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

Abstract

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.

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