DISAGGREGATION OF METHANOSARCINA SPP AND GROWTH AS SINGLE CELLS AT ELEVATED OSMOLARITY

The effect of medium osmolarity on the morphology and growth of Methan osarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Me thanosarcina vacuolata, and Methanosarcina acetivorans was examined. E ach strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonma rine sources exhibited similar growth characteristics at all NaCl conc entrations tested, demonstrating that these species are capable of ada pting 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 aggregat es. Aggregated cells had a methanochondroitin outer layer, while disag gregated 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 sin gle-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 aggregat ed cells by gradually readapting cultures to lower NaCl (<0.2 M) and M g2+ (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery r ates 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, deterg ent-sensitive, single cells on agar medium makes these species amenabl e to mutant selection and screening for genetic studies and enables ce lls to be gently lysed for the isolation of intact genetic material.