Lipoteichoic acids (LTAs) are key anionic polymers of the Gram-positive cell envelope, essential for cell growth, division, and stress responses. In Enterococcus faecalis, two putative LTA synthases, LtaS1 and LtaS2, have been identified, but their specific contributions remain unclear. Here, we constructed single (ΔltaS1, ΔltaS2) and double (ΔltaS1ΔltaS2) deletion mutants to investigate their roles in LTA biosynthesis, growth and response to envelope-targeting stressors.

Deletion of ltaS1 caused a marked loss of LTA, confirmed by Western blot, impaired growth, subtle changes in cell morphology, reduced surface hydrophobicity, and hypersensitivity to envelope targeting antibiotics, including daptomycin, vancomycin, and bacitracin, as well as to chemical stresses such as SDS and bile salts. In contrast, deletion of ltaS2 had minimal impact under standard laboratory conditions, and the double mutant exhibited phenotypes largely driven by the absence of LtaS1. These observations suggest a predominant role of LtaS1 over LtaS2 under the tested conditions. Conversely, acidic pH affected all strains similarly, suggesting that LTA is not a major determinant of acid tolerance in E. faecalis.

Overall, our findings highlight the central role of LtaS1 in LTA production and maintenance of cell envelope function in E. faecalis and provide a foundation for future studies on how LTA synthesis contribute to bacterial adaptability.