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 Mg or Ca promoted extreme spheroplast enlargement, synthesizing the outer membrane. Our findings strongly suggest that Mg or Ca enlarges spheroplasts, which could change the lipid composition of the spheroplast membrane.

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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.

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