Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae

Chu, Chien-Peng; Kariyama, Reiko; Daneo-Moore, L; Shockman, Gérald D.

doi:10.1128/jb.174.5.1619-1625.1992

Cited by 61 publications

(33 citation statements)

References 29 publications

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“…These data indicate that the protein, immunoreactivity, and enzymatic activity of extracellular M-2 are stable when it is incubated under these conditions. Consequently, proteinase inhibitors were not used in further experiments (e.g., purification to homogeneity of extracellular M-2 [3]). …”

Section: Resultsmentioning

confidence: 99%

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Kariyama

Shockman

1992

J Bacteriol

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A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcusfaecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) was shown to possess two separate and distinct peptidoglycan hydrolase activities; both enzymes are N-acetylmuramoylhydrolases (muramidases) (18,29). Both enzymes are high-molecular-weight, complex proteins that possess a number of rather unusual properties, especially in contrast to avian and bacteriophage lysozymes that hydrolyze the same bond in the cell wall peptidoglycan. Muramidase-1 (M-1) was isolated and purified to homogeneity and was shown to occur in a latent, 130-kDa form that can be proteolytically hydrolyzed to an active, 87-kDa form (17,24,30). M-1 was also shown to be a glycoenzyme containing covalently attached monomeric and oligomeric glucose (17) and to possess approximately 12 phosphodiester-linked monomeric 5-mercaptouridine monophosphate residues (8). In addition, M-1 was shown to processively hydrolyze linear, soluble, un-cross-linked peptidoglycan chains (1).Muramidase-2 (M-2) was partially purified from the supernatant culture medium and was shown to be a 70-to 75-kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (18). M-2 was also purified to apparent homogeneity from the insoluble pellet of disrupted E. hirae (7). Two polypeptide bands, one of about 125 kDa and the other of about 71 kDa, were show...

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Section: Resultsmentioning

confidence: 99%

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Kariyama

Shockman

1992

J Bacteriol

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“…The Ami protein is predicted to have a two-domain structure. The presence of two domains (a catalytic domain and a non-catalytic, cell-wall-binding domain) appears to be a common feature of autolysins (Chu et al, 1992;Kuroda et al, 1992;Romero et al, 1990). The high homology (49% identity over 183 aa) of the N-terminal domain of Ami to the amidase domain of the S. aureus autolysin At1 (Foster, 1995, Oshida et al, 1995, suggests that Ami also has amidase activity.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of an autolytic amidase of Listeria monocytogenes EGD

McLaughlan

Foster

1998

Microbiology

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The gene encoding a 102 kDa autolysin has been cloned from an expression library of Listeria monocytogenes EGD genomic DNA, using a direct screening protocol. The encoded protein has two domains, an N-terminal enzymic domain showing a high level of homology to the amidase domain of the major autolysin (at/) of Staphylococcus aureus, and a C-terminal, putative cell-wallbinding domain containing four imperfect direct repeats. In order to examine the role of the enzyme, the autolysin-encoding gene was insertionally inactivated by site-specif ic integration of a temperature sensitive plasmid. The enzyme accounts for 66% of the total lytic enzyme activity when L. monocytogenes walls are used as substrate and several of the major autolytic bands are missing on renaturing gels when compared to the wild-type. The enzyme does not appear to be directly involved in cell separation but has a role in motility. Characterization of the recombinant enzyme expressed in Escherichia coli has revealed it to be an amidase and to be able to hydrolyse a range of peptidoglycan substrates.

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“…3). Such amino acid repetitions have often been observed in the noncatalytic domains of several cell wall hydrolases (4,24,27,39). We have previously reported that the N-terminal to central region of the CwlM protein is a catalytic domain and that the C-terminal repetition may be involved in its substrate specificity, i.e., the CwlM protein hydrolyzes the Micrococcus cell wall preparation more efficiently than those of B. licheniformis and B. subtilis, but the truncated CwlM protein (lacking the C-terminal repetition) has lost this preference (27).…”

Section: Cw1bmentioning

confidence: 99%

Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis

1993

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Southern hybridization analysis ofBacillus subtilis 168S chromosomal DNA with a Bacilus licheniformis cell wall hydrolase gene, cwlM, as a probe indicated the presence of a cwlMf homolog in B. subtils. DNA sequencing of the cwLM homologous region showed that a gene encoding a polypeptide of 255 amino acids with a molecular mass of 27,146 Da is located 625 bp upstream and in the opposite direction of spoVJ. The deduced amino acid sequence of this gene (tentatively designated as cwlC) showed an overall identity of 73% with that of cwlM and of 40% with the C-terminal half of the B. subtiUis vegetative autolysin, CwIB. The construction of an in-frame cwlC-lacZ fusion gene in the B. subtils chromosome indicated that cwlC is induced at 6 to 7 h after sporulation (t6 to t7). The spoIHIC (d~") mutation and earlier sporulation mutations greatly reduced the expression of the cwlC-lacZ fusion gene. Northern hybridization analysis using oligonucleotide probes of the cwlC region indicated that a unique cwlC transcript appeared at t7*. and t9. Transcriptional start points determined by primer extension analysis suggested that the -10 region is very similar to the consensus sequence for the oK-dependent promoter. Insertional inactivation of the cwlC gene in the B. subtUis chromosome caused the disappearance of a 31-kDa protein lytic for Micrococcus cell walls, which is mainly located within the cytoplasmic and membrane fractions of cells at t9. The CwIC protein hydrolyzed both B. subtilis vegetative cell walls and spore peptidoglycan.From Bacillus subtilis, two vegetative autolysins, a 50-kDa N-acetylmuramoyl-L-alanine amidase (amidase) and a 90-kDa endo-3-N-acetylglucosaminidase (glucosaminidase), have been purified and characterized (16,38). The former occurs in large amounts (16,24), and the gene, cwlB, has recently been cloned and studied at the molecular level (24,29). The cwlB operon consists of three genes encoding a putative lipoprotein (LppX), a modifier protein (CwbA) that stimulates amidase activities, and CwlB, in that order (22,24,26,29). The transcription of the cwlB operon mainly depends on expression of the eD protein, which is responsible for cell motility and chemotaxis (26,32). Other cell wall hydrolases of B. subtilis have been described previously by us (23) and Foster (11,12). A 30-kDa amidase gene, cwl4, has been cloned, but its expression is very low under normal growth conditions (11,23). Foster demonstrated novel cell wall lytic activities of A3 (34-kDa) and A4 (30-kDa), which increases during sporulation, and also sporulation-specific activities of A5 (23-kDa) and A6 (41-kDa) by means of renaturing gel electrophoresis in substrate-containing gels (12). The physiological roles of these multiple autolysins, i.e., cell wall turnover (2, 5), cell separation (5, 10, 37), flagellation (10), and competence (3), have been suggested by many investigators, but there was little evidence because they used regulatory mutants [flaD (sin, lyt) and sacU (Hy)] with reduced autolysin levels (26). Asymmetric septum pep...

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Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae

Cited by 61 publications

References 29 publications

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Molecular characterization of an autolytic amidase of Listeria monocytogenes EGD

Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis

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