Author

Priya Kadam

Date

October 1996

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Graduate Institute of Science & Technology

Abstract

Several methanogens of the family Methhanosarcinaceae were characterized phenotypically and phylogenetically. Methanolobus bombayensis and Methanolobus vulcani were characterized physiologically and taxonomically. The influence of ammonia on cytosolic pH and ammonia toxicity was studied for Methanolobus bombayensis, Methanolobus taylorii and Methanohalophilus zhilinaeae. The first of these three methanogens, Methanolobus 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 H[subscript 2] + CO[subscript 2], acetate or formate. The cells were non motile, irregular coccoids (diameter, 1.0 to 1.5µm) 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 mM) concentrations of divalent cations (Mg[superscript 2+ and Ca[superscript 2+]). 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 ± 0.1%. The comparison of 16S rRNA sequences showed that the strain B-1 was phylogenetically related to Methanolobus vulcani, but the sequences of these organisms differed by 2%, indicating that they belong in separate species. Methanolobus vulcani PL- 12/M, was isolated by Konig, Stetter and Thomm. The characterization of this strain was rudimentary and thus it was further studied. It grew on methylamines and methanol but not on dimethyl sulfide, formate, acetate or H[subscript 2] + CO[subscript 2].The cells grew rapidly at mesophilic temperatures, at neutral pH (6 to 7.5), and in medium supplemented with 0.1 to 1.2 M NaCl and 13 rnM Mg[superscript 2+]. The cells grew in mineral medium plus biotin and catabolic substrate, but their growth was stimulated by yeast extract and peptones. Methanolobus vulcani was physiologically similar to Methanolobus tindarius 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, Methanolobus taylorii and Methanohalophilus 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 (ΔpH) at all pH values tested. The ΔpH of these three methanogens was in the range of -0.4 to -0.9 pH units, with cytosol more acidic than environment. Methanohalophilus zhilineae had the most negative ΔpH (-0.9 pH units). Consequence of these negative pH gradients was the accumulation of ammonium NH[subscript 4] [superscript +]),with cytosolic NH[subscript 4] [superscript +] concentration as high as 180 mM during growth. The high concentrations of cytosolic NH[subscript 4] [superscript +] were accompanied by higher ΔpH and lower concentrations of the major cytosolic cation K[superscript +]. Methanolobus bombayensis and Methanolobus taylorii 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 (IC [subscript 50]) was about 2-6 mM regardless of pH. This is consistent with arnrnonia inhibition of other bacteria. Methanohalophilus zhilineae on the contrary, was more resistant to un-ionized ammonia than any other known organism. It had an IC[subscript 50] for un-ionized ammonia of 13 mM 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.

Identifier

doi:10.6083/M43776P8

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