Physiology of halophilic, methylotrophic methanogens

Physiology of Halophilic, Methylotrophic Methanogens. Priya Kadam, Ph. D. Oregon Graduate Institute of Science & Technology, 1996 Supervising Professor: David R. Boone Several methanogens of the family Melharzosarcir2aceae were characterized phenotypically and phylogenetically. Methanolobus bombayensis and Methariolobza ~ui'cai~i were characterized physiologically and taxonomically. The influence of ammonia on cytosolic pH and ammonia toxicity was studied for Methanolobus bornbayensis, Methanolobzrs taylorii and Methar~ohalophiltrs zhiliriaeae. The first of these three methanogens, Methar~olobzls bombayensis B-1 (= OCM 438), was isolated from sediments of Arabian Sea near Bombay, India. This strain grew on methylamines, methanol, and dimethyl sulfide, but it did not catabolize Hz + COz, acetate or formate. The cells were non motile, irregular coccoids (diameter, 1.0 to 1.5 pm) that occurred singly. Electron micrographs showed a cell membrane and a protein cell-wall. The cells grew fastest at mesophilic temperatures, neutral pH, and salinity near that of the ocean, but they required higher (30 rnM) concentrations of divalent cations ( M ~ ~ ' and ca2+). The cells grew in mineral medium, but they were greatly stimulated by yeast extract and peptones. The guanine-plus-cytosine content of the DNA was 39.2 f 0.1%. The comparison of 16s rRNA sequences showed that the strain B-1 was phylogenetically related to Methai~olobus wrlcaiii, but the sequences of these organisms differed by 2%, indicating that they belong in separate species. Methanolobus vtilcani PL12/M, was isolated by Konig, Stetter and Thomm. The characterization of this strain was rudimentary and thus it was hrther studied. It grew on methylamines and methanol but not on dimethyl sulfide, formate, acetate or H2 + CO2. The cells grew rapidly at mesophilic temperatures, at neutral pH (6 to 7.9, and in medium supplemented with 0.1 to 1.2 M NaCl and 13 rnM ~ g ~ ' . The cells grew in mineral medium plus biotin and catabolic substrate, but their growth was stimulated by yeast extract and peptones. Methnrlolobzrs vrrlcaiti was physiologically similar to Methai~olobzrs tincjnrius and had a similar 16s rRNA sequence, although the results of DNA-DNA hybridization experiments indicate that these organisms should be considered separate species. Three halophilic, methylotrophic, methanogens, Methanolobus bombayensis, Mc~hczi~01oh11.s tnyforii and Methnr~ohalophilus zhilinaeae, which grew at environmental pH ranges that overlapped with each other and spanned the pH range from 7.0-9.5, had reversed membrane pH gradients (ApH) at all pH values tested. The ApH of these three methanogens was in the range of -0.4 to -0.9 pH units, with cytosol more acidic than environment. Methanohaloyhiii~s zhilinneae had the most negative ApH (-0.9 pH units). Consequence of these negative pH gradients was the accumulation of ammonium m'), with cytosolic NH4' concentration as high as 180 mM during growth. The high concentrations of cytosolic N-' were accompanied by higher ApH and lower concentrations of the major cytosolic cation K'. Methanolobzrs bombayensis and Mefhnr~okobri.~ tnylorii were more sensitive to total external ammonia at higher external pH, but the inhibitory concentration of un-ionized ammonia that resulted in 50% reduction of growth rate (1Cso) was about 2-6 rnM regardless of pH. This is consistent with arnrnonia inhibition of other bacteria. Methar~ohalophii~~s zhiliiiaeae on the contrary, was more resistant to un-ionized ammonia than any other known organism. It had an ICso for un-ionized ammonia of 13 rnM at pH 8.5 and 45 mM at pH 9.5. We examined the effect of pH on three ammonia-assimilating activities (glutamine synthetase, glutamate dehydrogenase and alanine dehydrogenase) in cell lysates, and found that the pH ranges were consistent with the observed ranges of intracellular pH.