Presentation Information
[P03-369]Analysis of Cell-Wall–Deficient Cells in Gram-Positive Bacteria
○Hibiki Umeyama1, Ikami Mitsue2, Toshiki Nagakubo3,4, Nobuhiko Nomura3,4,5,6, Masanori Toyofuku3,4,6 (1. Grad. Sch. Life Environ. Sci., Univ. Tsukuba (Japan), 2. Agro Biol. Res. Sci., Univ. Tsukuba (Japan), 3. Fac. Life Environ. Sci., Univ. Tsukuba (Japan), 4. TIAR, Univ. Tsukuba (Japan), 5. TARA, Univ. Tsukuba (Japan), 6. MiCS, Univ. Tsukuba (Japan))
Keywords:
cell wall deficient cell,autolysin,spherical cell,prophage
[Purpose]
Most extant bacteria possess a cell wall composed primarily of peptidoglycan. The cell wall plays essential roles in protecting cells from osmotic stress and maintaining cell morphology; however, cell wall-deficient forms such as L-forms have been reported and are capable of growth without a cell wall. These cell wall-deficient cells exhibit properties not observed in typical walled cells, including antibiotic resistance, transformation competence, and phage infection avoidance. Thus, adopting a cell wall-deficient state is considered an important survival strategy for bacteria.These phenomena, however, have largely been observed under artificially induced cell wall stress.
Our laboratory has identified that within a Bacillus subtilis population, spherical cell wall-deficient cells (Round cells, R-cells) emerge as a subpopulation. To date, we have identified two mechanisms responsible for R-cell formation in B. subtilis. These mechanisms involve either prophage-derived holin-endolysin encoded in the genome or autolysins, which are peptidoglycan-degrading enzymes broadly conserved among walled bacteria. In this study, we aimed to elucidate the biological significance of R-cells by analyzing their physiological states under each formation mechanism.
[Method]
To assess the membrane integrity of R-cells, we performed cell staining using SYTO9 and propidium iodide (PI), which are membrane-permeable and -impermeable fluorescent dyes, respectively.
Respiratory activity was assessed using CTC staining, which detects NADH primarily generated through bacterial electron transport.
To evaluate antibiotic susceptibility, cells were exposed to ß-lactam antibiotic imipenem.
[Results]
R-cells induced by holin-endolysin were stained with PI, indicating increased membrane permeability. In contrast, R-cells induced by autolysins were only stained with SYTO9, suggesting that membrane integrity was maintained. Regarding respiratory activity, CTC fluorescence was detected only in R-cells formed via autolysins, suggesting that these cells retain respiratory activity. Furthermore, R-cells exhibited resistance to antibiotics that target cell wall synthesis.
[Consideration]
Autolysin-induced R-cells appear to better maintain their physiological activity than holin-endolysin-induced R-cells, as indicated by intact membranes and detectable respiration. In addition, the antibiotic resistance of R-cells to cell-wall-targeting antibiotics is likely due to their lack of a cell wall.
[Conclusion]
These findings indicate that the physiological characteristics of R-cells vary depending on their formation mechanism, and that the absence of the cell wall confers properties distinct from those of walled cells. Collectively, these results indicate that cell wall-deficient R-cells may represent a previously unrecognized bacterial survival strategy whereby bacteria acquire resistance to antibiotics.
Most extant bacteria possess a cell wall composed primarily of peptidoglycan. The cell wall plays essential roles in protecting cells from osmotic stress and maintaining cell morphology; however, cell wall-deficient forms such as L-forms have been reported and are capable of growth without a cell wall. These cell wall-deficient cells exhibit properties not observed in typical walled cells, including antibiotic resistance, transformation competence, and phage infection avoidance. Thus, adopting a cell wall-deficient state is considered an important survival strategy for bacteria.These phenomena, however, have largely been observed under artificially induced cell wall stress.
Our laboratory has identified that within a Bacillus subtilis population, spherical cell wall-deficient cells (Round cells, R-cells) emerge as a subpopulation. To date, we have identified two mechanisms responsible for R-cell formation in B. subtilis. These mechanisms involve either prophage-derived holin-endolysin encoded in the genome or autolysins, which are peptidoglycan-degrading enzymes broadly conserved among walled bacteria. In this study, we aimed to elucidate the biological significance of R-cells by analyzing their physiological states under each formation mechanism.
[Method]
To assess the membrane integrity of R-cells, we performed cell staining using SYTO9 and propidium iodide (PI), which are membrane-permeable and -impermeable fluorescent dyes, respectively.
Respiratory activity was assessed using CTC staining, which detects NADH primarily generated through bacterial electron transport.
To evaluate antibiotic susceptibility, cells were exposed to ß-lactam antibiotic imipenem.
[Results]
R-cells induced by holin-endolysin were stained with PI, indicating increased membrane permeability. In contrast, R-cells induced by autolysins were only stained with SYTO9, suggesting that membrane integrity was maintained. Regarding respiratory activity, CTC fluorescence was detected only in R-cells formed via autolysins, suggesting that these cells retain respiratory activity. Furthermore, R-cells exhibited resistance to antibiotics that target cell wall synthesis.
[Consideration]
Autolysin-induced R-cells appear to better maintain their physiological activity than holin-endolysin-induced R-cells, as indicated by intact membranes and detectable respiration. In addition, the antibiotic resistance of R-cells to cell-wall-targeting antibiotics is likely due to their lack of a cell wall.
[Conclusion]
These findings indicate that the physiological characteristics of R-cells vary depending on their formation mechanism, and that the absence of the cell wall confers properties distinct from those of walled cells. Collectively, these results indicate that cell wall-deficient R-cells may represent a previously unrecognized bacterial survival strategy whereby bacteria acquire resistance to antibiotics.
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