Identification and Molecular Characterization of an <i>N</i>‐Acetylmuraminidase, Aml, Involved in <i>Streptococcus mutans</i> Cell Separation

Yoshimura, Goh; Komatsuzawa, Hitoshi; Hayashi, Ikue; Fujiwara, Takanori; Yamada, Sakuo; Nakano, Yoshio; Tomita, Yuko; Kozai, Katsuyuki; Sugai, Motoyuki

doi:10.1111/j.1348-0421.2006.tb03846.x

Cited by 17 publications

(21 citation statements)

References 31 publications

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“…Some N-acetylmuramoyl-L-alanine amidases have been reported to have Zn 2ϩ -dependent peptidoglycan recognition domains (21,24). ORF9 was also identified as the N-acetyl- did not seem to be essential in its activity, because ORF9 showed lytic activity in the turbidity assay in the presence of EDTA and in the zymographic resolution after renaturation without any ion supplementation.…”

Section: Discussionmentioning

confidence: 96%

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Characterization of Lytic Enzyme Open Reading Frame 9 (ORF9) Derived from Enterococcus faecalis Bacteriophage φEF24C

Uchiyama

Takemura

Hayashi

et al. 2011

Appl Environ Microbiol

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In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage EF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage EF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-L-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn 2؉ ions for its activity, contrary to the bioinformatics prediction of a Zn 2؉ ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-L-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N-or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage EF24C and can be a therapeutic alternative to antibiotics.

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Section: Discussionmentioning

confidence: 96%

“…Analysis of the ORF9 peptidoglycan cleavage site. The preparation of highperformance liquid chromatography (HPLC) samples generally followed the procedure of Yoshimura et al (24). Briefly, E. faecalis STF-3 peptidoglycan (0.8 mg) was treated with either ORF9-His (0.2 mg/ml) or mutanolysin (0.14 mg/ml; Sigma-Aldrich) overnight at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Lytic Enzyme Open Reading Frame 9 (ORF9) Derived from Enterococcus faecalis Bacteriophage φEF24C

Uchiyama

Takemura

Hayashi

et al. 2011

Appl Environ Microbiol

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“…In both bacteria, the cell length of a mutant increases in proportion to the number of genes inactivated, even though disruption of some cell wall hydrolases imparts no phenotypic change. In other bacteria, various other cell lytic enzymes have been identified, e.g., Aml from Streptococcus mutans, Cse from Streptococcus thermophilus, and AcmA from Lactococcus lactis (7,39,49).…”

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confidence: 99%

Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R

et al. 2008

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In previous work, random genome deletion mutants of Corynebacterium glutamicum R were generated using the insertion sequence (IS) element IS31831 and the Cre/loxP excision system. One of these mutants, C. glutamicum strain RD41, resulting from the deletion of a 10.1-kb genomic region (⌬cgR_1595 through cgR_ 1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_ 1596 in WT C. glutamicum R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In C. glutamicum R, CgR_ 0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in C. glutamicum.The gram-positive, high-GC-content bacterium Corynebacterium glutamicum has been one of the most important bacteria in industry for several decades due to its high production of amino acids such as glutamate (16). The bacterium often shows a V-shaped cell form under the microscope, and its coryneform rod-shaped cells have one side of their poles slightly wider than the other. Since its unique characteristics enable it to produce such large amounts of amino acids, most C. glutamicum research has remained focused on the creation of highly productive strains on the one hand and analysis of metabolic flow regulation on the other (14,15,32). Consequently, the unusual behavior of the bacterium to produce V-shaped cells has elicited less research attention. The cell division system resulting in V-shaped cells is known as snapping division (37). Besides C. glutamicum, other Actinomycetales such as Arthorobacter, Nocardia, and Mycobacteria are known to produce V-shaped cells (9,18,28,38). Although several genes involved in cell division (13,17,20,30,34,47) and cell morphology (21,35,44,45) in C. glutamicum have been characterized, the molecular mechanism of the snapping division is still largely unknown.Cell division is achieved by the consecutive actions of cell extension, chromosome replication and segregation, and cell separation. In most prokaryotic and eukaryotic species, cell separation starts by constriction and the subsequent formation of two equivalent daughter cells. After completion of chromosome replication and segregation of the daughter chromosomes to the two halves of the cell, constriction occurs at a predetermined site and two progeny cells are produced. ...

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“…A bactericide was prepared with an N-acetylmuraminidase, the auto-mutanolysin (Aml) strongly specific to Streptococcus sobrinus and Streptococcus mutans, two bacteria found in human oral cavity [89]. This preparation could be used to treat dental caries and periodontal disease and easily incorporated into gum, dentifrice or mouthwash.…”

Section: Virolysins Against Cariogenic Bacteriamentioning

confidence: 99%

Production and Application of Bacteriophage and Bacteriophage-Encoded Lysins

Courchesne

Parisien²,

Lan

2009

BIOT

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The widespread resistance to antibiotics among pathogenic bacteria has made development of alternatives to antibiotics a pressing public concern. Extensive studies have established bacteriophages (phages) and phage-encoded lytic enzymes (virolysins) as two of the most promising families of alternative antibacterials for the treatment and prophylaxis of bacterial infections. They have shown great potential in veterinary and human medicine for the treatment and prophylaxis of infections. Technologies have also been patented employing phages and virolysins in other pathogen related applications including detection and decontamination.

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Identification and Molecular Characterization of an N‐Acetylmuraminidase, Aml, Involved in Streptococcus mutans Cell Separation

Cited by 17 publications

References 31 publications

Characterization of Lytic Enzyme Open Reading Frame 9 (ORF9) Derived from Enterococcus faecalis Bacteriophage φEF24C

Characterization of Lytic Enzyme Open Reading Frame 9 (ORF9) Derived from Enterococcus faecalis Bacteriophage φEF24C

Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R

Production and Application of Bacteriophage and Bacteriophage-Encoded Lysins

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