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. 2009 Apr;75(8):2517-27.
doi: 10.1128/AEM.02367-08. Epub 2009 Feb 27.

Changes in biochemical and phenotypic properties of Streptococcus mutans during growth with aeration

Sug-Joon Ahn  1 , Sang-Joon Ahn, Christopher M Browngardt, Robert A Burne
Affiliations

Changes in biochemical and phenotypic properties of Streptococcus mutans during growth with aeration

Sug-Joon Ahn et al. Appl Environ Microbiol. 2009 Apr.

Abstract

Oxygen has a potent influence on the expression of genes and the activity of physiological and biochemical pathways in bacteria. We have found that oxygen significantly altered virulence-related phenotypic properties of Streptococcus mutans, the primary etiological agent of human dental caries. Transport of glucose, fructose, or mannose by the sugar:phosphotransferase system was significantly enhanced by growth under aerobic conditions, whereas aeration caused an extended lag phase and slower growth of S. mutans in medium containing glucose, fructose, or mannose as the carbohydrate source. Aeration resulted in a decrease in the glycolytic rate and enhanced the production of intracellular storage polysaccharides. Although aeration decreased the acid tolerance of S. mutans, aerobically grown cells had higher F-ATPase activity. Aeration altered biofilm architecture but did not change the ability of S. mutans to interact with salivary agglutinin. Growth in air resulted in enhanced cell-associated glucosyltransferase (Gtf) activity at the expense of cell-free Gtf activity. These results demonstrate that S. mutans can dramatically alter its pathogenic potential in response to exposure to oxygen, suggesting that the phenotype of the organism may be highly variable in the human oral cavity depending on the maturity of the dental plaque biofilm.

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Figures

FIG. 1.
FIG. 1.
Growth curves of S. mutans UA159 grown under aerobic or anaerobic conditions. Growth in TV medium supplemented with glucose (A), fructose (B), or mannose (C) was monitored in a Bioscreen C system that was set to continuously shake (aerobic conditions). For anaerobic conditions, sterile mineral oil was placed on top of the broth culture.
FIG. 2.
FIG. 2.
Confocal microscopic images of S. mutans UA159 biofilms grown under aerobic or anaerobic conditions. For biofilm formation, S. mutans was grown in BM with either 20 mM glucose (A) or sucrose (B) for 24 h. Cells were stained with SYTO 13 for 20 min. Data presented here are representative of at least three independent experiments.
FIG. 3.
FIG. 3.
Sugar transport activity by the PTS (A) or ATPase activity (B) in S. mutans UA159 grown under aerobic or anaerobic conditions. PTS activity is expressed as nanomoles of NADH oxidized in a phosphoenolpyruvate-dependent manner min−1 (mg protein)−1. ATPase activity is expressed as μmoles of released phosphate min−1 (mg protein)−1. Data are expressed as means for at least three independent experiments. Error bars represent standard deviations. ***, P < 0.001, Mann-Whitney U test.
FIG. 4.
FIG. 4.
Oxidative stress assays with S. mutans UA159 grown under aerobic or anaerobic conditions. For hydrogen peroxide killing (A), aerobically or anaerobically grown cells were harvested and suspended in BHI containing 0.2% hydrogen peroxide for 30, 60, or 90 min. The growth rate of S. mutans under 0.001% hydrogen peroxide (B) or 25 mM paraquat (C) was monitored using a Bioscreen C system to analyze the ability of cells to withstand oxidative stress under aerobic or anaerobic conditions. Data presented here are representative of at least three independent experiments.
FIG. 5.
FIG. 5.
Changes in oxidative tolerance in S. mutans UA159 when cells are reexposed to oxygen. Cells were grown under aerobic or anaerobic conditions, diluted into fresh BHI broth containing 0.001% hydrogen peroxide (A) or 25 mM paraquat (B), and regrown under anaerobic (overlaid with mineral oil) or aerobic (without mineral oil) conditions. The growth rate of S. mutans was monitored using a Bioscreen C system (solid black line, aerobically grown cells were diluted and regrown under aerobic conditions; dotted black line, anaerobically grown cells were diluted and regrown under aerobic conditions; solid gray line, aerobically grown cells were diluted and regrown under anaerobic conditions; dotted gray line, anaerobically grown cells were diluted and regrown under anaerobic conditions). Data presented here are representative of at least three independent experiments.
FIG. 6.
FIG. 6.
Acid tolerance assays with S. mutans UA159 grown under aerobic or anaerobic conditions. For killing assays (A), aerobically or anaerobically grown cells were exposed to pH 2.8 for 30, 60, or 90 min. The growth rate of S. mutans at pH 6.0 (B) was monitored using a Bioscreen C system under aerobic or anaerobic conditions. Data presented here are representative of at least three independent experiments.
FIG. 7.
FIG. 7.
Glycolytic acidification by S. mutans UA159 grown under aerobic or anaerobic conditions in the presence of added glucose (A) or from endogenous stores (B). Experiments were performed as detailed in Materials and Methods, and the data shown are representative of at least three independent experiments.
FIG. 8.
FIG. 8.
Bacterial aggregation (A) or adhesion (B) assay to analyze the interaction of S. mutans with salivary preparations. In bacterial aggregation assays, cell suspensions were mixed with SAG, UWS, or PBS and transferred to cuvettes. The OD600 of the samples was recorded at 10-min intervals at 37°C for 120 min in a spectrophotometer equipped with a temperature-controlled multicuvette positioner. Percentage aggregation (percent decrease in OD600) was calculated as follows: [(OD600 at 0 min − OD600 at 120 min)/(OD600 at 0 min)] × 100. For bacterial adhesion, aerobically or anaerobically grown cells were harvested at an OD600 of 0.5 and stained with SYTO 13. Adhesion assays were performed in two different ways: salivary preparations were added to each well with the cell suspensions (fluid-phase salivary preparations), or wells were first coated with salivary preparations before inoculation with cell suspensions (surface-adsorbed salivary preparations). For the experiments with surface-adsorbed salivary preparations, each well was conditioned with 100 μl of SAG, UWS, or PBS for 2 h, washed, and air dried. For the experiments with fluid-phase salivary preparations, 150 μl of the cell suspension was inoculated into the wells concurrently with 15 μl of SAG, UWS, or PBS. Plates were incubated for 3 h in an aerobic or anaerobic environment and washed, and adherent bacteria were measured using a BioTek microplate scanning spectrophotometer. The data represented herein were from incubation under aerobic conditions, because there was no significant difference in bacterial adhesion to polystyrene wells between aerobic and anaerobic incubation. The error bars represent standard deviations. Data presented here are representative of at least three independent experiments.
FIG. 9.
FIG. 9.
Results of glucan synthetic activities by an FTF-deficient mutant of strain UA159 grown under aerobic or anaerobic conditions. Cell-associated glucan synthetic activity (A) was measured using [14C]sucrose after homogenization of aerobically or anaerobically grown cells in the presence of glass beads, while glucan synthetic activity of cell culture supernatant fluids (B) was measured after 50-fold concentration. Enzyme activities from cells and supernatants were normalized to the total protein content as determined by a bicinchoninic acid assay with bovine serum albumin as the standard or to percentage cpm per ml culture fluid, respectively. Data presented here are representative of at least three independent experiments.
FIG. 10.
FIG. 10.
Changes in phenotypic and biochemical properties of S. mutans during biofilm maturation. S. mutans cells dramatically change core physiologic properties according to oxygen levels. Cells in immature biofilms (A) may show greater oxygen metabolism, adhesion capacity, PTS permease activity, F-ATPase activity, and intracellular polysaccharide storage to survive and persist in a higher oxygen concentration. As biofilms mature (B), redox potential and oxygen levels fall, and cells display more-efficient growth, improved biofilm formation, and enhanced acidogenic and acid tolerance properties.
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References

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