Abstract
A range of Bacillus thermophiles was tested for the ability to be converted to protoplasts with lysozyme and subsequently, to regenerate to bacillary form. Protoplast formation was straightforward but many of the strains failed to regenerate on commonly-used media. Two medium components were found to be causing the inhibition. Growth of protoplasts as L-forms only occurred if the medium lacked phosphate. However, reversion of L-forms to bacilli was asynchronous and infrequent. Regeneration of cell walls by protoplasts/L-forms to re-establish bacillary form was greatly improved when the medium was gelled with pluronic polyol F127 in place of agar or similar polysaccharides.
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Aiba S, Koizumi JI (1984) Effects of temperature on plasmid stability and penicillinase productivity in a transformant of Bacillus stearothermophilus. Biochem Bioeng 26: 1026–1031
Aiba S, Imanaka T, Koizumi JI (1981) Transformation of Bacillus stearothermophilus with plasmid DNA and its application to molecular cloning. Ann NY Acad Sci 57–70
Bingham AHA, Bruton CJ, Atkinson A (1981) Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli. J Gen Microbiol 114: 401–408
Bourne N, Dancer BN (1986) Regeneration of protoplasts of Bacillus subtilis and closely related strains. J Gen Microbiol 132: 251–255
Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168: 111–115
Clive D, Landman OE (1970) Reversion of Bacillus subtilis protoplasts to the bacillary from induced by exogenous cell wall, bacteria and by growth in membrane filters. J Gen Microbiol 28: 249–269
Elliott TSJ, Ward JB, Rogers HJ (1975) Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis. J Bacteriol 124: 623–632
Gardener S, Gwyfryn-Jones J (1984) A new solidification agent for culture media which liquefies on cooling. J Gen Microbiol 130: 731–733
Gray O, Chang S (1981) Molecular cloning and expression of Bacillus licheniformis-lactamase gene in Escherichia coli and Bacillus subtilis. J Bacteriol 145: 422–428
Heinen W, Heinen AM (1972) Characteristics and properties of a caldoactive bacterium producing extracellular enzymes and two related strains. Arch Microbiol 82: 1–23
Heinen W, Lauwers AM (1981) Growth of bacteria at 100°C and beyond. Arch Microbiol 129: 127–128
Imanaka T, Fujii M, Aiba S (1981) Two replication determinants of an antibiotic-resistant plasmid. J Bacteriol 147: 1091–1097
Imanaka T, Fujii M, Aramori I, Aiba S (1982) Transformation of Bacillus stearothermophilus with plasmid DNA and characterisation of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis. J Bacteriol 149: 824–850
Landman OE, Halle S (1963) Enzymically and physically induced inheritance changes in Bacillus subtilis. J Mol Biol 7: 721–738
Lin CC, Casida LE (1984) Gelrite as a gelling agent in media for the growth of thermophilic microorganisms. Appl Envir Microbiol 47: 427–429
Ljungdahl LG (1979) Physiology of thermophilic bacteria. Adv Microb Physiol 19: 149–156
Salton MRJ (1957) Properties of lysozymes. Bacteriol Rev 21: 82–99
Sharp RJ, Bown KJ, Atkinson A (1980) Phenotypic and genotypic characterisation of some thermophilic species of bacilli. J Gen Microbiol 117: 201–210
Shugar D (1952) Measurement of lysozyme activity and the ultraviolet inactivation of lysozyme. Biochim Biophys Acta 8: 302–309
Sonnleitner B (1983) Biotechnology of thermophilic bacteria, growth products and applications. Adv Biochem Eng Biotech 28: 69–138
Ward JB (1978) The reversion of bacterial protoplasts and L-forms. In: Stanier RY, Rogers HJ, Ward JB (eds) The Society of General Microbiology Symposium 28, Relations between structure and function in the prokaryotic cell. Soc Gen Microbiol 28:249–269
Wolf J, Sharp RJ (1981) Taxonomic and related aspects of thermophiles within the genus Bacillus. In: Berkeley RCW, Goodfellow M (eds) The aerobic endospore-forming bacteria: Classification and identification. Academic Press, New York, pp 251–296
Wyrick RB, Rogers HJ (1973) Isolation and characterisation of cell wall defective variants of Bacillus subtilis and Bacillus licheniformis. J Bacteriol 116: 456–465
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Dunn, R.M., Munster, M.J., Sharp, R.J. et al. A novel method for regenerating the protoplasts of thermophilic bacilli. Arch. Microbiol. 146, 323–326 (1987). https://doi.org/10.1007/BF00410929
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DOI: https://doi.org/10.1007/BF00410929