Sugar enhances outer membrane fusion in Deinococcus grandis spheroplasts to generate calcium ion-dependent extra-huge cells.

In our previous study, we showed that cell fusion occurred in spheroplasts of Deinococcus grandis at 200 mM calcium chloride in the incubation medium. Extra-huge cells (> 0.1 mm in diameter) were observed at this concentration with a low frequency of appearance. In this study, we showed that cell fusion occurred consecutively in D. grandis spheroplasts following an incubation for spheroplast enlargement using medium containing 16.2 mM calcium chloride and 333 mM sucrose. As a result, more extra-huge cells were generated, where cells had maximum diameter of > 1 mm. They can be observed with naked eyes in the incubation medium. The giant cells contained multiple cytoplasms covered by the plasma membrane, indicating that the cell fusion occurred only among the outer membranes. Thus, only the outer membrane and the periplasmic space are shared but not the cytoplasm, indicating that genome of each cell remains in its cytoplasm. Our findings indicate that sugar enhances outer membrane fusion in D. grandis spheroplasts to generate calcium ion-dependent extra-huge cells.

Calcium ion induces outer membrane fusion of Deinococcus grandis spheroplasts to generate giant spheroplasts with multiple cytoplasms.

Generally, enlarged spheroplasts of the Gram-negative bacterium Deinococcus grandis contain a single cytoplasm and a large periplasmic space. Enlargement of D. grandis spheroplasts requires the presence of divalent cation Ca2+ or Mg2+. In this study, we elucidated the effects of concentrations of these divalent cations on the enlargement of spheroplasts. We compared the cell sizes of the spheroplasts at five different concentrations (16.2, 62, 100, 200 and 333 mM) of CaCl2 or MgCl2. At the lowest concentration (16.2 mM) of CaCl2 or MgCl2, the inner membrane of D. grandis spheroplasts collapsed and the spheroplasts did not enlarge. At the highest concentration (333 mM) of CaCl2 or MgCl2, enlargement was inhibited. At 200 mM of CaCl2, the outer membranes of D. grandis spheroplasts were fused repeatedly, but the inner membranes were not fused. Thus, at 200 mM of CaCl2, giant cells that have multiple cytoplasms were observed and were ≥ 500 µm in diameter. However, cell fusions were not observed in any concentrations of MgCl2. This indicates that Ca2+ induces lipopolysaccharide dehydration more strongly than Mg2+ and outer membranes may be fused by hydrophobic bonding. Our findings show the different functions of Ca2+ and Mg2+ on the outer membrane stability.

Enlargement of Deinococcus grandis spheroplasts requires Mg2+ or Ca2.

While the cell wall strictly controls cell size and morphology in bacteria, spheroplasts lack cell walls and can become enlarged in growth medium under optimal conditions. Optimal conditions depend on the bacterial species. We frequently observed extreme enlargement of spheroplasts of the radiation-resistant bacterium Deinococcus grandis in Difco Marine Broth 2216, but not in TGY broth (a commonly used growth medium for Deinococcus). Thorough investigation of media components showed that the presence of Mg2+ or Ca2+ promoted extreme spheroplast enlargement, synthesizing the outer membrane. Our findings strongly suggest that Mg2+ or Ca2+ enlarges spheroplasts, which could change the lipid composition of the spheroplast membrane.

Comparison of gene expression levels of appA, ppsR, and EL368 in Erythrobacter litoralis spheroplasts under aerobic and anaerobic conditions, and under blue light, red light, and dark conditions.

We compared the gene expression levels of the blue-light-responsive genes, appA (encoding photosynthesis promoting protein AppA), ppsR (encoding photosynthesis suppressing protein PpsR), and EL368 (encoding a blue-light-activated histidine kinase with a light, oxygen, or voltage domain) between aerobic and anaerobic conditions in spheroplasts of the aerobic photosynthetic bacterium Erythrobacter litoralis. The spheroplasts conducted photosynthesis under red light but not under blue light. All three blue-light-responsive genes showed higher expression under aerobic conditions than under anaerobic conditions under blue light. In contrast, under red light, although the expression level of appA was higher in the presence of oxygen than in the absence of oxygen, the expression levels of ppsR and EL368 were similar in the presence and absence of oxygen. Our findings demonstrate that the expression of blue-light-responsive genes is strongly affected by oxygen in E. litoralis spheroplasts.