Biopolym. Cell. 1988; 4(3):151-157.
Gene-Engineering Biotechnology
Obtaining of spheroplasts in nodule bacteria of lucerne, clover, bean and pea
1Bazhenova O. V., 1Potokin I. L., 1Simarov B. V.
  1. All-Union Research Institute of Agricultural Microbiology
    Leningrad, USSR


A simple universal procedure has been developed for obtaining stable spheroplasts from the fast-growing species of Rhizobium (R. meliloti, R. phaseoli, R. trifolii, R. leguminosarum). Spheroplasts were prepared by culturing cells in the osmotically stable medium with a decreased content of calcium cations in the presence of penicillin, followed by treatment with lysozyme. The electron-microscopic analysis of the process of spheroplasts formation was performed. The average effectiveness of the cell wall regeneration is 1.4-5.0 %.


[1] Yakovenko KN, Troitskiy NA. Protoplasts microorganisms. Minsk: Nauka i tekhnika, 1985; 159 p.
[2] Chang S, Cohen SN. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979;168(1):111-5.
[3] Akamatsu T, Sekiguchi J. Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts. Arch Microbiol. 1983;134(4):303-8.
[4] Staniewski R, Rugala A. Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts. Acta Microbiol Pol A. 1975;8(3):151-60.
[5] Berry JO, Atherley AG. Spheroplasts from Rhizobium Japonicum. Can J Microbiol. 1984; 30(2):415-419.
[6] Child JJ, Sietsma J. N. Spheroplasts from Rhizobium japonicum. Plant Sci Lett. 1975; 4(4):267-271.
[7] Filatova OV, Simarov BV, Kuchko VV. Preparation of protoplasts in alfalfa nodule bacteria Rhizobium meliloti. Izv akad nauk SSSR. Ser Biol. 1982; (1):140-2.
[8] Jouanin L, De Lajudie P, Bazetoux S, Huguet T. DNA sequence homology in Rhizobium meliloti plasmids. Mol Gen Genet. 1981;182(2):189-95.
[9] Allen ON. Experiments in soil bacteriology. Minneapolis: Burgess publ. co., 1959. 59 p.
[10] Franklin TJ, Snow GA. Biochemistry of antimicrobial action. New York, Chapman and hall, 1981; 201 p.
[11] Maksimova NP, Zheldakova RA, Fomichev IuK. Preparation of protoplast-like structures of Pseudomonas putida and their reversion to bacterial forms. Mikrobiologiia. 1982;51(4):628-31.
[12] Bergersen FJ. The growth of Rhizobium on synthetic media. Austr J Biol Sci. 1961; 14(1):349-60.
[13] McQuillen K. Bacterial protoplasts. The Bacteria. New York: Acad, press, 1981. Vol. 1:249-360.
[14] Tsenin AN, Karimov GA, Rybchin VN. Recombination during fusion of Escherichia coli K 12 protoplasts. Dokl Akad Nauk SSSR. 1978;243(4):1066-8.
[15] Weiss RL. Protoplast formation in Escherichia coli. J Bacteriol. 1976;128(2):668-70.
[16] Leive L. Release of lipopolysaccharide by EDTA treatment of E. coli. Biochem Biophys Res Commun. 1965;21(4):290-6.
[17] Goldschmidt MC, Wyss O. Chelation effects on Azotobacter cells and cysts. J Bacteriol. 1966;91(1):120-4.