Ten strains of obligate methanotrophs were screened for the presence of plasmid DNA using a variety of methods. Plasmids were detected in all strains except Methylococcus capsulatus Bath. No significant similarity between plasmids was observed with respect to size or restriction digest patterns except for three strains of Methylosinus trichosporium, which appeared to contain the same three plasmids. Nitrocellulose filter hybridization revealed that the plasmid DNA from the M. trichosporium strains shared a small region of homology with the plasmid DNA from Methylosinus sporium 5. All of the plasmids remain cryptic. As the first step in characterization, a restriction digest map of the 55 kb plasmid found in Methylomonas albus BG8 was constructed.
Two new facultative methane-oxidizing bacteria have been isolated from lake water enrichments. The organisms have been characterized in terms of colony types, growth characteristics, the guanine plus cytosine content of their deoxyribonucleic acid, thin sections, oxidation rates, and carbon assimilation pathways. Methane-grown cells of both organisms contained intracytoplasmic membranes similar to those described as type II in other methanotrophic bacteria. Neither organism had such membranes when grown heterotrophically. Both organisms assimilated methane by way of the isocitrate lyase-negative serine pathway for formaldehyde incorporation. The enzymes of this pathway were high in specific activity in cells grown on methane and were at low levels in cells grown either on heterotrophic substrates or on heterotrophic substrates plus methane. It is proposed that both organisms be classified in the genus Methylobacterium as two new species, Methylobacterium ethanolicum and Methylobacterium hypolimneticum.
S U M M A R YSeveral mutants have been isolated from the facultative methylotroph, Methylobacterium organophilum, using either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light as mutagens. One of these isolates, a glutamate auxotroph lacking isocitrate dehydrogenase, has been transformed to prototrophy, using wild-type DNA, at a frequency of 0.5 %. Competence and DNA uptake occur only in cultures which are near the end of exponential growth, and maximal transformation requires a DNA concentration of loo pg ml-l. I N T R O D U C T I O NMethylobacterium organophilum is a facultative methylotroph. It can utilize C1 compounds as sole carbon and energy sources but unlike all previously isolated methane oxidizers it can also grow on more complex organic substrates (Patt et al., 1974). The isolation and characterization of this organism has greatly increased the potential for physiological studies of sterol synthesis (T. E. Patt, unpublished results), membrane biogenesis and C1 metabolism. However, these studies can only be adequately supported with the benefit of genetic analysis. This depends on the availability of mutants, but mutants of methane oxidizers have been difficult to obtain for reasons that are not clear (Williams & Bainbridge, 1970. M E T H O D SOrganism. Methylobacterium organophilum (~~c c 2 7 8 8 6 , Patt et al., 1974) was used in all experiments.Media and growth conditions. The modified nitrate mineral salts medium (NMS) previously described (Patt, Cole & Hanson, 1976) was used for all studies. All supplements except methanol were dissolved in distilled water, sterilized separately and added directly to the growth medium at the following concentrations (w/v): succinate, 0-I %; Casamino acids, 0.05 %; gluta am ate, ob5 % (unless stated otherwise). Methanol (96 %, v/v) was filtersterilized and added separately to the growth medium at a concentration of 0.5 % (v/v).All cultures were incubated at 30 "C with methanol as the carbon source unless stated otherwise. Liquid cultures were incubated on rotary shakers. The large batches of cells used for DNA isolation were grown and harvested as described previously (O'Connor & Hanson, Sunyival curvesUltraviolet (u.v.) light. An exponential-phase culture (40 ml) was harvested and resuspended in 2 ml sterile NMS medium. The suspension was placed in a sterile Petri dish in the dark and exposed to U.V. light (Westinghouse Sterilamp, at a distance of 40 cm. At intervals, samples were withdrawn and plated on NMS/methanol agar. 1975).
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