ISOLATION AND CHARACTERIZATION OF METHANOHALOPHILUS-PORTUCALENSIS SP-NOV AND DNA REASSOCIATION STUDY OF THE GENUS METHANOHALOPHILUS

Six strains of coccoid, halophilic methanogens were isolated from various salinaria and natural hypersaline environments. These isolates (strains FDF-1T [T = type strain], FDF-2, SF-2, Ret-1, SD-1, and Cas-1) grew on media containing methanol and mono-, di-, and trimethylamines as catabolic substrates, but not on media containing dimethyl sulfide, methane thiol, H-2, formate, or acetate; when cells were provided with H2 in addition to methanol or trimethylamine, they grew on the medium containing a methyl substrate but did not catabolize H-2. All of the strains were capable of growth in mineral medium to which trimethylamine was added as a catabolic substrate, although some strains were greatly stimulated by biotin or p-aminobenzoate. DNA reassociation and denaturing electrophoresis of whole-cell proteins indicated that strains FDF-1T, FDF-2, SF-2, and Ret-1, together with previously described strains SF-1, Z-7302, Z-7401, Z-7404, and Z-7405, belong to a new taxon named Methanohalophilus portucalensis sp. nov; FDF-1 (= OCM 59) is the type strain. These strains grew fastest at temperatures near 40-degrees-C and, in medium containing 0.5 to 2.5 M NaCl, at pH values near 7. The two new strains excluded from the species on the basis of the results of phylogenetic tests, strains Cas-1 and SD-1, also differed from M. portucalensis in some minor physiological characteristics. Strain Cas-1 was less halophilic (fastest growth occurred in the presence of 0.5 to 1 M NaCl), and strain SD-1 was slightly alkaliphilic (fastest growth occurred at pH 7.8). The DNA reassociation study also showed that Methanohalophilus mahii SLP(T) exhibited 52% sequence similarity with Methanohalophilus halophilus Z-7982T, supporting the classification of these organisms as separate but closely related species.