Download Relationship between Chromosome Replication and Induction of

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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
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540th MEETING, OXFORD
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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
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