Novobiocin inhibits membrane synthesis and vacuole formation of Enterococcus faecalis protoplasts.

We demonstrate that plasma membrane biosynthesis and vacuole formation require DNA replication in Enterococcus faecalis protoplasts. The replication inhibitor novobiocin inhibited not only DNA replication but also cell enlargement (plasma membrane biosynthesis) and vacuole formation during the enlargement of the E. faecalis protoplasts. After novobiocin treatment prior to vacuole formation, the cell size of E. faecalis protoplasts was limited to 6 µm in diameter and the cells lacked vacuoles. When novobiocin was added after vacuole formation, E. faecalis protoplasts grew with vacuole enlargement; after novobiocin removal, protoplasts were enlarged again. Although cell size distribution of the protoplasts was similar following the 24 h and 48 h novobiocin treatments, after 72 h of novobiocin treatment there was a greater number of smaller sized protoplasts, suggesting that extended novobiocin treatment may inhibit the re-enlargement of E. faecalis protoplasts after novobiocin removal. Our findings demonstrate that novobiocin can control the enlargement of E. faecalis protoplasts due to inhibition of DNA replication.

Species-dependent protoplast enlargement involves different types of vacuole generation in bacteria.

Vacuole generation occurs frequently during the enlargement of bacterial protoplasts and spheroplasts. Gram-positive Enterococcus faecalis protoplasts and gram-negative Lelliottia amnigena spheroplasts had large and small vacuoles inside the cytoplasm, respectively. Although no vacuoles were found at the early stage of cell enlargement, all enlarged cells used in the microinjection procedures had vacuoles. The plasma membrane of L. amnigena was more flexible than that of E. faecalis. In addition, E. faecalis protoplasts had unique discoidal structures as well as spherical structures in the cytoplasm. Our findings showed that the number of vacuoles increased as the L. amnigena plasma membrane expanded and that the size of vacuoles increased as the E. faecalis plasma membrane expanded, suggesting that bacterial cell enlargement involved vacuole generation. Thus, biosynthesis of the plasma and vacuolar membranes was synchronous with the bacterial cell enlargement. Differences in the plasma membrane flexibility might influence the different types of vacuole generation.

DNA replication and cell enlargement of Enterococcus faecalis protoplasts.

Protoplasts of Enterococcus faecalis did not divide but enlarged in Difco Marine Broth containing penicillin. Our previous studies have demonstrated that transcription and translation were essential for bacterial cell enlargement. However, it was uncertain whether replication was also essential. In this study, we measured the amount of DNA in E. faecalis cells during the course of enlargement using quantitative polymerase chain reaction. The growth of normally divided cells (native forms) of E. faecalis exhibited a log phase before 6 h of incubation was reached. Although a difference in quantitation cycle (Cq) values between the replication initiation and termination regions was observed in the log phase, it was not present in the stationary growth phase. On the other hand, the amount of DNA in E. faecalis protoplasts increased during the cell enlargement incubation. The difference of Cq values between the protoplasts at 0 and 96 h of incubation was 8-9, indicating that the DNA amount at 96 h was 200-500 times higher than that at 0 h. The Cq values differed between the replication initiation and termination regions, indicating that the replication level was high. When novobiocin, a DNA replication inhibitor, was added to the medium at 24 h of incubation, DNA replication and cell enlargement were almost stopped. Thus, replication plays an important role in the enlargement of E. faecalis protoplasts.