Formation and Regeneration of Methanococcus voltae Protoplasts

Abstract

Methanococcus voltae cells were converted into protoplasts by suspension in anaerobic 0.1 M Tris-HCl buffer containing 0.4 M sucrose and 0.05 M NaCl as osmoprotectants. Protoplast formation was monitored microscopically by observing the conversion of the typical irregularly shaped (uneven peripheries) coccoid whole cells to rounded forms with smooth peripheries. Although the procedure resulted in about 50%Yo lysis of the initial number of cells, the remainder were converted to the rounded form. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy of negatively stained cell preparations indicated that the treatment removed the wall layer from whole cells to yield protoplasts. Protoplast regeneration was evaluated by using optimized plating conditions and an anaerobic microplating technique. Between 50 and 63% of the initial number of protoplasts regenerated as colonies on agar medium (35°C, 7 days). The colony and cell morphologies of the regenerated protoplasts were indistinguishable from those of whole cells plated under identical conditions.

Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae

“…Protoplast formation and transformation M. voltae cells were protoplasted (Patel et al, 1993) and transformed using the method of Patel et al (1994) as slightly modified by Berghöfer and Klein (1995). Approximately 25 m g of the insertional vector pKJ19 were used in the transformation procedure.…”

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts

“…BD broth medium was similar to the BV medium of Patel et al (15) except that molybdate, tungstate, and selenite were omitted and yeast extract and tryptone were replaced by L-leucine (50 mg/liter), L-isoleucine (100 mg/liter), and sodium pantothenate (5 mg/liter). The medium was reduced and dispensed anaerobically by previously described techniques (14).…”

“…BD agar medium (BD broth supplemented with 1% [wt/vol] Noble agar [Difco Laboratories]) was reduced and dispensed (20 ml per 60-ml serum vial) anaerobically before autoclaving. The agar plates (20 ml of agar per petri plate [100 by 15 mm]) were prepared and dehydrated (30°C, 18 to 24 h) inside an anaerobic chamber filled with 85% N2-10% H2-5% CO2 as described previously for BV agar (15).…”

“…In bacteria with which low to negligible transformation frequencies are obtained, it may be possible to increase these rates by using protoplasts or spheroplasts (4). Although the production of either protoplasts or spheroplasts has been reported for several methanogens, these could not be successfully regenerated prior to our recent demonstration with M. voltae (15). Regeneration is essential for success in transformation with protoplasts and spheroplasts (11).…”

“…In M. voltae, the only barrier to the penetrating DNA, external to the cytoplasmic membrane, is a paracrystalline array (S-layer) consisting of a 76-kDa protein as the major component (10). On the basis of the sensitivity of M. voltae to lysis in hypotonic solutions, we described a gentle, simple technique for forming protoplasts and for regenerating them at high frequencies into cells with the normal S-layer wall (15). In this report, we demonstrate that increases in transformation frequency of 2 to 3 orders of magnitude can be achieved with the vector Mipl by using M. voltae protoplasts.…”