Role of VltAB, an ABC transporter complex, in viologen tolerance in Streptococcus mutans

Abstract

Streptococcus mutans, a Gram-positive organism, 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 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. Here, we wanted to understand the mechanisms by which S. mutans withstands exposure to methyl viologen (MV), a quaternary ammonium compound (QAC) that generates superoxide radicals in the cell. To elucidate the essential genes for MV tolerance, screening of ∼3,500 mutants generated by ISS1 mutagenesis, revealed 15 MV-sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906 genes, respectively. These two genes are appeared to be organized in an operon and encode a putative ABC transporter complex; we designated the genes as vltA and vltB, for viologen transporter. To verify our results, vltA was deleted by using an antibiotic resistance marker; the mutant was just as sensitive to MV as the ISS1 insertion mutants. Furthermore, vltA and vltB mutants were also sensitive to other viologen compounds such as benzyl and ethyl viologens. Complementation assays were also carried out to confirm the role of VltA and VltB in viologen tolerance. Sensitivity to various drugs, including a wide range of QACs, was evaluated. It appears that a functional VltA is also required for full resistance toward acriflavin, ethidium bromide, and safranin; all are well-known QACs. These results indicate that VltA/B constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that homologs of VltA/B are widely present in streptococci, enterococci, and other important Gram-positive pathogens.

FIG. 1.

Organization of vltA and vltB loci in S. mutans. The predicted topology and the conserved motifs in VltA and VltB are shown. The conserved motifs of the ABC transporter superfamily are indicated for both proteins. The ABC membrane superfamily domain contains six and five predicted α-helices for VltA and VltB, respectively. The upstream gene, SMU.902, corresponds to YpjG in the NCBI database. Boxes above the diagram show the site of ISS1 insertions and their relative positions. The designation within each box indicates the name of the mutant.

FIG. 2.

Linage analysis of vltA and vltB loci. (A) Genetic map of the vltA and vltB loci in S. mutans UA159 with the relative positions of the primers used for the PCR analysis to determine potential linkage of the genes. (B) Results of PCR analysis showing linkages of the various genes that are cotranscribed. RNA was used as a template to produce cDNA. PCR was then performed on RNA (control), cDNA, and chromosomal DNA (gDNA), using the primer pairs depicted in panel A, to determine which of the genes are cotranscribed with vltA. As a control, PCR was also performed on cDNA using the A+F primers, which did not produce any fragment; however, the A+G primers produced a fragment of the expected size (data not shown). The primers are as follows: A, Smu905-9 BamCF; B, Lnk-905R; C, Lnk 905-906F; D, Lnk 905-906R; E, Lnk906-MF; F, Lnk906-MF; and G, Smu.906DR.

FIG. 3.

Verification of viologen-sensitive phenotype. (A) ISS1 transposon mutants that displayed an initial MV-sensitive phenotype were further verified by spotting 10.0 μl from a 10-fold dilution series, with a starting optical density (A₆₀₀) of 2.0 made in 0.85% NaCl, onto THY agar plates containing 5 mM MV (THY+MV). As a control, cultures were also spotted on plain THY agar plates with no additions (THY). UA159 is the wild-type strain, while IBSA34 and its plasmid cured derivative IBSA42 are independent MV-sensitive mutants. IBSA43 is a plasmid-cured derivative of IBSA32, another MV-sensitive mutant. IBSA26 is a deletion mutant of vltA locus, which was identified by inverse PCR. (B) Complementation analysis to verify MV-sensitive phenotypes. The complemented strains were generated as described in the text. Experiments A and B were repeated at least three times, and the relevant areas of the representative plates are shown. (C) PCR verification of the complemented strains. Sample strains are numbered as follows: 1 and 4, UA159; 2, IBSA26; 3, IBSA50; 5, IBSA43; and 6, IBSA51. M, GeneRuler 1-kb-Plus DNA ladder (Fermentas). The primers Smu.905-6BamCF and Lnk905-906R were used for lanes 1 to 3, while the primers Smu.906F and Smu.906SR were used for lanes 4 to 6.

FIG. 4.

A disk diffusion assay was used to measure the susceptibility of S. mutans to viologen compounds. Lawns of wild-type (UA159) and vltA mutant (IBSA26) strains were prepared by overlaying THY agar plates with 10 ml of soft THY agar containing 0.5-ml overnight cultures. Portions (10 μl) of ethyl viologen (EV; 1.5 M) or benzyl viologen (BV; 50 mg/ml) were spotted onto a filter paper disk (6 mm) and placed on the plates. The plates were then incubated under microaerophilic conditions at 37°C for 16 h. The inhibitory-zone diameters for both cultures were measured and compared.

FIG. 5.

Susceptibility of S. mutans to QAC, as determined by disk diffusion assay. Plates with overlaid cultures were prepared as described in the text. Ethidium bromide (EB; 10 mg/ml), safranin (SF; 1.5%), or acriflavin (AF; 100 mg/ml) was spotted onto a paper disk, and the plates were incubated overnight under microaerophilic conditions and photographed.

FIG. 6.

Verification of SMU.902 mutant MV sensitivity. Dilutions of fresh overnight cultures were spotted onto THY agar plates with or without MV (5 mM). The plates were incubated at 37°C under microaerophilic conditions. Experiments were repeated no fewer than three times, and relevant areas of representative plates are shown. Strains are indicated as follows: wild type, UA159; SMU.905 mutant, IBSA26; and SMU902 mutants, IBSA31 and IBSA44. Note that there was a slight increase in resistance in IBSA44, a plasmid-cured derivative, compared to IBSA31.