Characterization of Two New Facultative Methanotrophs

Martha J. Lynch, Ann E. Wopat, Mary L. O'Connor
1980 Applied and Environmental Microbiology  
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
more » ... en 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. Methane-utilizing bacteria have been known to exist for over 70 years (25). However, until recently all isolates were obligate C-1 utilizers; that is, unable to utilize compounds containing carbon-carbon bonds as sole sources of carbon and energy (6, 27, 28). Since 1974, two facultative methanotrophs have been reported, both of which contained peripheral (type II) internal membrane systems and utilized the serine pathway for formaldehyde fixation (20, 22) . The isolation of these organisms has opened the way for detailed study of C-1 assimilation processes in methanotrophs (17) (18) (19) . However, neither facultative methanotroph has proven suitable for genetic studies of methane oxidation, nitrogen fixation, and related functions. This study was undertaken to isolate and characterize new strains of facultative methanotrophs which might prove more amenable to genetic analysis. Two such strains have been isolated and described in terms of their characteristics and taxonomic position. MATERIALS AND METHODS Media and growth conditions. The modified mineral salts medium (NMS) of Patt et al. (22) was used for most experiments. In some cases, a nitrogenfree mineral salts medium (NFMS) was used, which consisted of NMS excluding KNO3 and substituting K2HPO4 (0.28 g/liter) for Na2HPO4. Cells for deoxyribonucleic acid (DNA) isolation were grown on nutrient broth. Succinate, acetate, glucose, xylose, galactose, sucrose, lactate, malate, and fructose were sterilized separately and added directly to the growth medium at 0.1% (wt/vol). Methanol (96%, vol/vol), ethanol (95%, vol/vol), and propanol (100%, vol/vol) were filter sterilized and added directly to the growth medium at 0.5% (for methanol) or 0.2% (for ethanol and propanol). A vitamin supplement (in,g/liter: biotin, 20; folic acid, 20; thiamine hydrochloride, 50; D-calcium pantothenate, 50; B12, 1; riboflavin, 50; nicotinamide, 50) was routinely included in the medium for isolate H4-14. Although vitamins were not required by this isolate, they stimulated growth, especially after transfer from one substrate to another. Yeast extract (0.005%, wt/vol) was routinely included in all media for the growth of isolate 37W. Liquid cultures were incubated at 30°C on rotary shakers. Methane-grown cultures were initially gassed with a methane-air mixture of 90%-10%. As turbidity increased, the percentage of air was gradually increased to 50%. Cultures were gassed daily. Cultures were maintained on NMS plates, incubated under methane-air (90%-10%), and transferred weekly. Isolation. Samples were taken aseptically throughout the water columns by using Cobet samplers (12). Portions (10 ml) were placed in flasks containing 30 ml of NFMS medium, gassed with methane-air mixtures (90%-10%), and incubated at 30°C with shaking for 2 to 3 weeks until turbid. Samples were streaked onto Millipore membrane filters soaked in NFMS medium and incubated under a methane-air atmosphere as before. In some cases a small amount of yeast extract (0.005%) was included in the medium. Colonies were alternately placed into liquid medium and streaked onto filters until pure cultures which grew well under both conditions were obtained. Preparation of crude extracts. Frozen cells were thawed and suspended in 4 volumes of potassium phosphate buffer (20 mM, pH 6.8). Disruption was effected by ultrasonification (MSE, model MT20) using six 30-s bursts.
doi:10.1128/aem.40.2.400-407.1980 fatcat:6heceytqy5egpep35ebbmesg5m