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. 2003 Apr;71(4):1972-9.
doi: 10.1128/IAI.71.4.1972-1979.2003.

Mutation of luxS affects biofilm formation in Streptococcus mutans (VSports最新版本)

Justin Merritt  1 , Fengxia Qi, Steven D Goodman, Maxwell H Anderson, Wenyuan Shi
Affiliations

VSports - Mutation of luxS affects biofilm formation in Streptococcus mutans

V体育安卓版 - Justin Merritt et al. Infect Immun. 2003 Apr.

Abstract (VSports手机版)

Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www. genome. ou. edu/smutans. html). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5alpha. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S VSports手机版. mutans. .

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Figures

FIG. 1.
FIG. 1.
Alignment of the S. mutans LuxS protein and several other representative LuxS proteins. Residues that coordinate a Zn2+ ion and comprise the catalytic center of LuxS (H, H, and C) are printed in bold. 1, V. harveyi; 2, S. mutans; 3, E. coli; 4, S. pyogenes; 5, B. subtilis.
FIG. 2.
FIG. 2.
S. mutans luxS complements a frameshift mutation in E. coli DH5α. E. coli DH5α was transformed with an E. coli-S. mutans shuttle vector containing an intact copy of the S. mutans luxS gene (pLuxSm). This strain was examined for AI-2 production using the luminescence-based AI-2 reporter assay. V. harveyi strain BB170 (sensor 1−, sensor 2+) served as a positive control, while E. coli DH5α served as a negative control. Luminescence is expressed as fold induction relative to the background values.
FIG. 3.
FIG. 3.
AI-2 induction in the presence or absence of sugar. S. mutans was grown and assayed as described in Materials and Methods. Cells were grown overnight and resuspended to an OD600 of 0.4 in reporter assay (AB) medium and incubated with aeration for 3 h at 37°C. One sample was incubated in AB alone, while for the other, sucrose was added to a final concentration of 1%. The presence of sucrose in the medium caused a potent reduction in luminescence to below background values.
FIG. 4.
FIG. 4.
luxS knockout in S. mutans. (A) Illustration of the knockout procedure. Plasmids containing cloned fragments of S. mutans DNA as well as the erythromycin cassette were cut using the indicated restriction sites and then ligated into a linearized pUC19 backbone. The resulting construct was linearized with the unique AatII site and transformed into S. mutans for double crossover. (B) Confirmation of crossover event. In lanes 1 to 4, wild-type DNA was amplified with primers: internal to luxS, the erythromycin cassette, upstream external luxS plus erythromycin, or downstream external luxS plus erythromycin. In lanes 5 to 8, luxS mutant DNA was amplified using the same primer combinations. External luxS primers bind to sites that were not subject to crossover. (C) AI-2 production was assayed to confirm that activity was lost in the mutant.
FIG. 5.
FIG. 5.
Mutation of luxS causes an alteration in S. mutans biofilm. (A) Two images of in vitro biofilms representing both the wild type and the mutant using dark-field microscopy at magnifications ×20 and ×40. Cells were grown on glass coverslips as described in Materials and Methods. Similar results were obtained for more than 100 independent wild-type and luxS mutant biofilms examined. (B) Biofilms were tested for their ability to resist detergent treatment with SDS. Three samples of wild-type and mutant biofilms grown on glass coverslips were shaken at 150 rpm for 1 h. The OD600s of the resulting supernatants were compared.
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References

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