Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity

The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.