants, agr and sar, showed that expression of /ytM is increased about twofold in these mutants as compared with the parents. Protein homology searches revealed that LytM could be a member of the zinc protease family, as it contained a homologous 38-amino-acid motif, Tyr-X-His-X,,-Val-X, , , -Gl y-X, -Hi s.Atomic absorption spectrometric analysis of LytM revealed the presence of 0 9 mol zinc (mol LytM)".Keywords : autolysin, glycylglycine endopeptidase, lytM gene, transmission electron microscopy, Staphylococcus aureus INTRODUCTIONPeptidoglycan hydrolases are enzymes that are ubiquitous in bacteria and which hydrolyse the peptidoglycan of the bacterial cell envelope (Ghuysen et al., 1966). There are five main classes of peptidoglycan hydrolase, the N-acetylmuramidases, N-acetylmuramoyl-L-alanine amidases, endopeptidases, glucosaminidase and transglycosylases (Actor et al., 1988;Holtje & Tuomanen, 1991;Rogers et al., 1980; Tomasz, 1984). Several roles have been proposed for these enzymes in cell wall growth, cell separation, cell wall turnover, lysis initiated by antibiotics affecting the cell wall, competence for genetic transformation, flagellum formation, sporulation arid pathogenicity (Actor et al., 1988;Berry et al., 1989;Rogers et al., 1980;Shockman & Barrett, 1983;Shockman & Holtje, 1994; Ward & Williamson, 1984).Abbreviations: DEPC, diethyl pyrocarbonate; DNP, 2,4-dinitrophenyl; PCW, purified cell wall.Since the first report on Staphylococcus aureus autolysins (Welsch & Salmon, 1950), a variety of intracellular and extracellular lytic enzymes have been reported and characterized in this bacterium (Sugai, 1997). Tipper (1969) demonstrated the presence of amidase, glucosaminidase and endopeptidase activities in isolated cell walls of S. aureus. A bifunctional structural lytic gene encoding the S. aureus N-acetylglucosaminidase and N-acetylmuramoyl-L-alanine amidase was later characterized (Foster, 1995 ;Oshida et al., 1995;Oshida & Tomasz, 1992). Recently, we identified a lytic gene, l y t M , from an autolysis-defective mutant (Lyt-) of S. aureus (Ramadurai & Jayaswal, 1997). The deduced amino acid sequence of LytM showed significant homology to lysostaphin...

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Section: Zinc Ligands In Lytmmentioning

confidence: 81%

Section: Qualitative Effects Of Physical and Chemical Factors On The mentioning

confidence: 99%

Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus

Ramadurai¹,

Lockwood²,

Lockwood³

et al. 1999

Microbiology

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“…Transformation of S. aureus by electroporation was performed as described (25). Southern blotting of DNA and hybridization were performed as described previously (24). Primers used in this study are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization of an atl Null Mutant of Staphylococcus aureus

Takahashi

Komatsuzawa

Yamada

et al. 2002

Microbiology and Immunology

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atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non‐covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non‐growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin‐induced lysis of the cells.

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“…3. The N-terminal sequence of enterolysin A contains a domain which is found in members of the M37 family of metallopeptidases which have Zn 2+ in their catalytic sites (Recsei et al, 1987;Sugai et al, 1997). Members of this family include lysostaphin (Schindler & Schuhardt, 1964;Schleifer & Kandler, 1972;Schleifer & Fischer, 1982;Baba & Schneewind, 1996) and zoocin A (Simmonds et al, 1997) whose conserved N-terminal domain provides the enzymic functions of the proteins and contains a His-Xxx-His motif which may serve as the Zn 2+ ligand (Sugai et al, 1997).…”

Section: Dpc5280 Contains the Genetic Determinants For Cytolysin Prodmentioning

confidence: 99%

Production of enterolysin A by a raw milk enterococcal isolate exhibiting multiple virulence factors

et al. 2003

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Even though enterococci are a common cause of human infection they can readily be isolated from a range of food sources, including various meat and dairy products. An enterococcal strain, DPC5280, which exhibits a broad spectrum of inhibition against many Gram-positive bacteria was recently isolated from an Irish raw milk sample. Characterization of the inhibition revealed that the strain exhibits haemolytic activity characteristic of the two-component lantibiotic cytolysin and also produces a heat-labile antimicrobial protein of 34 kDa. The latter protein displayed cell wall hydrolytic activity, as evidenced by zymogram gels containing autoclaved lactococcal cells. N-terminal sequencing of the purified protein yielded the sequence ASNEWS which is 100 % identical to enterolysin A (accession no. AF249740), a protein which shares 28 and 29 % identity to the Gly-Gly endopeptidases, lysostaphin and zoocin A, respectively. Indeed, amplification of entL from DPC5280 and sequencing revealed that the protein is 100 % identical to enterolysin A. The DPC5280 strain also contained the determinants associated with multiple virulence factors, including gelatinase, aggregation substance and multiple antibiotic resistance. The linkage of this cell-wall-degrading enzyme to other virulence factors in enterococci may contribute to the competitiveness of pathogenic enterococci when found in complex microbial environments such as food and the gastrointestinal tract. INTRODUCTIONDespite the fact that foods containing enterococci have a long history of safe use, these organisms are not considered as GRAS (generally recognized as safe) organisms. They can readily be isolated from a range of food sources, including raw milk, and are often a constituent of some mixed starter strains used commercially. However, many strains can act as opportunistic pathogens, causing a variety of infections such as urinary tract infections, bacteremia and infective endocarditis (Murray, 1990;Jett et al., 1994), and are of major importance in community-acquired and in hospital-acquired (nosocomial) infections (Jett et al., 1994;Low et al., 1994;Jones et al., 1997;Simjee & Gill, 1997). A contributing factor to their pathogenesis is their evolving resistance to antibiotics. For example, resistance to the antibiotic vancomycin is now widespread among members of the genus, which leaves few options for disease management (Aguirre & Collins, 1993;Knudtson & Hartman, 1993;Facklam & Sahm, 1995;Klein et al., 1998). Considerable progress has recently been made in determining the traits responsible for pathogenesis of enterococci.For example, studies have shown that phenotypes such as production of cytolysin, which has both haemolytic activity (by lysing a broad spectrum of cells, including human, horse and rabbit erythrocytes) and bactericidal activity (against Gram-positive bacteria) play a role in the progression of enterococcal infection (Ike et al., 1984;Jett et al., 1992). Cytolysin enhances the virulence of Enterococcus faecalis in animal models, such as muri...

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Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1

Cited by 97 publications

References 66 publications

Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus

Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus

Molecular Characterization of an atl Null Mutant of Staphylococcus aureus

Production of enterolysin A by a raw milk enterococcal isolate exhibiting multiple virulence factors

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