ROLE OF AN ABC TRANSPORTER COMPLEX IN VIOLOGEN TOLERANCE IN STREPTOCOCCUS MUTANS

Abstract

The gram-positive bacterium Streptococcus mutans is the primary causative agent in the formation of dental caries in humans. To persist in the oral cavity, S. mutans must be able to tolerate rapid and substantial environmental fluctuations and exposure to various toxic chemicals. However, the mechanisms underlying the ability of this cariogenic pathogen to survive and proliferate under harsh environmental conditions remain largely unknown. In the current study we wanted to understand the mechanisms by which S. mutans withstand exposure to various quaternary ammonium compounds (QAC) such as methyl viologen (MV) that also generates superoxide radicals in the cell. To elucidate the genes that are essential for MV tolerance, sensitive mutants of S. mutans were generated via ISS1 mutagenesis. Screening of approximately 3,500 mutants revealed fifteen MV sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906, respectively. These two genes are organized in an operon and encode a putative ABC-transporter complex. Linkage PCR analysis supports the operon organization of these two genes and also indicates that the transcription start site maps further upstream of SMU.905. To confirm our results, SMU.905 was deleted using an antibiotic resistance marker; the SMU.905 deletion mutant was just as sensitive to MV as the ISS1 insertion mutants. Furthermore, SMU.905 and SMU.906 mutants were sensitive to other viologen compounds such as benzyl- and ethyl- viologen. Sensitivity to various drugs including a wide range of QACs was tested. It appears that a functional SMU.905 is also required for full resistance towards acriflavin, ethidium bromide, and safranin; all are well known QAC. These results indicate that SMU.905/SMU.906 probably constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that this ABC-type efflux pump is widely present in streptococci, enterococci, and clostridia including some important human pathogens.