Spheroplasts of Methanospirillum hungatii formed upon treatment with dithiothreitol.

Abstract

Strains of Methanospirillum hungatii, when treated with dithiothreitol at alkaline pH, formed spheroplasts which lysed in the absence of an osmotic stabilizer. Other methanogenic bacteria showed no response to this treatment, including Methanobacterium thermoautotrophicum. Methanobacterium strain M.o.H., Methanobacterium strain G2R, and Methanosarcina barkeri. The reaction with M. hungatii proceeded slowly at pH 8.0, but increased dramatically up to a pH of 10. Spheroplasts did not form in the presence of MgCl2 or if large amounts of FeS precipitate had been deposited on the cells during growth. Ultra-thin sections of M. hungatii GPI confirmed that individual cells, surrounded by a membrane and an inner wall, were contained within an outer wall or sheath. Adhesive material, which stained in the region of the cell spacer, was observed to bind together the inner and outer wall layers. After treatment with dithiothreitol, the spheroplasts, which retained the flexible inner wall and membrane, were released from the rigid outer wall. Examination of the isolated outer cell wall revealed that dithiothreitol (and dodecyl sodium sulfate) dissolved the adhesive material and damaged the cell spacer at the attachment site to the outer wall. It is proposed that dithiothreitol at alkaline pH loosens both the cell spacer and inner wall attachments to the outer wall, thereby allowing the osmotic pressure of the cytoplasm to eject the spheroplast. Resistance of the cells to lysis by Triton X-100 is lost upon spheroplast formation, indicating that protection to the cell is conferred by the outer cell wall.