Abstract
This protocol defines conditions under which the germination of spores can be used to synchronize Bacillus subtilis cells, utilizing the time-ordered sequence of events taking place during the transition from spore to vegetative cells. The transition stages involve: phase change, swelling, emergence, initial division, and elongation. By using this method we have obtained two distinctive synchronized cell cycles, while the synchrony faded away in the third cycle. The advantage of using spore outgrowth and germination is that a highly synchronized population of bacterial cells can be obtained. Non-dividing spores stay synchronized, while synchrony rapidly decays during a few divisions. The limitations of this method are that it can be applied only for sporulating bacteria and synchrony lasts for only a limited period of time exceeding not more than two cycles.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Graumann P (ed) (2007) Bacillus: cellular and molecular biology, 1st edn. Caister Academic Press, Haverhill, UK, http://www.horizonpress.com/bac
Hansen JN, Spiegelman G, Halvorson HO (1970) Bacterial spore outgrowth: its regulation. Science 168:1291–1298
Keynan A (1973) The transformation of bacterial endospores into vegetative cells. Symp Soc Gen Microbiol 23:85–123
Mandelstam J (1976) Bacterial sporulation: a problem in the biochemistry and genetics of a primitive developmental system. Proc R Soc B 193:89–106
Nelson DL, Kornberg A (1970) Biochemical studies of bacterial sporulation and germination. XVIII. Free amino acids in spores. J Biol Chem 245:1128–1136
Setlow P, Kornberg A (1970) Biochemical studies of bacterial sporulation and germination. XXIII. Nucleotide metabolism during spore germination. J Biol Chem 245:3645–3652
Balassa G (1965) Synthesis macromoleculaires au cours de la germination des spores de B. Subtilis. I. Cinctique. Ann De I’lnstitut Pasteur 109:13–35
Balassa G (1969) Biochemical genetics of bacterial sporulation. I. Unidirectional pleiotropic interactions among genes controlling sporulation in Bacillus subtilis. Mol Gen Genet 104:73–103
Kobayashi I, Steinberg W, Higa A, Halvorson HO, Levinthal C (1965) Sequential synthesis of macromolecules during outgrowth of bacterial spores. In: Campbell LL, Halvorson HO (eds) Spores IZZ. American Society for Microbiology, Washington, DC, pp 200–212
Torriani A, Levinthal C (1967) Ordered synthesis of proteins during outgrowth of spores of B. cereus. J Bacteriol 94:176–183
Armstrong RL, Sueoka N (1968) Phase transition in ribonucleic acid synthesis during germination of B. Subtilis spores. Proc Natl Acad Sci U S A 59:153–160
Steinberg W, Halvorson HO (1968) Timing of enzyme synthesis during outgrowth of Bacillus cereus. I. Ordered enzyme synthesis. J Bacteriol 95:469–478
Kennett RN, Sueoka N (1971) Gene expression during outgrowth of B. subtilis spores. The relationship between order on the chromosome and temporal sequence of enzyme synthesis. J Mol Biol 60:31–44
Campbell LL Jr (1957) Bacterial spore germination. Definitions and methods of study. In Spores. HO Halvorson (ed.). American Institute of Biological Sciences, Washington, DC, pp. 33–38
Levinson HS, Hyatt MT (1956) Correlation of respiratory activity with phases of spore germination and growth in Bacillus megaterium as influenced by manganese and L-alanine. J Bacteriol 72:176–183
Halvorson HO (1959) Symposium on initiation of bacterial growth. Bacteriol Rev 23:267–272
Harrell WK, Halvorson H (1955) Studies on the role of L-alanine in the germination of spores of Bacillus terminalis. J Bacteriol 69:275–279
Yasuda Y, Tochikubo K (1984) Relation between D-glucose and L- and D-alanine in the initiation of germination of Bacillus subtilis spores. Microbiol Immunol 28:197–207
McCann KP, Robinson C, Sammons RL, Smith DA, Corfe BM (1996) Alanine germination receptors of Bacillus subtilis. Letts Appl Microbiol 23:290–294
Liang L, Gai Y, Hu K, Liu G (2008) Te gerA operon is required for spore germination in Bacillus thuringiensis. Wei Sheng Wu Xue Bao 48:281–286 (article in Chinese)
Powell JF (1950) Factors affecting the germination of thick suspensions of Bacillus subtilis spores in L-alanine solution. J Gen Microbiol 4:330–338
Pulvertaft RJV, Haynes JA (1951) Adenosine and spore germination; phasecontrast studies. J Gen Microbiol 5:657–663
Powell JF, Strange RE (1953) Biochemical changes occurring during the germination of bacterial spores. Biochem J 54:205–209
Murrell WG, Scott WJ (1958) The permeability of bacterial spores to water. In: Proceedings of the 7th international congress of microbiology, Stockholm, p. 26
Powell JF (1957) Biochemical changes occurring during spore germination in bacillus species. J Appl Bacteriol 20:349–358
Bhattacharya S, Sarkar N (1981) Study of deoxyribonucleic acid replication in permeable cells of Bacillus subtilis using mercurated nucleotide substrates. Biochemistry 20:3029–3034
Woese C, Morowitz HJ (1958) Kinetics of the release of dipicolinic acid from spores of Bacillus subtilis. J Bacteriol 76:81–83
Cutler RG, Evans JE (1966) Synchronization of bacteria by a stationary-phase method. J Bacteriol 91:469–476
Maruyama Y, Yanagita T (1956) Physical methods for obtaining synchronous culture of Escherichia coli. J Bacteriol 71:542–546
Abbo FE, Pardee AB (1960) Synthesis of macromolecules in synchronously dividing bacteria. Biochim Biophys Acta 39:473–485
Lark KG, Lark C (1960) Changes during the division cycle in bacterial cell wall synthesis, volume, and ability to concentrate free amino acids. Biochim Biophys Acta 43:520–530
Helmstetter CE, Uretz RB (1963) X-ray and ultraviolet sensitivity of synchronously dividing Escherichia coli. Biophys J 3:35–47
Helmstetter CE, Cummings DJ (1963) Bacterial synchronization by selection of cells at division. Proc Natl Acad Sci U S A 50:767–774
Lark KG, Maaloe O (1954) The induction of cellular and nuclear division in Salmonella typhimurium by means of temperature shift. Biochem Biophys Acta 15:345–356
Ron EZ, Rozenhak S, Grossman N (1975) Synchronization of cell division in Escherichia coli by amino acid starvation: strain specificity. J Bacteriol 123:374–376
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Banfalvi, G. (2017). Synchronization of Bacillus subtilis Cells by Spore Germination and Outgrowth. In: Banfalvi, G. (eds) Cell Cycle Synchronization. Methods in Molecular Biology, vol 1524. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6603-5_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6603-5_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6602-8
Online ISBN: 978-1-4939-6603-5
eBook Packages: Springer Protocols