Zymographic Characterization of Bacteriolytic Enzymes Produced by Oral Streptococci

Yoshimura, Goh; Komatsuzawa, Hitoshi; Kajimura, Junko; Fujiwara, Takanori; Ohara, Masaru; Kozai, Katsuyuki; Sugai, Motoyuki

doi:10.1111/j.1348-0421.2004.tb03537.x

Cited by 7 publications

(9 citation statements)

References 16 publications

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“…In other cases, bacterial cells were suspended in 4% SDS and incubated for 30 min at room temperature. After centrifugation, the supernatant was used as the SDS extract (69,89). To extract surface-associated proteins (84), the cells were suspended in phosphate-buffered saline (PBS) containing 0.2% (wt/vol) N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent; Sigma), incubated at 28°C with shaking at 80 rpm for 1 h, and centrifuged.…”

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confidence: 99%

The atlA Operon of Streptococcus mutans : Role in Autolysin Maturation and Cell Surface Biogenesis

Ahn

Burne

2006

J Bacteriol

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The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlAdeficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.Bacteria produce a variety of enzymes involved in the modification and degradation of peptidoglycan, including N-acetyglucosaminidases, N-acetylmuramidases, N-acetylmuramyl-Lalanine amidases, endopeptidases, and transglycosylases (31,54,71,80). Some of these enzymes are known as autolysins because they digest the cell wall when cells are exposed to unfavorable conditions (70,71,80). The presence of these enzymes may not be sufficient for lysis, and additional factors may be required to activate or regulate the activity of the autolysins (44), presumably to protect the cells from the lytic activity of the enzymes. Peptidoglycan hydrolases have also been demonstrated to play critical roles in cell wall turnover, cell growth, antibiotic resistance, cell-to-surface adhesion, genetic competence, and protein secretion (9,23,28,51,74), as well as contributing to virulence (8, 86). In many bacteria, a correlation of a lack of peptidoglycan hydrolase(s) activity and a failure in cell separation has been reported (35,60,77,87).An increasing number of peptidoglycan hydrolases have been identified in gram-positive bacteria, including Bacillus subtilis (37,39,48,59,74), Lactococcus lactis (11,12,22,34,75), Listeria monocytogenes (57, 79, 86), Staphylococcus aureus (19,36,53,78), Enterococcus faecalis (15,16,55,56,72,73), and Streptococcus pneumoniae (8,18,20,83). Recently, during a search for surface prote...

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The atlA Operon of Streptococcus mutans : Role in Autolysin Maturation and Cell Surface Biogenesis

Ahn

Burne

2006

J Bacteriol

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“…A zymogram analysis of autolysins was carried out by using an SDS-10% polyacrylamide gel (14) containing 1% (wet weight) S. mutans cells. The preparation of S. mutans cells for incorporation into polyacrylamide gels was performed as described previously (28,44). Briefly, cell cultures (800 ml) of S. mutans Xc were harvested by centrifugation, and the pellet was washed three times with distilled water and then resuspended in 60 ml of 4% SDS.…”

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Identification and Characterization of an Autolysin-Encoding Gene of Streptococcus mutans

et al. 2005

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We identified a gene (atlA) encoding autolytic activity from Streptococcus mutans Xc. The AtlA protein predicted to be encoded by atlA is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved ␤-1,4-N-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated atlA. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of atlA resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by S. mutans. The antibody produced against the C-terminal peptide containing the ␤-1,4-N-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to S. mutans. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.

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“…The bacteriolytic enzyme profile was analyzed using zymography with polyacrylamide gels containing heat-killed cells of S. mutans MT703R as described elsewhere (31).…”

Section: Methodsmentioning

confidence: 99%

“…We previously demonstrated S. mutans produces two major bacteriolytic bands at 80 and 100 kDa using zymographic analysis (31). These bands were visible in zymograms using SDS-boiled S. mutans or S. sobrinus cells as substrates; and not visible using SDS-boiled S. mitis, S. salivarius or S. sanguinis cells as substrates.…”

Section: Identification Of 80 and 100 Kda Bacteriolytic Enzymesmentioning

confidence: 97%

“…These species use fermentable sugars including sucrose to produce several kinds of glucan that generate dental plaque (3,9); and also produce lactic acid as a metabolite that destroys dental enamel and dentin (21). We have previously characterized the bacteriolytic enzyme profiles of S. mutans, S. sobrinus, S. salivarius, S. sanguinis and S. mitis using zymography; and showed S. mutans produces two bacteriolytic enzymes with molecular masses of 100 kDa and 80 kDa (31). Here, we identified the proteins corresponding to these enzymes and found that they are from a single gene product.…”

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

Yoshimura

Komatsuzawa

Hayashi

et al. 2006

Microbiology and Immunology

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Bacteria produce several peptidoglycan hydrolases (PGHs). There are at least five types of PGHs: endo-β-N-acetylglucosaminidase, endo-β-N-acetylmuraminidase, N-acetylmuramyl-L-alanine-amidase, endopeptidase and transglycosylase; each known for the specific bond they split in the cell wall (1,19). They are ubiquitous in bacteria and show functions in restructuring the cell wall where they have many important roles in cell wall metabolism including nicking peptidoglycan for insertion of a new peptidoglycan monomer, cell division, cell separation, and cell wall turnover (1,19,26). Many PGHs are termed bacteriolytic because the action of the enzyme often leads to bacterial cell lysis. Some enzymes are able to disintegrate their own peptidoglycan saccules when the cells are placed in unfavorable conditions; and such enzymes are called autolysins.Streptococci are Gram-positive bacteria where individual cells are connected with each other forming a chain morphology. Among the streptococci, S. pneumoniae, the leading cause of pneumonia, blood stream infections and otitis media, is the most extensively studied concerning the biochemistry and physiological functions of the PGHs. S. pneumoniae produces at least three types of PGHs: LytA amidase (16), LytB endo-β-N-acetylglucosaminidase (4, 7) and LytC lysozyme (6). Their roles in cell separation and viru-

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Zymographic Characterization of Bacteriolytic Enzymes Produced by Oral Streptococci

Cited by 7 publications

References 16 publications

The atlA Operon of Streptococcus mutans : Role in Autolysin Maturation and Cell Surface Biogenesis

The atlA Operon of Streptococcus mutans : Role in Autolysin Maturation and Cell Surface Biogenesis

Identification and Characterization of an Autolysin-Encoding Gene of Streptococcus mutans

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

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