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.