Isolation and Characterization of <i>Staphylococcus aureus</i> Mutants Which Form Altered Cell Clusters

Sugai, Motoyuki; Komatsuzawa, Hitoshi; Ooku-Inomata, Kaoru; Miyake, Yoichiro; Ishida, Eisaku; Suginaka, Hidekazu

doi:10.1111/j.1348-0421.1994.tb02158.x

Cited by 7 publications

(7 citation statements)

References 14 publications

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“…This suggests that glycylglycine endopeptidase(s) is also involved in cell separation. Our proposed model, together with previous studies, suggests that the atl gene products act at the last step of cell separation after completion of septum separation (30)(31)(32). On the other hand, ultra thin section of a femB mutant revealed retarded septum separation, implying that glycylglycine endopeptidase(s) has a role in septum separation (16).…”

Section: Discussionmentioning

confidence: 86%

“…Antiimmunoglobulin G (IgG) generated against the purified 62-kDa AM or 51-kDa GL inhibited cell separation of S. aureus and induced cells to form giant cell clusters (30). Mutants with little or no 62-kDa AM and 51-kDa GL activity grew in clusters (31). These results suggest that the 62-kDa AM and the 51-kDa GL is involved in cell separation after cell division.…”

mentioning

confidence: 69%

“…In many bacteria, correlation of a lack of peptidoglycan hydrolase(s) activity and a failure in cell separation has been reported (4,5,7,8,10,11,23,26,27,31,34,35). Recent studies have identified peptidoglycan hydrolases involved in cell separation in Listeria monocytogenes and in Lactococcus lactis (3,36).…”

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confidence: 99%

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An autolysin ring associated with cell separation of Staphylococcus aureus

et al. 1996

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atl is a newly discovered autolysin gene in Staphylococcus aureus. The gene product, ATL, is a unique, bifunctional protein that has an amidase domain and a glucosaminidase domain. It undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, a 51-kDa endo-␤-N-acetylglucosaminidase and a 62-kDa N-acetylmuramyl-L-alanine amidase. It has been suggested that these enzymes are involved in the separation of daughter cells after cell division. We recently demonstrated that atl gene products are cell associated (unpublished data). The cell surface localization of the atl gene products was investigated by immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-␤-Nacetylglucosaminidase immunoglobulin G. Protein A-gold particles reacting with the antigen-antibody complex were found to form a ring structure on the cell surface at the septal region for the next cell division site. Electron microscopic examination of an ultrathin section of the preembedded sample revealed preferential distribution of the gold particles at the presumptive sites for cell separation where the new septa had not been completed. The distribution of the gold particles on the surface of protoplast cells and the association of the gold particles with fibrous materials extending from the cells suggested that some atl gene products were associated with a cellular component extending from the cell membrane, such as lipoteichoic acid. The formation of a ring structure of atl gene products may be required for efficient partitioning of daughter cells after cell division.Peptidoglycan hydrolases are a group of enzymes which catalyze the turnover or degradation of peptidoglycan in bacteria. They include N-acetylglucosaminidases, N-acetylmuramidases, N-acetylmuramyl-L-alanine amidases (AMs), endopeptidases, and transglycosylases (for reviews, see references 1, 18, 22, 25, 26, and 34). These enzymes degrade peptidoglycan saccules, resulting in cell lysis. Therefore, the activities of these enzymes should be strictly regulated. Various physiological functions of these enzymes have been proposed, including their roles in cell separation, wall growth, wall turnover, muropeptide recycling, sporulation, formation of flagella, and transformation (for reviews, see references 22, 25, 26, and 34). Peptidoglycan hydrolases have also been implicated in autolysis induced by ␤-lactams or in bacterial pathogenesis (1, 2, 13-15, 19; for reviews, see references 22 and 26).Bacterial cell separation is a dynamic event in the cell cycle and requires cleavage of peptidoglycan connecting daughter cells. In many bacteria, correlation of a lack of peptidoglycan hydrolase(s) activity and a failure in cell separation has been reported (4,5,7,8,10,11,23,26,27,31,34,35). Recent studies have identified peptidoglycan hydrolases involved in cell separation in Listeria monocytogenes and in Lactococcus lactis (3, 36).We have previously described the identification of a 51-kDa endo-␤-N-acetylglucosaminidase (GL) and a 62...

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Section: Discussionmentioning

confidence: 86%

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confidence: 69%

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An autolysin ring associated with cell separation of Staphylococcus aureus

et al. 1996

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“…Antiimmunoglobulin G (IgG) generated against purified 51-kDa GL or 62-kDa AM (anti-AM IgG or anti-GL IgG) inhibits cell separation of S. aureus and induces cells to form giant clusters. Mutants with little or no 51-kDa GL and 62-kDa AM activity grow in clusters (13,14). Furthermore, immunoelectron microscopic studies have demonstrated that atl gene products are localized on the cell surface at the presumptive sites of cell separation (17).…”

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Localized perforation of the cell wall by a major autolysin: atl gene products and the onset of penicillin-induced lysis of Staphylococcus aureus

et al. 1997

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We investigated the cell surface localization of the atl gene products of Staphylococcus aureus exposed to a lytic concentration (4 MIC) of penicillin G (PCG) by means of immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-␤-N-acetylglucosaminidase immunoglobulin G. Protein A-gold conjugates reacting with antigen-antibody complex localized at sites of defects of the cell wall at the nascent cross wall. Anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-␤-N-acetylglucosaminidase immunoglobulin G inhibited the decreased turbidity caused by PCG-induced lysis and the formation of defects in the wall. The autolysis-defective mutant, S. aureus RUSAL2 (atl::Tn551), exposed to 4 MIC of PCG resisted autolysis and formation of the wall defect. These results suggest that activation or deregulation of the atl gene products at localized sites where formation of new cross wall was disturbed by PCG causes small defects in the cell wall in situ, eventually leading to general autolysis.

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“…Ampicillin-resistant transformants containing recombinant plasmids, as indicated by the inactivation of lacZЈ, were used for screening. To select clones expressing staphylolytic activity, agar plates containing heat-killed S. aureus were used as described elsewhere (8,20,41,55). E. coli cells were plated on TSB solidified with 1.5% (wt/vol) agar containing heat-killed S. aureus FDA209P (0.5 mg [dry weight]/ml), lysozyme (4 mg/ml), and IPTG.…”

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

et al. 1997

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A novel staphylolytic enzyme, ALE-1, acting on Staphylococcus aureus, was purified from a Staphylococcus capitis EPK1 culture supernatant. The optimal pH range for staphylolytic activity was 7 to 9. ALE-1 contains one Zn 2؉ atom per molecule. Analysis of peptidoglycan fragments released by ALE-1 indicated that the enzyme is a glycylglycine endopeptidase. The effects of various modulators were determined, and we found that o-phenanthroline, iodoacetic acid, diethylpyrocarbonate, and Cu 2؉ reduced the staphylolytic activity of ALE-1. ␤-Casein, elastin, and pentaglycine were poor substrates for ALE-1. Molecular cloning data revealed that ALE-1 is composed of 362 amino acid residues and is synthesized as a precursor protein which is cleaved after Ala at position 35, thus producing a mature ALE-1 of 35.6 kDa. The primary structure of mature ALE-1 is very similar to the proenzyme form of lysostaphin. It has the modular design of an N-terminal domain of tandem repeats of a 13-amino-acid sequence fused to the active site containing C-terminal domain. Unlike lysostaphin, ALE-1 does not undergo processing of the N-terminal repeat domain in broth culture. ale-1 is encoded on the plasmid. Protein homology search suggested that ALE-1 and lysostaphin are members of the novel Zn 2؉ protease family with a homologous 38-amino-acid-long motif, Tyr-X-His-X 11 -Val-X 12/20 -Gly-X 5-6 -His.A number of bacterial species produce peptidoglycan hydrolases that preferentially lyse staphylococcal species. They include Pseudomonas (5-7, 28, 35, 40), Aeromonas (10,25,35,68), Clostridium (36), Actinomyces (1), Streptomyces (64, 70), Chalaropsis (17), Flavobacterium (27), and Staphylococcus (45, 46, 56) species. The physiological functions of these enzymes remains largely unknown. Lysostaphin is a staphylolytic enzyme with a molecular weight of 25,000 secreted by only one strain of Staphylococcus simulans bv. staphylolyticus (45,46). Although its catalytic properties are not well characterized, it is apparent that lysostaphin hydrolyzes glycylglycine bonds in interpeptide bridges of Staphylococcus aureus peptidoglycan (24). Herein, we describe the purification and molecular characterization of the novel staphylolytic enzyme ALE-1, which is produced by Staphylococcus capitis EPK1 that was originally isolated from a contaminated nutrient agar plate (56). We show that ALE-1 is a glycylglycine endopeptidase that is structurally and functionally related to lysostaphin and is a member of the novel Zn 2ϩ protease family. MATERIALS AND METHODSBacterial strains and plasmid. S. capitis EPK1 (56) and 10 clinically isolated S. capitis strains were used. Clinical strains were obtained from normal human skin. DNA from S. capitis EPK1 was cloned in Escherichia coli JM109 (69). Subcloning of the ALE-1 gene containing DNA fragment was carried out with pUC19 as vector and E. coli XL1-Blue (4). Heat-killed S. aureus FDA209P was used for the staphylolytic assay. Staphylococcus and Escherichia were grown in Trypticase soy broth (TSB) (Becton Dickinson Microbiolog...

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Isolation and Characterization of Staphylococcus aureus Mutants Which Form Altered Cell Clusters

Cited by 7 publications

References 14 publications

An autolysin ring associated with cell separation of Staphylococcus aureus

An autolysin ring associated with cell separation of Staphylococcus aureus

Localized perforation of the cell wall by a major autolysin: atl gene products and the onset of penicillin-induced lysis of Staphylococcus aureus

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

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