The pseudomurein-degrading enzyme from autolysates of Methanobacterium wolfei was purified approximately 500-fold to electrophoretic homogeneity by ion-exchange chromatography under anaerobic conditions. Analysis of the soluble cell wall fragments produced by the pure enzyme from a cell wall preparation of M. thermoautotrophicum indicated that it is a peptidase hydrolyzing the e-Ala-Lys bond of pseudomurein. A partially purified preparation of pseudomurein endopeptidase was free of nuclease activity and thus proved useful for the preparation in high yields of undegraded chromosomal and plasmid DNA from M. thermoautotrophicum. The partially purified enzyme was also used for the preparation of protoplasts, which were stabilized by 0.8 M sucrose. Under growth conditions the protoplasts produced methane and increased up to 100-fold in size, but failed to regenerate a cell wall.Methanogenic bacteria exhibit a striking heterogeneity with respect to the composition of their cell wall sacculus in that four basic types of cell walls are found in this physiological group of archaebacteria (3, 5). Methanosarcina spp. possess a methanochondroitin cell wall (15). Methanococcus spp. have a sodium dodecyl sulfate (SDS)-sensitive cell wall of regularly arranged protein subunits (24). Tubelike sheaths, also consisting of proteins, are isolated from Methanospirillum spp. (9), and the cell wall sacculi of the gram-positive genera Methanobacterium, Methanobrevibacter, Methanothermus, and Methanosphaera consist of pseudomurein, which, in contrast to the murein of eubacteria, contains N-acetyl-L-talosaminuronic acid instead of Nacetyl-D-muramic acid (12, 13). Also, the peptide moieties of pseudomurein consist exclusively of L-amino acids.Several reports describe gentle lysis of methanogen cell walls by either chemical or enzymatic agents. Methanococcus vannielii lysed within minutes in solutions containing 0.001% SDS (7). Methanobacterium formicicum was lysed by proteinase K, by pronase, and by an extracellular serine protease produced by a Streptomyces isolate, two enzymes acting on the cytoplasmic membrane but not on the pseudomurein sacculus (2). Spontaneous spheroplast or protoplast formation has been reported for Methanobacterium bryantii (6) and Methanosarcina barkeri (4). Dithiothreitol treatment at alkaline pH has been used to form spheroplasts of Methanospirillum hungatii (25), and in the case of Methanobacterium thermoautotrophicum protoplasts were obtained by treating cells that were grown in the presence of 50 mM sorbitol with lysozyme (22). The methods described for the lysis of Methanobacterium spp. cell walls have certain disadvantages for practical use in the preparation of lysates or protoplasts. The organisms must be grown under stress conditions or else the enzymes applied, such as proteinase K, may damage cell compounds.We therefore have explored whether the lytic activity that has recently been detected in Methanobacterium wolfei may be of use as an experimental tool. The lytic activity of this organism is formed...