For successful colonization and maintenance of a dominant presence in the oral cavity, S. mutans
has developed multiple strategies. These strategies help this organism to grow under nutrition-limiting conditions and protect it from various environmental insults (28
). Although most of the previous studies were focused on understanding the mechanisms of acid tolerance and oxidative-stress responses, our knowledge of the mechanisms of tolerance to various toxic chemicals remains limited. To obtain further insight into this process, a collection of random insertion mutants of S. mutans
UA159 was screened to select clones with high sensitivity to MV, a charged dipyridyl-ring-containing QAC that also generates ROS. This approach allowed us to identify genes that may be responsible for defense against QAC, without prior knowledge of the genes' function(s). In the present study, we only screened approximately 3,500 such mutants; therefore, the screening process was not particularly exhaustive since S. mutans
genome encodes about 1,900 genes. Of the five loci that were identified in our analysis, at least one gene, ciaH
, was previously reported as an important player in the oxidative-stress response in streptococcus and other bacteria (2
), signifying that the screening method used here is a viable approach. Four unique loci were identified by our search: three ABC transporter-encoding genes (SMU.902, vltA
, and vltB
) and a hypothetical protein (SMU.283). In the present study, we further characterized the vltAB
loci in order to better understand the mechanism and substrate specificity of these ABC transporters.
Sequence analysis and genome organization strongly suggest that VltA and VltB encode a heterodimeric ABC-type exporter pump. Our linkage analysis also demonstrated that these two genes are transcriptionally linked. S. mutans
genome analysis indicates that this organism encodes several ABC transporters, of which at least 42 are putative exporter pumps (3
). Since we specifically obtained multiple insertions in vltA
(five in vltA
and four in vltB
), this ABC transporter appears to be the most important for viologen tolerance in S. mutans
. Analysis of the sequences by a transmembrane (TM) helices prediction program, TMHMM (www.cbs.dtu.dk/services/TMHMM
), of both VltA and VltB revealed that these two proteins contain 6 (residues 1 to 294) or 5 (residues 1 to 382) TM helices, respectively. Both VltA and VltB also contain putative nucleotide-binding domains, Walker A and Walker B motifs, and ABC signature sequences (16
) (Fig. ). We also found that both VltA and VltB were necessary for viologen resistance, supporting our notion that VltA and VltB is a heterodimeric ABC-type multidrug efflux pump.
The ABC transporter that we identified export, in addition to viologen compounds, some other QACs such as acriflavin, ethidium bromide, and safranin. Analysis of the structures of these compounds failed to identify any common structural moiety that could easily explain the substrate specificity (Table ). However, all of the compounds are charged heterocyclic molecules. Definitely charge plays a role in the substrate recognition since the ABC transporter complex did not recognize dipyridyl and benzidine, which are structurally very similar to viologen but uncharged. Similarly, VltA expelled DQ, which is structurally very similar to phenanthroline but charged, out from the cell, whereas phenanthroline was not recognized. On the other hand, charged alone is not sufficient to explain the substrate specificity as well. This is because we also tested several QACs ranging from compounds that contain simple structures such as tetraethylammonium bromide to compounds that contain heterocyclic rings such as malachite green and crystal violet; VltA recognized none of these QACs. Thus, in addition to charge, other physical characteristics such as hydrophobicity or amphiphilicity may be also very important.
Although IBSA43 (vltB mutant) displayed increased sensitivity to viologen compounds, this mutant, surprisingly, did not show any significant differences compared to the wild type when tested for sensitivity to acriflavine, ethidium bromide, and safranin. Thus, it appears that VltB is not involved in the resistance of these compounds. Although VltA and VltB are expected to interact with each other to form a functional heterodimeric ABC transporter, it is possible that VltA and VltB can each form homodimers and that these homodimers have different substrate specificities. For example, while VltA homodimer is involved in QAC resistance, VltB homodimer does not take part in QAC resistance. Future studies will address the question of whether VltA and VltB can also form homodimers and, if so, to what extent their substrate specificities differ from that of the VltA/B heterodimer.
BLAST searches using protein sequences as a query against the nonredundant database at the National Center for Biotechnology Information showed that homologues of VltA/B are widely present in streptococci, enterococci, and clostridia (see Fig. S2 in the supplemental material). In all cases, two open reading frames were located in tandem, and many genes seemed to encode multidrug resistance ABC-type proteins. The closest homologues (>90% identity), SAG1338 and SAG1337, are found in group B streptococci (GBS), and all of the sequenced GBS strains encode these ABC transporter genes. In contrast to other organisms in which VltA/B homologues are found, the genomic locus for this ABC transporter is somewhat conserved in GBS. Specifically, the four upstream genes are highly conserved, including the SMU.902 homologue, SAG1340. On the other hand, two genes immediately downstream of SAG1337 are homologues of SMU.911 and SMU.913. However, homologues of SMU.909 that encode a malate permease and SMU.910, which encodes glucosyltransferase, are absent in GBS. Our BLAST search also identified two ABC transporter proteins from Enterococcus faecalis
, EfrA (EF2920) and EfrB (EF2919), which showed >80% identity to SMU.905 and SMU.906, respectively (26
). It has been demonstrated that expression of both efrAB
genes confers resistance to many drugs, including acriflavin, ethidium bromide, and safranin (26
). Unfortunately, MV or other quat compounds were not tested in that study; thus, whether EfrAB is involved in viologen efflux remains to be seen.
Although we have identified VltA/B as an ABC transporter involved in the efflux of viologens and QACs, the organism may not encounter these chemicals frequently during its growth in natural habitat in the dental plaque. Dental plaque is a polymicrobial community that harbors more than 500 species or phylotypes (1
), and the cell density can reach as high as 1011
). The oral biofilm is continuously challenged by changes in the environmental conditions and, as a response to such challenges, the bacterial community evolved, with individual members with specific functions such as primary or secondary colonizers, including the ability to metabolize or tolerate toxic excreted products produced by other species (25
). About 20% of the oral bacteria are streptococci (32
), and these organisms with their specific spatial and temporal distribution determine the development of the biofilms. When present in high numbers, the pioneer colonizers can antagonize S. mutans
, as suggested by clinical studies (6
). However, S. mutans
can become dominant in oral biofilms, which leads to dental caries development. This dominance depends on competition with other organisms and is influenced by various factors. We speculate that the presence of numerous transporters, such as VltA/B, allows S. mutans
to withstand toxic compounds produced by competing species or present in the plaque environment.