The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Heinrich, Peter C.; Rosenstein, Ralf; Böhmer, Maria; Sonner, Peter; Götz, Friedrich

doi:10.1007/bf00331163

Cited by 84 publications

(63 citation statements)

References 42 publications

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“…The N-terminus of the mature lysostaphin (band 3 in Fig. 2) was uniform with the sequence 248A-A-T-H-E-H, which corresponds to the previously determined N-terminus of mature lysostaphin (Heinrich et a/., 1987). The cleavage products that were smaller than mature lysostaphin and that appeared after longer incubation did not exhibit staphylolytic activity in an activity gel (not shown) and did not have a uniform N-terminus (band 4 in Fig.…”

Section: Processing Of Prolysostaphin By the Cysteine Proteasesupporting

confidence: 65%

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus

Thumm¹,

Götz²

1997

Molecular Microbiology

155

162

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SummaryLysostaphin is an extracellular glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphyIolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan. Renewed analysis of the sequence of the lysostaphin gene (Iss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36aa, a propeptide of 21 1 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246aa. Prolysostaphin is processed in the culture supernatant of S. simulans biovar StaphyIoIyficus by an extracellular cysteine protease. Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active. The controlled expression in Staphylococcus carnosus of lss and lss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state. lntracellularly expressed pro-and mature lysostaphin exert staphylolytic activity in cell-free extracts, but do not affect growth of the corresponding clones. We characterized a lysostaphin immunity factor gene ( / i f ) which is located in the opposite direction to lss. The expression of /if in s. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to /if, Iss is coexpressed the serinelglycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation. Lif shows similarity to FemA and FemB

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Section: Processing Of Prolysostaphin By the Cysteine Proteasesupporting

confidence: 65%

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus

Thumm¹,

Götz²

1997

Molecular Microbiology

155

162

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“…Propeptides are also found in extracellular proteins of various staphylococci, for example, in lysostaphin of S. simulans biovar staphylolyticus (14) and the lipase of S. hyicus (41). The lengths of the propeptides vary, ranging from 10 to over 200 amino acids.…”

Section: Discussionmentioning

confidence: 99%

“…Lysostaphin is a zinc metalloendopeptidase (34) which hydrolyzes the glycine interpeptide bridge of staphylococci, causing cell lysis. The lysostaphin gene has been recently cloned and sequenced (14,21). According to its deduced amino acid sequence and processing, lysostaphin appears to be organized as preproenzyme.…”

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confidence: 99%

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Teufel

Götz

1993

J Bacteriol

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The gene sepA from Staphylococcus epidermidis TU3298-P, encoding the extracellular neutral metallopro- carnosus(pT181mcsPl) corresponded to the extracellular S. epidermidis T03298-P protease. The partially purified protease had a pH optimum between 5 and 7, and its activity could be inhibited by zinc-and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, indicating that it is a neutral metalloprotease. The protease had a low substrate specificity. Glucagon was cleaved preferentially between aromatic (Phe) and hydrophobic (Val) amino acids. The protease hydrolyzed casein and elastin. The amino acid sequence of the mature form of SepP1 revealed pronounced similarities with the thermolabile and thermostable neutral proteases of various bacilli (44 to 55% identity) and a central part of the mature form of the Pseudomonas aeruginosa elastase (31% identity). From homology comparison with the Baciflus thermoproteolyticus thermolysin, we predict that mature SepP1 binds one zinc ion at a conserved zinc-binding site.Most Staphylococcus aureus strains secrete proteolytic enzymes; three different types of proteases have been studied in detail. Serine protease from S. aureus V8, commonly referred to as V-8 protease or protease I, is an endoprotease which cleaves peptide bonds on the carboxy-terminal side of glutamic acid and to a lesser extent of aspartic acid (8). The enzyme, having a molecular weight of 12,000, is inhibited by diisopropyl fluorophosphate (DFP) but not by EDTA. Similar serine proteases with the same cleavage preference are also found in other S. aureus strains; however, they differ from the above-described enzyme in their size and sensitivity to DFP (2, 24). The amino acid sequence of a 27-kDa serine protease of S. aureus revealed a 43-residue C-terminal peptide composed nearly exclusively of aspartic acid, asparagine, and proline (6), proposed to be involved in the transport of the enzyme through the cytoplasmic membrane (7).The second type of protease from S. aureus V8, thiol protease (protease II), is characterized by an isoelectric point of pH 9.4, a molecular weight of 12,500, and a very broad specificity; it also exhibits esterase activity with N-benzoyl-L-tyrosine ethyl ester as a substrate (2). The enzyme is active only in the presence of reducing agents and is inactivated by heavy metals such as Hg2+, Zn2+, or Ag', indicating that protease II must possess free SH groups to be active and that it is a cysteine enzyme.The third protease of S. aureus V8, a metalloprotease (2), is completely inactivated by EDTA and is insensitive to sulfhydryl reagents and DFP. Calcium is essential for the stability of the protease. The molecular weight is 28,000, and the pH optimum for hydrolysis of casein is 7.4. Like the thiol * Corresponding author.protease, the metalloprotease has a rather broad proteolytic specificity, cleaving preferentially at the N-terminal side of hydrophobic residues (1). A metalloprotease with a molecular weight of 38,000 and a pH optimum of 7.0 has been isolated from a mut...

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“…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, encoded by plasmids of Staphylococcus simulans (Heinrich et al, 1987;Recsei et al, 1987), and ALE-1, encoded by Staphylococcus capitis purification and biochemical characterization of the lytM gene product. This is the first report on a chromosomally encoded, probable glycylglycine endopeptidase in S. aureus.…”

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confidence: 97%

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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The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Cited by 84 publications

References 42 publications

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus

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