The presence of at least two binding sites for MleR within the coding region of Smu.136c suggests a complex ARRY-438162 solubility dmso regulatory mechanism, which has to be elucidated further by means of DNase footprinting and mutagenesis. Conclusion In summary, we showed that

the mle genes including oxdC are under the control of acid inducible promoters and that they are induced within the first 30 minutes upon acid shock. Therefore they are part of the early acid tolerance response in S. mutans, which is induced within 30 minutes after acidification [8]. Further enhancement of their transcription can be obtained by MleR and L-malate in an acidic environment. The use of gel retardation assays showed the presence of multiple binding sites for MleR, even in the coding sequence of another gene, suggesting a complex regulatory mechanism. We clearly showed that the presence of L-malate Selleckchem 4EGI-1 contributed strongly to the survival this website of S. mutans under low pH conditions. MLF is one of the strategies aciduric bacteria have evolved to cope with low pH and to compete with other bacteria in dental plaque. S. mutans is able to carry out MLF under more acidic conditions than other Streptococci [17], thus emphasizing the dominant role of S. mutans in the oral

cavity. Methods Bacterial strains, plasmids, and culture conditions Bacterial strains and plasmids and their relevant characteristics are listed in table 2. Escherichia coli was routinely cultured in Luria Bertani (LB, Carl-Roth, Karlsruhe, Germany) medium at 37°C. E. coli strains carrying plasmids were selected with 100 μg ml-1 ampicillin, or 50 μg ml-1 spectinomycin. All Streptococcus mutans

strains were cultivated in Todd Hewitt Broth medium supplemented with 0.1% (w/v) yeast extract (THBY, Becton Dickinson, Heidelberg, Germany) or in BM [27] medium containing 0.5% sucrose (BMS) or 1% (w/v) glucose (BMG). S. mutans strains were grown at 37°C without agitation aerobically (5% CO2 enriched) in THBY or in BM medium under anaerobic conditions (80% N2, 10% H2, 10% CO2). Pre-cultures were grown in THBY medium. Selection of mutant strains was carried out with 10 μg ml-1 erythromycin, or 500 μg ml-1 spectinomycin. Table 2 Bacterial strains and plasmids used in this study. Strain/plasmid Relevant Characteristicsa Methane monooxygenase Reference/source Strains        E. coli     DH5α General cloning strain   Tuner(DE3) Expression strain Novagen    S. mutans   ATCC 700610 UA159 Wild-type, Erms, Sps This study ALSM3 UA159ΔmleR, Ermr This study ALSM20 UA159::ϕ(mleR P-luc), Spr This study ALSM13 UA159ΔmleR::ϕ(mleR P-luc), Ermr, Spr This study ALSM33 UA159::ϕ(mleS P-luc), Spr This study ALSM34 UA159ΔmleR::ϕ(mleS P-luc), Ermr, Spr This study     This study     This study Plasmids        pFW5 Suicide vector, Spr A. Podbielski [29]    pHL222 Apr, luc H.