Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation - PubMed (original) (raw)

Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation

Akihiro Yoshida et al. Appl Environ Microbiol. 2002 Dec.

Abstract

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.

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Figures

FIG. 1.

FIG. 1.

Bacterial growth and biofilm formation of S. mutans GS-5 in media with different carbon sources. Bacteria were inoculated in either CDM, CDM supplemented with glucose (0.5 or 1.0%), CDM-sucrose (0.5 or 1.0%), or THB. Growth (black bars) and biofilm formation (white bars) were measured under anaerobic conditions. The data are the averages of three samples, and standard errors are shown.

FIG. 2.

FIG. 2.

Bacterial growth and biofilm formation of S. mutans GS-5 and biofilm-defective mutants. Assays were performed using CDM-0.5% glucose and polystyrene plates under anaerobic conditions. Growth (black bars) and biofilm formation (white bars) were measured. The data are averages of three samples, and error bars are shown.

FIG. 3.

FIG. 3.

Biofilm formation of S. mutans GS-5 and comA, comB, comC, comD, and comE mutants. Growth (black bars) and biofilm formation (white bars) were measured using CDM-0.5% glucose and polystyrene plates under anaerobic conditions. The data are averages of three samples, and error bars are shown.

FIG. 4.

FIG. 4.

Scanning electron micrographs comparing biofilm formation of S. mutans GS-5 (left) and CC1301 (right) accumulated on polystyrene tips after 48 h of inoculation. Images were obtained at ×1,500 magnification.

FIG. 5.

FIG. 5.

Biofilm formation of sgp antisense strains. (A) Bacterial growth (black bars) and biofilm formation (white bars) of control and sgp antisense strains. Assays were performed using CDM-0.5% glucose and polystyrene plates under anaerobic conditions. The data are averages of three samples, and error bars are shown. (B) Biofilm formation of control strain (left) and sgp antisense strain (right) in CDM supplemented with 0.5% glucose. Biofilm formation was demonstrated using minimal medium supplemented with 0.5% glucose and six-well polystyrene plates under anaerobic conditions. CV-stained biofilm on polystyrene plate wells is shown. (C) Scanning electron micrographs of control strain (left) and sgp antisense strain (right) biofilms accumulated on polystyrene tips after 48 h of inoculation. Images were obtained at ×3,500 magnification.

FIG. 6.

FIG. 6.

Biofilm formation by the dgk mutant. (A) Bacterial growth (black bars) and biofilm formation (white bars) of S. mutans GS-5 and its dgk mutant TnSp-1. Assays were performed as described in the text. The data are averages of three samples, and error bars are shown. (B) Biofilm formation of S. mutans GS-5 (left) and TnSp-1 dgk mutant (right) in CDM-0.5% glucose in six-well polystyrene plates under anaerobic conditions. CV-stained biofilm on polystyrene plate wells is shown.

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