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. 2007 Sep;189(17):6293-302.
doi: 10.1128/JB.00546-07. Epub 2007 Jul 6.

Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans

Sang-Joon Ahn  1 , Robert A Burne
Affiliations

Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans

Sang-Joon Ahn et al. J Bacteriol. 2007 Sep.

Abstract

The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.

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Figures

FIG. 1.
FIG. 1.
Transcriptional analysis of the SMu0629 gene locus in S. mutans. (A) Schematic diagram of the atlA region and RT-PCR analysis. Gene assignments and gene numbers above the schematic diagram are based on the genomic sequence information available for S. mutans UA159. Arrows indicate the direction of transcription. The numbers inside the arrows and between open reading frames indicate the sizes of the open reading frames and intergenic regions, respectively, in base pairs. Following reverse transcription with a reverse primer (630-antisense), PCR amplification was performed with the primer set 629-sense and 630-antisense. The annealing sites of the primers are shown on the diagram. The PCR products were run in a Tris-acetate-EDTA gel as follows: lane M, size marker; lane 1, RT-PCR product; lane 2, negative control without RT; lane 3, positive control of PCR with chromosomal DNA of UA159. (B) Protein sequence of SMu0629, consisting of 165 amino acids. The sequence contains eight conserved cysteines (in bold) that may form a metal binding site and an FX4CXXC motif (above the sequence; amino acids 48 to 56) typical of the active sites of several members of the thioredoxin superfamily.
FIG. 2.
FIG. 2.
Growth of S. mutans strains (wild type and 630NP). (A) Growth in BHI broth was monitored in a Bioscreen C system which was set to shake for 15 s every 30 min (aerobic conditions). For anaerobic growth, sterile mineral oil was placed on top of the broth cultures (w/oil). (B) Biofilm formation of S. mutans UA159 and 630NP in BM medium supplemented with sucrose for 48 h. The culture was grown aerobically on a shaker (150 rpm). The anaerobic culture was overlaid with mineral oil. See the text for more details. Data are representative of at least two separate experiments performed in triplicate or greater. The error bars represent standard deviations (n = 6).
FIG. 3.
FIG. 3.
Phenotypic characterization of SMu0629 mutants (629NP [nonpolar] and 629P [polar]). (A) Expression of atlA monitored by real-time PCR. For measurements of atlA mRNA, total RNAs from UA159 (wild type), 629NP, and 629P were used for reverse transcription with the 630-antisense primer. (B) Biofilm formation. The cultures were grown in BM medium supplemented with glucose for 1 or 2 days. Data are representative of at least two separate experiments performed at least in triplicate. The error bars represent standard deviations. *, P < 0.01 (for 1 day) or P < 0.001 (for 2 days) (Student's t test). (C) SDS-PAGE analysis of bead-beaten SDS-boiled extracts from the wild type (WT) and two SMu0629 mutants (629NP and 629P) of S. mutans. Following SDS-PAGE, proteins were either stained with Coomassie blue (top) or transferred to a nitrocellulose membrane and subjected to Western blotting using an anti-630D1 polyclonal antiserum at a dilution of 1:350 (bottom). Lane M, size marker. (D) Autolysis assay. The autolytic activities of strains were monitored in a Bioscreen C system that was set to shake for 15 s before measurement every 30 min. The cell suspension was incubated at the optimum temperature for the autolytic activity of AtlA (44°C). Black line, UA159; gray line, 629NP; gray dotted line, 629P.
FIG. 4.
FIG. 4.
Effects of aerobic growth of S. mutans strains (UA159, 629NP, and 629P). For aerobic growth, the cultures were grown on a rotary shaker (150 rpm). (A) Growth curves. The cultures were grown in BHI medium at 37°C. The data shown are from a single experiment representative of three independent experiments that yielded the same outcome. (B) Biofilm formation. The cultures were grown in BM medium supplemented with glucose at a final concentration of 20 mM. The culture was grown aerobically on a shaker (150 rpm). See the text for more details. Data are representative of at least two separate experiments that were performed in triplicate or greater. The error bars represent standard deviations. *, P < 0.001 (Student's t test).
FIG. 5.
FIG. 5.
Phenotypic characterization of the vicK mutant (vicK-NP). (A) Expression of the SMu0629 and atlA genes by real-time PCR. For measurements of SMu0629 and atlA mRNAs, total RNAs from the UA159 (wild type) and vicK-NP strains were used for reverse transcription with the 629-antisense and 630-antisense primers, respectively. Data presented are means ± standard deviations (error bars) for three independent experiments. *, P < 0.05 (Student's t test). (B) SDS-PAGE analysis of two different cell extracts from wild-type (WT) and vicK-NP strains of S. mutans. Following SDS-PAGE, proteins were either stained with Coomassie blue (left) or transferred to a nitrocellulose membrane and subjected to Western blotting (whole-cell lysates) using an anti-630D1 polyclonal antiserum at a dilution of 1:350 (right). Lane M, size marker. Bands of interest were excised from the stained gel and subjected to mass spectrometric analysis. (C) Autolysis assay. The autolytic activities of strains were monitored in a Bioscreen C system that was set to shake for 15 s before measurement every 30 min. The cell suspension was incubated at the optimum temperature for the autolytic activity of AtlA (44°C). Thick line, UA159; thin gray line, vicK-NP.
FIG. 6.
FIG. 6.
Effects of aerobic growth of S. mutans strains (UA159 and vicK-NP). For aerobic growth, the cultures were grown on a rotary shaker (150 rpm). (A) Biofilm formation. The cultures were grown in BM medium supplemented with 20 mM sucrose. Biofilm formation was assayed on polystyrene microtiter plates after staining with crystal violet. (B) Differential expression of SMu0629, atlA, and vicR genes in response to aerobic growth of S. mutans UA159, using real-time PCR. Data shown are means ± standard deviations for three independent experiments performed in triplicate or greater. *, P < 0.02 (Student's t test). See the text from more details.
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References

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