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Journal of Bacteriology, September 2007, p. 6293-6302, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00546-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Effects of Oxygen on Biofilm Formation and the AtlA Autolysin of Streptococcus mutans{triangledown}

Sang-Joon Ahn and Robert A. Burne*

Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida 32610

Received 10 April 2007/ Accepted 25 June 2007


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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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INTRODUCTION
 
Streptococcus mutans is considered the principal etiological agent of human dental caries (32). A critical virulence property of S. mutans is its ability to become established as part of the structurally and compositionally complex biofilms on tooth surfaces (28, 58). Once established at a site, oral biofilms remain relatively stable over time, despite continuously changing environmental conditions. The abilities to survive these environmental challenges and to emerge as a numerically significant member of stable oral biofilm communities are essential elements for the persistence and cariogenicity of S. mutans. Recently, we identified a surface-associated protein (SMu0630) that was required for normal biofilm formation by S. mutans UA159 (8). In subsequent studies, the protein was revealed to be essential for maturation of biofilms and autolysis of cells (1, 48). The gene product was designated AtlA (48) because of its autolytic activity. AtlA is required for biogenesis of a normal cell surface in that AtlA-deficient strains have a greatly diminished complement of surface proteins extractable with nonionic detergents (1). In addition, AtlA was shown to be required for full expression of genetic competence by S. mutans (1).

Bacterial autolysins are capable of hydrolyzing the peptidoglycan component of the cell wall, which is a highly dynamic structure that expands as the cells grow and is reshaped when cells divide or differentiate (16, 19, 43, 49, 56). Autolysins are often produced throughout the growth cycle and have been shown to play central roles in many critical functions, including cell wall turnover, cell growth, antibiotic resistance, cell-to-surface adhesion, genetic competence, protein secretion, and pathogenicity (6, 7, 17, 18, 36, 50, 57). Regulation of autolysin activity is believed to occur most commonly at the posttranslational level, through substrate conformation or modification, differential binding to the cell via various cell wall-binding domains, topological arrangement of enzyme complexes in the cell wall, and control of the site of export (20), although transcriptional control of autolysins has been demonstrated (50). In some gram-positive bacteria, autolysis occurs spontaneously when the cells reach the late stationary phase of growth. This lethal event has been proposed as a meaningful biological phenomenon because the release of DNA during cell lysis contributes to survival and the genetic diversity of naturally competent bacteria (19). The irreversible effects caused by ß-lactam antibiotics, such as penicillin-induced bacteriolysis, are also well described (15, 55). Other factors shown to affect autolysin activity or activation include nutrient limitation (45), the proton motive force (23, 25), and a number of factors that affect the physicochemical properties of the cell wall (10, 11, 13).

A critical environmental factor affecting the composition and activity of dental biofilms is oxygen. In the human oral cavity, oxygen is abundant, but the biofilms colonizing the various surfaces of the mouth support a variety of aerobes, facultative anaerobes, and obligately anaerobic bacteria. The redox potential in dental plaque falls during the development of oral biofilms on a clean enamel surface, and the deep layers of dental plaque are considered anaerobic (26). Thus, oxygen tension and the oxidizing environment of oral biofilms vary widely with the site and the characteristics of the biofilm. Not surprisingly, oral bacterial biofilms have relatively active oxygen metabolism and have developed defenses against the presence of oxygen or a wide variety of redox environments (34). Notably, exposure of bacteria to oxygen has significant impacts on sugar metabolism, acid production, stress tolerance, and factors related to persistence in dental plaque (22, 52, 53).

In the current study, we demonstrate profound effects of oxygen on the capacity of S. mutans to form biofilms and demonstrate that oxygen affects the expression and maturation of AtlA, which is critical for biofilm formation and biogenesis of a normal cell surface. Novel insights into a linkage between oxidative environments, AtlA biogenesis, autolysis of cells, a two-component signal transduction pathway (TCS), and virulence expression by S. mutans are revealed.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, media, and growth conditions. Escherichia coli DH10B was grown in Luria broth, and S. mutans UA159 and its derivatives were grown in brain heart infusion (BHI) broth (Difco). For selection of antibiotic-resistant colonies after genetic transformation, ampicillin (100 µg ml–1 for E. coli), erythromycin (300 µg ml–1 for E. coli and 10 µg ml–1 for S. mutans), and kanamycin (50 µg ml–1 for E. coli and 1 mg ml–1 for S. mutans) were added to media as needed. For biofilm formation assays, S. mutans strains were grown in microtiter plates in the semidefined medium BM (33) supplemented with glucose or sucrose at a final concentration of 20 mM. Gene numbers correspond to those annotated at the Los Alamos Oral Pathogens website (http://www.oralgen.lanl.gov/).

Construction of mutant strains. Primers used for deletion mutagenesis are listed in Table 1. To make deletions in the SMu0629, vicR, and vicK genes, 5'- and 3'-flanking regions of each gene were amplified from chromosomal DNA of S. mutans UA159, ligated together using BamHI sites designed into each primer set, and cloned into a pGEM-T Easy vector (Promega, Madison, WI). Plasmids were digested with BamHI and ligated to a nonpolar (NPKm) or polar ({Omega}Km) kanamycin cassette, from pALH124 or pVT924 (3), respectively, digested with the same enzyme (3). The mutagenic plasmids were used to transform S. mutans UA159. Transformants were selected on BHI agar containing kanamycin, and double-crossover recombination into each gene was confirmed by PCR and sequencing. The mutant strains of S. mutans constructed in this study are listed in Table 2.


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  		TABLE 1. Primers used for construction of deletion mutants and real-time PCR


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  		TABLE 2. S. mutans strains used in this study

Growth, biofilm, and autolysis assays. For growth rate comparisons, fresh medium was inoculated with 1:100 dilutions of overnight cultures of S. mutans. The optical density at 600 nm (OD600) was measured at 37°C at routine time intervals or monitored using a Bioscreen C lab system (Helsinki, Finland) (3). The Bioscreen C system was set to shake for 15 s every 30 min. For anaerobic conditions, sterile mineral oil (50 µl per well) was placed on top of the cultures. The cultures were also observed by phase-contrast microscopy to record the chain length, as previously detailed (1). The ability to form stable biofilms in microtiter plates was measured as previously described (3). Biofilms were allowed to form by incubating the plates statically at 37°C in air, in an aerobic atmosphere with 5% CO2, or on a rotary shaker at 150 rpm in an aerobic atmosphere, with or without an overlay of mineral oil. An autolysis assay was carried out as described previously, with minor modifications (48). Briefly, S. mutans cells in the late exponential phase of growth (OD600 = 0.7) were harvested by centrifugation and washed twice with phosphate-buffered saline. The cells were resuspended in 20 mM potassium phosphate buffer (pH 6.5) containing 1 M KCl, 1 mM CaCl2, 1 mM MgCl2, and 0.4% sodium azide to an OD550 of 0.9. The cell suspension was incubated at 44°C, and autolysis was monitored by measuring the OD550 of the cell suspension, using a Bioscreen C lab system.

SDS-PAGE and Western blotting. Protein extracts from S. mutans were prepared from cell pellets that were harvested from BHI cultures at mid-exponential phase (OD600 = 0.5) and washed twice with Tris-buffered saline (10 mM Tris-Cl, 0.9% NaCl, pH 7.4). Whole-cell lysates for protein analysis were obtained by homogenization with a bead beater (Biospec, Bartlesville, OK) in sodium dodecyl sulfate (SDS) boiling buffer (60 mM Tris, pH 6.8, 10% glycerol, and 5% SDS) in the presence of glass beads, as previously described (9). Bacterial cells were also suspended in 4% SDS and incubated for 1 h at room temperature to extract surface-associated proteins. After centrifugation, the supernatant was used as the 4% SDS extract. Proteins (10 µg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 10% polyacrylamide gel with a 4.5% stacking gel, as described by Laemmli (29). The proteins were then stained with Coomassie blue or blotted onto Immobilon-P membranes (Millipore, Bedford, MA) and subjected to Western blot analysis by standard techniques (46). Membranes were incubated with the anti-630D1 polyclonal antiserum, which was raised against full-length, purified, recombinant AtlA (1). The protein concentrations in samples were determined by a bicinchoninic acid assay (Sigma). Bands of interest were excised from the stained gel and sent to the Proteomics and Mass Spectrometry Facility at the Donald Danforth Plant Science Center (Washington University, St. Louis, MO) for identification.

Transcriptional analysis. The potential for cotranscription of two genes was examined by reverse transcriptase PCR (RT-PCR). Levels of mRNA were quantified by real-time RT-PCR. Extraction of RNA, RT-PCR, and real-time RT-PCR were performed, and data were analyzed and normalized as previously described (2). The primers used for reverse transcription reactions and real-time PCR are shown in Table 1.


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RESULTS
 
The atlA operon. We previously demonstrated that the Streptococcus mutans atlA gene is transcribed from at least three promoters, PatlA, PpepT, and PthmA, as part of a four-gene operon (atlA-SMu0631-pepT-thmA) (1). During further characterization of the atlA region, we identified a gene (designated the SMu0629 gene), located 249 bp upstream of the start codon of atlA, that is shown here to be capable of being cotranscribed with atlA (Fig. 1A). This finding suggested that there may be a functional connection between the gene products of the SMu0629 gene and atlA. The SMu0629 gene is 495 bp long and encodes a conserved hypothetical protein with a predicted mass of 19,504 Da. A Pfam search revealed that residues 51 to 155 placed SMu0629 in an uncharacterized UPF0153 protein family that contains eight conserved cysteines that are proposed to form a metal binding site (Fig. 1B). Notably, SMu0629 also contains an F-X4-C-X-X-C motif (Fig. 1B). The C-X-X-C motif is known to be the active site of members of the thiol-disulfide oxidoreductase family, which are involved in diverse redox activities, including reduction of proteins and oxidative stress tolerance (4, 24). In light of the essential role played by AtlA in biofilm formation and the potential for a functional connection between the gene products of atlA and the SMu0629 gene, we explored the effects of oxygen on the growth and biofilm formation of various S. mutans strains.


Figure 1
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  		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.

Effects of oxygen on autolysis and biofilm formation. To compare the growth of S. mutans in the presence and absence of oxygen, growth was monitored in BHI medium under aerobic and anaerobic conditions, as detailed in Materials and Methods. As shown in Fig. 2A, no differences were observed in the growth rates of the wild-type and AtlA-deficient (630NP) strains. However, the atlA mutant displayed significant resistance to autolysis in late stationary phase under anaerobic conditions (Fig. 2A). Light microscopic observation revealed that the formation of long chains, a characteristic of atlA-deficient strains, was not observed when the mutant was grown under aerobic conditions (data not shown).


Figure 2
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  		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).

For an in vitro biofilm assay, microtiter plates were grown in air on an orbital shaker (150 rpm) under aerobic and anaerobic conditions (overlaid with mineral oil). Interestingly, the capacity to form biofilms in BM medium supplemented with sucrose was dramatically impaired when UA159 was grown with aeration for 48 h. Also, the atlA mutant accumulated a greater biomass in biofilms than did the wild-type strain growing under the same conditions (Fig. 2B). When S. mutans was cultivated with sucrose, the Gtf enzymes produced substantial quantities of water-insoluble glucans that promote adhesion and accumulation of biofilms. Biofilm formation in BM medium supplemented with glucose could not be studied because the biofilm-forming capacity of 630NP under these conditions is so poor (8). The impact of oxygen on biofilm formation and the restoration of biofilm formation in air by mutation of atlA suggest that autolysis of cells can be influenced by oxygen, leading to a change in the ability of S. mutans to form biofilms. We reasoned that these phenotypes associated with growth in oxygen may be connected with the function of SMu0629 as a potential oxidoreductase. Therefore, we characterized AtlA biogenesis and the growth characteristics of strains lacking the SMu0629 gene.

Construction and characterization of SMu0629 gene mutants. The SMu0629 gene was disrupted by deletion and insertion of a nonpolar or a polar kanamycin cassette to create strains 629NP and 629P, respectively (Table 2). The nonpolar insertion into the SMu0629 gene was confirmed to allow efficient readthrough to atlA by real-time RT-PCR (Fig. 3A). The atlA mRNA that arose from transcription immediately upstream of atlA, not as a result of cotranscription with the SMu0629 gene, was measured using total RNA from strain 629P. As shown in Fig. 3A, atlA mRNA in 629P was about 50% less abundant than that in 629NP or the wild type, confirming that the genes could be cotranscribed and providing evidence for a possible functional connection between SMu0629 and AtlA.


Figure 3
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  		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.

No obvious differences were observed in the growth rates of these strains in BHI medium (data not shown). It is also noteworthy that clumping and increases in chain length, which are characteristic of the atlA-deficient mutant, were not observed in either the 629NP or 629P mutant (data not shown). Interestingly, the SMu0629 gene mutant exhibited significant increases in biofilm formation in BM medium supplemented with glucose, of 25% after 24 h and 55% after 48 h, compared with the wild-type strain (Fig. 3B). However, no obvious differences were observed in biofilm formation in BM-sucrose (data not shown).

SMu0629 is required for normal production of AtlA. SDS-PAGE and Western blot analysis using whole-cell lysates revealed that disruption of the SMu0629 gene dramatically impacted the production and processing of AtlA (Fig. 3C). In particular, the amount of the processed form of AtlA (79 kDa) was significantly reduced in both the 629NP and 629P mutants compared to that in UA159, so a reduction in transcription of atlA was probably not responsible for the observations. Since AtlA is considered a major autolysin of S. mutans, we examined the autolytic activity of the mutant strains at 44°C (48). As shown in Fig. 3D, both the 629NP and 629P mutants exhibited lower autolysis rates, similar to that of the atlA-deficient mutant (630NP). It is likely, therefore, that the reduced efficiency of maturation of AtlA in the strains lacking SMu0629 accounts for reduced autolysis, unless SMu0629 can participate directly in autolysis of cells or influences the activities of other peptidoglycan hydrolases.

We demonstrated previously that the defects of the AtlA-deficient strain could be corrected to wild-type levels by adding as little as 2 ng ml–1 of purified AtlA protein (630D1) to the culture of the atlA mutant and that the full-length protein (107 kDa) could be processed to the 79-kDa form under these conditions (1). When a purified His-tagged AtlA protein (630D1) was added to the cultures of 630NP (deficient in atlA) and SAB95 (deficient in the SMu0629 gene and atlA) at a concentration of 2.0 ng ml–1, the ability to produce chains of a normal length was restored, and the strain lacking SMu0629 could still convert the 107-kDa form to the mature form of AtlA (data not shown), suggesting that SMu0629 may be important for AtlA biogenesis during secretion or localization but that it is not directly involved in the processing of AtlA at the cell surface. Also, although SMu0629 is required for efficient expression and processing of AtlA, a sufficient level of properly processed AtlA may be available to express the phenotypes assessed under the conditions tested.

The SMu0629 gene mutant is more sensitive to oxygen. One hypothesis for the function of SMu0629 is that it participates in adaptation of S. mutans to an oxidative environment by virtue of its sequence similarity to thiol-disulfide oxidoreductase, perhaps by sensing the redox state and modulating the maturation of AtlA. Since some members of this protein family protect cells against oxidative stress or participate in cellular redox activities, the SMu0629 gene mutants were tested for their response to oxidative stress. H2O2 killing experiments performed by treatment with 0.2% H2O2 did not reveal significant differences in the survival of mid-exponential-phase 629NP, 629P, or the parent strain (data not shown). Interestingly, though, when grown under aerated conditions, the strains lacking SMu0629 showed significantly lower growth rates and formed more clumps than did the wild type (Fig. 4A). Results obtained using a Bioscreen system were confirmed by growth in test tubes to ensure that the lower growth rate was not an artifact of cell aggregation (data not shown). Also, disruption of the SMu0629 gene allowed the organisms to form more biofilms in the presence of oxygen than did the wild-type strain (Fig. 4B), similar to the atlA mutant strains. Taken together, our results suggest that there are significant phenotypic differences between the SMu0629 gene mutants and the wild-type strain when cells are exposed to oxygen.


Figure 4
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  		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).

The VicK sensor kinase affects SMu0629 gene expression and AtlA maturation. The VicRK TCS was investigated as a possible regulatory system for the atlA operon or AtlA activity. The Vic system of S. mutans, designated CovRS for some related species, was recently shown to regulate the expression of glucosyltransferase genes (gtfBCD), the gene for fructosyltransferase (ftf), and gbpB, which encodes glucan-binding protein B (47). These gene products are intimately involved in exopolysaccharide production, and the Gtfs and Gbps in particular are critically important for sucrose-dependent adherence and biofilm formation. Loss of Vic also affects S. mutans growth, sucrose-dependent adhesion, biofilm formation, and development of genetic competence (47). A deletion mutation was generated in the vicK gene of S. mutans UA159. All three genes in the vicRKX locus were shown to be in an operon, so vicK was disrupted by a nonpolar insertion (vicK-NP). Because we were unable to isolate a vicR null mutant, vicK-NP was used as the Vic-deficient strain in this study. The mutant strain had an altered growth rate and aggregated at the bottom of the test tube during growth under static conditions (data not shown). The ability of the mutant to form biofilms was dramatically reduced in BM-glucose and BM-sucrose medium (data not shown), confirming observations made in a previous study (47).

To investigate the regulatory function of VicK over the atlA operon, we first compared the expression of the SMu0629 gene and atlA in the vicK mutant and wild-type strains (Fig. 5A). The expression of the SMu0629 gene was reduced about 40% in the vicK-NP strain (P < 0.02), and the expression of atlA was reduced about 35% (P < 0.007). More importantly, as shown in SDS-PAGE and Western blot assays using whole-cell lysates and 4% SDS extracts of surface proteins, a deficiency of vicK caused almost complete inhibition of the processing of AtlA (107 kDa) to its mature form (79 kDa) (Fig. 5B) and led to substantial resistance to autolysis (Fig. 5C).


Figure 5
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  		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.

Interestingly, it was evident from the 4% SDS extract fraction that either the expression or localization of several proteins was dramatically affected in the vicK mutant (Fig. 5B). Of particular note, two bands, of approximately 150 kDa and 70 kDa, were present in much greater quantities in the mutant than in the wild-type strain (Fig. 5B). To identify these two bands, they were excised from the gel and the trypsin digest pattern was analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The larger band was identified as GtfC, and the smaller protein was identified as a cell wall protein precursor protein (SMu0555) with homology to a putative N-acetylmuramidase/lysin of Streptococcus gordonii and to choline binding protein D of Streptococcus pneumoniae. Thus, these results indicate that the Vic system either directly or indirectly controls the localization or expression of virulence factors, including Gtfs and autolysins.

Effects of oxygen on the vicK mutant. Interestingly, similar to the case with the atlA mutant, the long-chain-length and cell-clumping characteristics of the vicK mutant (47) were dramatically lessened, to near-wild-type levels, when the cells were grown under aerated conditions (data not shown). Also, under the same conditions, the vicK mutant showed significantly increased biofilm formation in BM-sucrose medium compared to the wild type (Fig. 6A). Although the phenotypes of the vicK mutant were substantially affected by oxygen, expression of the vicK gene was not different in the presence or absence of oxygen (Fig. 6B). However, the expression of the SMu0629 gene (P = 0.05) and atlA (P = 0.003) was significantly reduced in the presence of oxygen (Fig. 6B).


Figure 6
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  		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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DISCUSSION
 
S. mutans AtlA is critical for proper cell separation, biofilm formation, and autolysis (1, 8, 48) as well as for the full expression of genetic competence (1). A remarkable finding that emerged from previous work was that AtlA plays a critical role in the biogenesis of a variety of other surface-associated proteins (1). Specifically, Zwittergent extracts of cells lacking AtlA have a dramatically reduced number of proteins compared with extracts from the wild-type strain. In addition, the loss of AtlA also affects proper localization of the major adhesin P1, which is normally covalently coupled to the cell wall by sortase (1). Thus, AtlA is a central protein in the genesis of a normal cell surface.

The results presented herein reveal that the exposure of S. mutans to oxygen has a profound effect on cell surface biogenesis and on the expression or localization of known virulence attributes. The ability of S. mutans to form biofilms is severely impaired by oxygen exposure, which may be due to alterations in exopolysaccharide metabolism or in cell-to-surface or cell-to-cell adherence. These findings imply that the behaviors of S. mutans in vivo in early biofilms versus mature biofilms may be tremendously different. Clearly, the AtlA and Vic pathways are central to the response of S. mutans to an oxidative environment. In this study, SMu0629, the first gene product from the atlA operon, appears to have the potential to act as a redox sensor and to regulate the activity, maturation, or localization of AtlA and possibly other gene products encoded in the atlA operon or other autolytic enzymes. Additionally, AtlA activity and atlA operon expression are subject to control by the VicRK TCS. The VicK sensor kinase protein was reported to harbor a PAS domain, which is a sensor of oxygen and redox potential (54). Thus, strategies to disrupt the establishment and persistence of S. mutans that capitalize on the Vic or AtlA regulatory pathway may be promising in subverting the maturation of cariogenic biofilms. Notably, although there are some proteins with weak similarity to AtlA, this protein is an attractive target for anticaries therapies because of its unique features, the critical role it plays in allowing S. mutans to properly elaborate and localize virulence factors, and its central role in biofilm maturation (1).

SMu0629 is likely to function as a thiol-disulfide oxidoreductase because the predicted SMu0629 protein contains the highly conserved FX4CXXC motif that is typical of the active sites of several members of the thioredoxin superfamily. These cysteine residues are often arranged in a C-X-X-C motif in which the two cysteine residues reversibly cycle between oxidized disulfide and reduced dithiol forms and thus participate in redox reactions and electron flow. Using the Protein Motif search tool, a total of 10 proteins, including SMu0629, were found to contain this FX4CXXC motif in the S. mutans UA159 genome. They include HopD (SMu0490), NrdH (SMu0611), CitX2 (SMu0932), an ABC transporter permease protein (SMu1094), HemN (SMu1293), TrxA (SMu1699), TrxH (SMu1789), and conserved hypothetical proteins (SMu0860 and SMu1044). Unlike the cytoplasmically located thiol-disulfide oxidoreductases, such as thioredoxin, which have low redox potentials and are involved in maintaining the reducing environment of the cytoplasm, S. mutans SMu0629 is predicted to be located outside the cytoplasmic membrane according to the program HMMTOP (http://www.enzim.hu/hmmtop/). Other extracytoplasmic thiol-disulfide oxidoreductases include the recently discovered Escherichia coli DsbA protein, which catalyzes the formation of disulfide bonds in proteins transported across the membrane (5), and E. coli DsbC, which functions in the redistribution of disulfide bonds among the cysteine residues of target proteins (51). It is notable that SMu0629 also contains eight conserved cysteines, which can oxidize to form disulfide (S-S) bonds. Interestingly, a gene (fer) immediately downstream of the atlA operon encodes a ferredoxin that also contains a C-X-X-C motif. Ferredoxin proteins usually contain a [4Fe-4S] cluster(s) that can transfer electrons in a wide variety of metabolic reactions. Given that fer is so tightly linked to the atlA operon, there may also be a functional connection with SMu0629. Notably, it is unlikely that SMu0629 or Fer binds directly with AtlA through disulfide bonding because AtlA has no cysteines.

SMu0629, alone or in concert with other proteins, appears to modulate AtlA levels. A lack of SMu0629 leads to significant reductions in detectable AtlA protein, which in turn translate to markedly enhanced resistance to autolysis. The effects of SMu0629 on AtlA probably do not occur after AtlA is presented on the cell surface, since treatment of the 629NP or 629P strain and SAB95 (SMu0629– atlA–) with exogenous His-tagged AtlA protein (630D1) resulted in complete restoration of normal chain length (data not shown) and normal maturation of AtlA. These findings suggest that SMu0629 may be involved, directly or indirectly, in the control of AtlA activity or maturation before AtlA is targeted to the cell surface and that this effect is dependent on the redox environment or the presence of adequate levels of oxygen. Notably, the SMu0629 protein does not appear to be required for oxidative stress tolerance in the form of H2O2, but diminished growth of the strains lacking SMu0629 and modification of the biofilm-forming capacity of the mutant by air imply that the activity of the protein is essential for allowing the cells to cope with growth in oxidizing environments. We are currently in the process of defining the underlying basis for these observations.

An additional significant finding of this study is that there is a linkage between the VicRK TCS and autolysis of S. mutans. This finding is primarily supported by the observation that the phenotypes of the VicK-deficient S. mutans strain are due in large part to improper processing of AtlA. The expression of the atlA operon is influenced by the Vic system, although it is not clear whether the decreased transcription in the vicK background is a direct result of a lack of activation by VicR. Loss of VicK resulted in alterations in chain length and morphology of cells that resembled those reported previously for AtlA-deficient mutants (630NP) (8, 48). Strikingly, the processing of AtlA to its mature, 79-kDa form is almost completely inhibited in the vicK mutant, leading to a level of resistance to autolysis that is, in fact, much greater than that observed for the atlA-deficient mutant. Moreover, the response of the vicK-deficient mutant (vicK-NP) to oxygen exposure is very similar to that of atlA-deficient strains in terms of the morphology of cells and the capacity to form biofilms. The vic genes are constitutively expressed regardless of the presence of oxygen, while the SMu0629 gene and atlA are differentially expressed in response to aerobic conditions. Thus, the Vic system may be involved in the regulation not only of gene products required for processing of AtlA in response to oxygen concentration but possibly of other autolysin networks or of genes that modify the envelope in a way that enhances resistance to autolysis. SMu0629 and other redox-sensing proteins may exert their influence by modifying the activity of the gene product required for processing of AtlA.

In our current work, the amounts of two proteins in particular were dramatically elevated in 4% SDS extracts of the vicK mutant. One was identified as a cell wall protein precursor (SMu0555) which has homology with a putative N-acetylmuramidase/lysin of S. gordonii and with choline binding protein D of Streptococcus pneumoniae. The other protein associated with the cells in the strain lacking VicK was the glycosyltransferase-SI enzyme (GtfC), which plays an essential role in the formation of the water-insoluble glucans that form the adhesive scaffolding of recalcitrant S. mutans biofilms (39, 58). Our preliminary results indicate that the transcription of gtfC is not dramatically altered under these conditions. In addition, our SDS-PAGE and Western blot assays showed that both GtfB and GtfC were elevated in whole-cell lysates of the vicK mutant (unpublished data), suggesting a posttranscriptional effect on Gtf localization. We propose that a loss of VicK causes substantial remodeling of the cell surface, probably as a direct result of its impact on AtlA maturation. In turn, the changes in the envelope known to occur in strains lacking AtlA (1) allow enzymes like GtfC to become preferentially associated with the cell. This finding may have particular relevance to early biofilm formation and to biofilm maturation and persistence in the oral cavity in that the cell surfaces of organisms exposed to higher levels of air may have different adhesive and exopolysaccharide-forming potentials than those of cells in a more anaerobic environment. Thus, Vic and AtlA likely play a central role in legislating cell surface composition in response to the redox environment.

The VicKR TCS is known to play critical roles in pneumococcal virulence (40-42). Defects in cell wall synthesis, biofilm formation, and competence development, as well as sensitivity to antibiotics and attenuated virulence, have been associated with the loss of the Vic system (12, 14, 21, 38, 41, 47). Two genes, pspA (surface virulence factor) and pcsB (murein biosynthetic gene), are known to be under the control of VicRK in S. pneumoniae (40, 42). The PcsB homologue of S. mutans acts as a glucan-binding protein and is responsible for normal cell wall synthesis (35). Interestingly, the proteins encoded by genes annotated as the SMu0555 and SMu0760 genes, which contain the I-III-I-II motif also found in AtlA (8), are paralogously related to PcsB (BLAST P value, <1e–3). Thus, Vic may have the potential to function broadly in the regulation of lytic pathways and the governance of cell surface composition and architecture.

Our results have addressed several key issues regarding the regulation of AtlA and provided further support for the interconnectedness of this pathway with the expression of other virulence attributes, including biofilm formation and exopolysaccharide production by S. mutans. Perhaps most importantly, we revealed that oxygen is a key environmental factor that strongly influences cell envelope composition and biofilm development. Our results provide direct evidence that the VicRK TCS and the AtlA pathway, including SMu0629, lie at the center of the responses of this organism to atmospheric composition to control gene regulation and cell surface composition and architecture in a way that would strongly impact establishment, persistence, and virulence expression in a human host. Studies are ongoing to further dissect the regulation and processing of AtlA and to evaluate the potential utility of AtlA as a target to disrupt S. mutans pathogenesis.


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ACKNOWLEDGMENTS
 
This study was supported by NIDCR grant DE13239.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Oral Biology, University of Florida College of Dentistry, Gainesville, FL 32610. Phone: (352) 392-4370. Fax: (352) 392-7357. E-mail: rburne{at}dental.ufl.edu Back

{triangledown} Published ahead of print on 6 July 2007. Back


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Journal of Bacteriology, September 2007, p. 6293-6302, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00546-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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