Genetic Transformation in Methylobacterium organophilum

Abstract

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of this organism has greatly increased the potential for physiological studies of sterol synthesis (T. E. Patt, unpublished results), membrane biogenesis and C1 metabolism.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).

Enzyme Regulation in Methylobacterium organophilum

“…The other two, which are revertible by NTG and EMS, might contain nonsense mutations or mutations in a regulatory gene or a gene coding for an inducer. The question of operonic control can only be answered by genetic mapping, a tool which is now available in this organism (O'Connor et al, 1977 …”

Evolutionary Origin and Adaptive Function of Meiosis

“…We can assess the plausibility of the idea that such an ancestor was capable of transformation by knowing whether present day αproteobacteria are capable of transformation. In fact, several present day α-proteobacteria are capable of natural transformation, including Agrobacterium tumefaciens (Demaneche et al, 2001), Methylobacterium organophilum (O'Connor et al, 1977) and Bradyrhizobium japonicum (Raina and Modi, 1972).…”

Bacterial gene transfer by natural genetic transformation in the environment.

“…In Haemophilus influenzae, the development of competence starts when the cells are transferred to defined media which do not allow growth or when cell division is blocked under conditions permissive for protein synthesis (325). Competence develops as cells begin to grow and reaches its maximum during early to late log phase in Acinetobacter calcoaceticus (272), Azotobacter vinelandii (266), Staphylococcus aureus (306), Streptococcus pneumoniae (325), and Anacystis nidulans R2 (43) or during the transition from log phase to stationary phase as observed in Bacillus subtilis (325), B. stearothermophilus (reviewed in reference 150), Chlorobium limicola (258), Methylobacterium organophilum (255), Pseudomonas stutzeri (38,201), Synechocystis spp. (115,206), and Vibrio sp.…”