Objectives: Undecaprenyl pyrophosphate phosphatases (UppPs) have been implicated in bacitracin resistance in some bacterial genera and the aim of this study was to determine the role of UppPs in mediating low-level bacitracin resistance in Enterococcus faecalis.
Methods: The uppP gene was identified in the genomes of laboratory (JH2-2) and clinical (V583) strains of E. faecalis. Gene fusions (uppP-lacZ) and 5'-RACE were used to study uppP expression. The uppP gene in both strains was inactivated and mutants were studied for antimicrobial susceptibility and their susceptibilities to various stress agents.
Results: The UppP protein from E. faecalis showed high sequence identity to the Escherichia coli BacA-type UppP and was predicted to be a hydrophobic protein with eight transmembrane helices. The expression of uppP-lacZ was constitutive and not affected by bacitracin or cell wall-active antimicrobials. E. faecalis uppP mutants showed no significant changes in growth rate, colony morphology and biofilm formation. The uppP mutants exhibited increased susceptibility to bacitracin (MICs=3-6 mg/L) relative to the isogenic wild-type (MICs=32-48 mg/L). When uppP was expressed in a wild-type background, the MIC of bacitracin increased to 128-≥256 mg/L. The MICs of cefoxitin, teicoplanin, vancomycin, gentamicin, enrofloxacin and d-cycloserine were unaltered in the uppP mutant relative to the wild-type, as were susceptibilities to other stress agents (glycine, lysozyme, NaCl, SDS, low and high pH, oxidative stress and ethanol).
Conclusions: The results demonstrate that low-level bacitracin resistance in E. faecalis is mediated by a BacA-type UppP.
Keywords: antimicrobials; cell wall inhibitors; gene expression.