Abstract
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.
Highlights
Vacuole generation occurs frequently during the enlargement of bacterial protoplasts and spheroplasts
In Difco Marine Broth 2216 (DMB), the plasma membrane expanded with vacuole generation in the cytoplasm[8] (Fig. 1)
Fluorescence microscopy showed that blue fluorescent protein (BFP) was contained in the cytoplasm but not in the vacuoles, and no fluorescence was observed in the debris of cells broken during the injection (Fig. 7b). These results showed that both E. faecalis and L. amnigena enlarged cells could be used for microinjection and the vacuolar membranes were devoid of the fluorescent protein
Summary
Vacuole generation occurs frequently during the enlargement of bacterial protoplasts and spheroplasts. Electron microscope observations revealed that the vacuoles in the E. coli and B. subtilis protoplasts were surrounded by single membranes, which did not contain DNA2,3 These vacuoles could be isolated from the protoplasts by the removal of the plasma membrane[2,3]. Patch clamp analyses showed that the E. coli and B. subtilis vacuoles have everted membrane[2,3] These results strongly suggested that the vacuolar membrane was generated by endocytosis of the plasma membrane[2,3]. In order to examine the effects of metal salts on the plasma membrane expansion and vacuole generation, the enlargement was performed under 16 different conditions, based on the reported effects of metal salts on the enlargement of D. grandis spheroplasts[7] (Supplementary Table 1)
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