Genetic and Phenotypic Analysis of Genes Involved in Bacitracin Resistance in Streptococcus mutans

Abstract

Streptococcus mutans is considered to be the primary causative agent of dental caries in humans. In the oral cavity, S. mutans frequently exposed to antimicrobial compounds secreted from competing species and from the host. To survive in the oral cavity, S. mutans must be able to sense and respond to these antimicrobial compounds. For example, S. mutans is well known to resist bacitracin; however, only a few studies have explored the molecular mechanisms of resistance of S. mutans to bacitracin or other related antibiotics. By screening a high-density transposon mutant library constructed previously in our laboratory, we identified a transposon insertion mutant in SMU.244 gene that was sensitive to bacitracin. The objective of this study was to characterize potential roles of SMU.244 in response and resistance to bacitracin and other cell wallacting antibiotics. We also extended our investigation into another genetic locus SMU.862-864 that encoded an ABC transporter (exporter) and might be involved in bacitracin response in S. mutans. Our results confirmed that SMU.244 encoded a homolog of BacA or UppP protein that is involved in the cell wall biosynthesis. Deletion of SMU.244 resulted in Sm?bacA mutant that was 32-fold more sensitive to bacitracin and 2-fold more sensitive to penicillin G, vancomycin, and nisin than the parent strain UA159. This defect in antibiotic resistance was completely restored in the complement strain (Sm-pCpbacA). Sm?bacA also showed a slower growth rate (Td = 1:22 h-1) than the parent (Td = 1:05 h-1) and its growth nearly ceased in the presence of 0.48 U/ml of bacitracin. In addition, Sm?bacA mutant formed a biofilm with reduced biomass, especially in the presence of bacitracin (0.48 U/ml), suggesting that it may play a role in biofilm formation. The work using qRT-PCR revealed that the bacA gene might not be directly regulated by the BceABRS system and its expression appeared to be independent from induction by bacitracin. Taken together, we conclude that SMU.244 encodes a BacA homolog that plays important roles in resistance to cell wall-acting antibiotics in S. mutans. Our work also showed that SMU.862-864 might not be involved in resistance to cell wall-acting antibiotics. With the methods used in this study, there was no sufficient evidence to support that this ABC transporter played direct roles in biofilm formation, acid resistance and stress responses in S. mutans.