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1022 BIOCHEMICAL SOCIETY TRANSACTIONS on the purified vegetative RNA polymerase. The enzyme modified in vitro apparently retains the same capacity to transcribe $e bacteriophage DNA or poly[d(A-T)] as does the vegetative enzyme. Kerjan, P. & Szulmajster, J. (1969) FEBS Lett. 5, 288-290 Losick, R. & Sonenshein, A. L. (1969) Nature (London) 224, 35-37 Maia, J. C. C., Kerjan, P. & Szulmajster, J. (1971) FEBS Lett. 13, 269-274 Millet, J., Kerjan, P., Aubert, J. P. & Szulmajster, J. (1972) FEBSLert. 23, 47-50 Relationship between Chromosome Replication and Induction of Spore Formation in Bacillus subtilis J. MANDELSTAM and SONIA A. HIGGS Microbiology Unit, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, U.K. Sporulation in Bacillus subtilis occurs at very low frequencies (less than 1:lo6)in good growth media, but is appreciable in a variety of poorer media (Schaeffer et al., 1965). With better-defined conditions in a chemostat it has been shown that limitation of either carbon or nitrogen will induce sporulation and that the incidence increases progressively as the bacteria are made to grow more slowly (Dawes & Mandelstam, 1970). These findings are consistent with the assumption that sporulation is a function that is controlled by catabolite repression (Schaeffer et al., 1965). Now, it is a characteristic of inducible enzymes that are subject to catabolite repression (e.g. the lac operon in Escherichia coli, which is the classic example) that the repression is relieved as soon as the repressor is removed. However, there were indirect reasons for supposing that sporulation might not be immediately inducible in this way at any time. These were based on experiments in which bacteria were subjected to intermittent periods of acute starvation in a continuous-culturevessel. When this was done it appeared that only a fraction of the competent cell population could be induced to sporulate at any one time (Dawes & Mandelstam, 1970). From this it followed that cells might have to be in a particular physiological state before sporulation could be induced, and that this state might be related to the growth cycle. To test this Dworkin et al. (1972) set up synchronized cultures of B. subtilis in a rich medium and tested the cells at intervals for their ability to sporulate when transferred to a poor medium. The experiments showed that the susceptibility to sporulation was indeed related to the growth cycle and that peaks in susceptibility were correlated with the peaks in cell division. However, the coincidencewas never altogether reproducible, and susceptibility to sporulation was displaceable relative to cell division by about 15min in different experiments. We therefore examined the possibility that induction of sporulation was related to DNA replication rather than to cell division. A mutant, ts-134, was used that is temperature-sensitive for initiation of DNA replication (Mendelson & Gross, 1967). If the growth temperature is raised from 35°C to 44°C chromosome replications that are already in train go to completion but new rounds do not begin. If the temperature is then restored to 35°C a new and fairly well synchronized replication occurs during about 40-45min. Cells taken at intervals from such a culture and transferred to poor medium were found to have the capacity to sporulate during the first 20min. After this it was too late to transfer them, and the capacity to sporulate declined rapidly to almost zero. This apparent linking of sporulation capacity to the DNA replication cycle was supported by the fact that cells that had passed the critical point could again be made susceptible to spore formation by initiating a new round of chromosome replication. We conclude that the control of sporulation is not analogous to that of simpler 1973 540th MEETING, OXFORD 1023 catabolite-repressed functions of the bacterial cell, and that, if induction is to occur at all, it is necessary not only to remove catabolite repressors but to do so during the first part of the DNA replication cycle. The facts are consistent with the assumption that spore induction is linked either to a specific stage in chromosome replication itself or possibly to an unidentified event that occurs at the same time. Dawes, I. W. & Mandelstam, J. (1970) J. Bacteriol. 103, 529-535 Dworkin, M., Higgins, J., Glenn, A. & Mandelstam, J. (1972) in Spores V (Halvorson, H. O., Hanson, R. & Campbell, L. L., eds.), pp. 233-237, American Society for Microbiology, Washington Mendelson, N. H. & Gross, J. D. (1967) J. Bacteriol. 94, 1603-1608 Schaeffer, P., Millet, J. & Aubert, J.-P. (1965) Proc. Nut. Acad. Sci. US.54, 704-711 Germination of Bacterial Endospores and the Development of New Vegetative Forms G. W. GOULD Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, U.K. Bacterial endospores characteristically differ from vegetative forms in being metabolically inert, dormant, refractile and resistant to heat, radiation, pressure, chemical reagents, antibiotics, stains and enzymes. They also differ from vegetative forms in structure, in particular in having more complex integuments, including a thick loosely cross-linked peptidoglycan layer (the cortex), which surrounds the protoplast, and an outermost multi-layered and mainly protein coat, which surrounds the cortex, and sometimes additionally a further layer (the exosporium) and complex appendages beyond the coats. A number of components (e.g. coat protein, dipicolinic acid, sulpholactic acid, high contents of calcium) are chemically characteristic or even unique to spores. During germination and the development of new vegetative forms the characteristic properties, structures and special components of spores are lost and new syntheses of vegetative-specificmaterial occur in a well-defined sequence of stages (see Gould, 1969; Strange & Hunter, 1969). The first stage, activation, is reversible and is thought to result from changes in tertiary structure of macromolecules rather than from metabolism in spores. Activation can be caused by heat or ionizing radiation or by exposure to acid, reducing agents and other chemicals, and is detected as an increase in the sensitivity of the spores to the environmental triggers that will initiate the next stage, germination. Germination can be initiated by diverse nutrients, which are nevertheless usually quite specific for spores of a particular species or strain. For example, commonly germination can be initiated by a single amino acid such as L-alanine, or by a riboside such as inosine, or less commonly by glucose or lactate or other metabolites. It is often found that a combination of two or more such germinants is necessary to trigger the initiation of germination and there is normally also a non-specific requirement for ions. A number of ‘failsafe’ pathways are known to operate that tend to decrease the extent of germination in spore populations at any one time (e.g. racemization of germinative L-alanine by spore alanine racemase to form the inhibitory D-isomer, the destruction of germinative ribosides by spore ribosidase). There is evidence that any metabolism during the initiation of germination is very slight (i.e. there are less than about 100 molecules of alanine metabolized/spore), and also evidence that generation of reducing power and transport of ions are essential for germination to occur. In addition to the nutrient germinants, a number of non-nutrient reagents will also initiate germination, e.g. some cationic surface-active agents such as n-dodecylamine and some chelates such as the spore component calcium dipicolinate. Further, enzymes Vol. 1