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

Abstract

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.

Sensitivity of <em>Deinococcus grandis rodZ</em> deletion mutant to calcium ions results in enhanced spheroplast size

“…The size of outer membrane can be measured, but the size of the cytoplasm cannot be measured in D. grandis spheroplasts. This is because a part of the cytoplasm is attached to the outer membrane [7]–[9]. Therefore, in this study, we measured the length between the edges of cytoplasmic areas attached to the outer membrane (Figure S6).…”

“…The enlargement requires calcium or magnesium ions [7]. Outer membrane fusion occurs in the presence of calcium ions [8],[9]. Transcription or translation inhibitors prevents the spheroplast enlargement [7].…”

Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts

“…Interestingly, outer membrane fusion occurs when incubation is done in a medium containing calcium ions [ 26 , 37 ], but not when incubated in a medium containing magnesium ions. Outer membrane fusion was observed at 200 mM of calcium ions, but was not observed at 100 mM of both calcium and magnesium ions as well as at 200 mM of magnesium ions [ 26 ].…”

“…Outer membrane fusion was observed at 200 mM of calcium ions, but was not observed at 100 mM of both calcium and magnesium ions as well as at 200 mM of magnesium ions [ 26 ]. These results indicate that calcium and magnesium ions do not have the same function in the enlargement of the outer membrane of D. grandis [ 26 , 37 ].…”

“…During D. grandis spheroplast enlargement, the synthesis of the outer membrane is much faster than that of the inner membrane in the presence of calcium ions [ 26 , 28 , 37 ]. This strongly suggests that the outer membrane has a calcium ion-binding component associated with its expansion and fusion.…”