Sep 15, 2013 - concentration of 1 - 10 mM or by a mixture of inosine and. L-alanine at about one hundredth of this concentration. Some preliminary evidence ...
Spore germination mutants of Bacillus cereus S.C. Warren (Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford) Journal of General Microbiology 04/1969; 55(3):18-9. Paper delivered at S.G.M. Symposium, Edinburgh Sept. 1968 Abstract Germination of spores of Bacillus cereus strain T can be initiated either by 0.1 - 10 mM lalanine or by a combination of 0.005 - 0.1 mM inosine and an equimolar concentration of lalanine. We have isolated some mutants of B. Cereus T whose spores show an altered response to either or both of these germinants. Two of the mutants germinate only very slowly in l-alanine alone but do so at a normal rate in inosine and alanine. Eight mutants which germinate normally in l-alanine do so normally in inosine + l-alanine. Six mutants germinate very slowly in either l-alanine alone or inosine + l-alanine. Four of these also show reduced rates of germination in n-dodecylamine but all six germinate normally in 30 mM calcium 2,6dipcolinate (Riemann, M. & Ordal, J. (1961), Science, N.Y., 133, 1703) and also; after treatment with thioglyoolic acid and urea, in lysozyme and in a spore-lytic enzyme. Some tentative conclusions can be drawn from these results. At least one reaction involved in germination by l-alanine alone is not essential for germination in inosine + alanine and at least one reaction involved in germination in inosine + alanine is not essential for germination in l-alanine alone. At least two reactions are essential for germination in both l-alanine and inosine + alanine but only one of these is essential for germination in n-dodecylamine. None of these reactions is essential for germination in calcium 2,6-dipicolinate, lysozyme or spore-lytic enzyme. Biochemical studies of the mutants may throw some light on the nature of the germination reactions and possibly also on the nature of the 'superdormant fraction of spores which invariably remain after the majority of a population have germinated. Introduction The germination of Bacillus cereus spores can be initiated by L-alanine alone at a concentration of 1 - 10 mM or by a mixture of inosine and. L-alanine at about one hundredth of this concentration. Some preliminary evidence suggests that two separate initiating pathways exist (Warren & Gould, 1968) because the two germinants have markedly different characteristics, as summarised in Table 1. Table 1
Characteristics of germination of B. cereus spores by l-alanine in the presence and absence of inosine Germinant Characteristic
l-alanine
l-alanine + 0.1 mM inosine
Substitution by other amino acids
Only l-α-amino butyric acid
Glycine or any neutral l-amino acid
D-alanine
Strongly inhibits germination
No effect
Km (approximate)
-4
5 x 10 M
-6
4.8 x 10 M
The decisive difference is the failure of D-alanine to inhibit germination by l-alanine in the presence of inosine, whereas the D-isomer completely inhibits germination by L-alanine alone.
We therefore sought mutants of B. cereus whose spores had lost the ability to respond to either or both of these germinants. Methods Vegetative cells of the organism were treated with sodium nitrite, centrifuged, washed and resuspended in a minimal sporulation medium, allowing no vegetative growth, where the surviving cells were allowed to form spores. The spores were separated by centrifugation, washed, re-suspended and divided into about 50 aliquots. This procedure maximised the chance that each aliquot would contain different mutants and minimised the risk that a single vigorous strain would dominate the population and dilute to invisibility the mutants that we sought. Samples of each aliquot were then activated by heat shock and incubated with each of six germinants: L-alanine; L-alanine + inosine; N-dodecylamine - (a surfactant) (Rode & Foster, 1961); Calcium 2,6-dipicolinate (Riemann & Ordal 1961); Lysozyme (an enzyme that breaks down bacterial cell walls); Spore lytic enzyme, an extract of germinating spores that completes the breakdown of the external structure of the spore (Gould, Hitchins & King, 1966). In each case the culture was then heated to kill the vegetative cells and the surviving ungerminated spores removed, washed, activated by heat shock and incubated with the same germinant again. After heating to kill germinated spores the survivors were plated out on rich medium and single colonies selected for testing against a variety of germinants. Results Some twenty mutants were isolated with impaired germination characteristics. Two mutant strains, A10 and A11, germinated normally in l-alanine + inosine but only very slowly in l-alanine alone. Eight mutants, from which data for D1 and D3 are shown, germinated normally in l-alanine alone but only very slowly in l-alanine + inosine. Six mutants, of which data are shown only for A7, C5 and D5, germinated only slowly in either of these two germinants. The rates are shown in Table 2. Table 2
Germination rates (% fall in OD580μ /min.) of sporulation mutants in l-alanine and l-alanine + inosine Germinant
Strain
l-alanine
l-alanine + inosine
Parent (wild type)
5.3 - 7.0
3.4 - 6.2
A10, A11
0.27 - 0.6
4.4 - 4.6
D1, D3
4.9 - 5.6
0.8 - 1.3
A7
0.8
0.6
C5, D5
0.6 - 0.9
0.8 - 0.9
This is consistent with other evidence that two separate pathways exist for the initiation of B. cereus spore germination (Warren and Gould (1968).
Six mutants that were resistant to both of the natural germinants were then exposed to four different "unnatural" germinants. Only two of the mutants were able to germinate in n-dodecylamine but all six germinated in 30 mM calcium dipicolinate and, after presensitisation with thioglyoolic acid and urea, which renders the spore coats permeable to large molecules, in lysozyrne and in a lytic enzyme extracted from spores. The rates are shown in table 3. Table 3
Germination rates (% fall in OD580μ /min.) of sporulation mutants in 4 non-natural germinants Germinant n-dodecylamine
Calcium DPA
lysozyme
Spore lytic enzyme
Parent (wild type)
2.0
1.7
6.7
4.2
A7
2.4
2.5
5.5
3.5
C5
0.4
3.3
6.8
4.3
D5
0.2
1.7
6.8
5.1
Strain
Discussion 0n the basis of these results we can tentatively construct a hypothetical pathway for the germination of B. cereus spores, as shown in Fig. 1. Figure 1
Hypothetical pathway for germination of B. cereus spores
In both pathways the initiating reactions would lead to activation of lytic enzymes that break down the outer layers of the spore, allowing rehydration and loss of material to the medium. A surfactant such as n-dodecylamine might be able to bypass the initiation reactions by damaging the spore membrane and allowing rehydration.
Mutants like C5 and D5, which do not respond to the surfactant might have defects in their endogenous lytic mechanism, since they germinate normally in the presence of exogenous enzymes. Further study of the mutants may provide more information on the nature and consequences of the lesions and thus about the nature of the initiating stages of spore germination and on the nature of the small fraction of "superdormant" spores that always remain after the majority of a population have germinated (Gould et al., 1968). Note added later Professor Gale suggested that a critical reaction might involve donation of an amino group. This could be derived from alanine by the action of alanine dehydrogenase or, alternatively, inosine might be able to carry an amino group as suggested in Fig 2, Figure 2 Amino acid Keto acid
Possible mechanism for donation of an amino group inosine adenosine
XNH2 X
This is an interesting idea but this mechanism is difficult to reconcile with the fact that adenosine is less effective than inosine as an adjunct to alanine as a spore germinant. Furthermore the germination rate in inosine + alanine is reduced in the presence of adenosine. References Gould, G.W., Jones, A. and Wrighton, C. (1968), J. Appl. Bact. 31, in press Gould, G.W., Hitchins A.D. and King W.L. (1966), J. Gen. Microbiol. 44, 293 Riemann, M. & Ordal, J. (1961), Science, N.Y., 133, 1703 Rode, L.J. & Foster J.W. (1961), J. Bact. 81, 768 Warren, S.C. & Gould, G.W. (1968), in preparation Author's note added 15.09.2013 The reference to this paper is recognised but has no text, probably because it was never published as a paper, only as a contribution to a Symposium. I have recovered the original text from my own archives, tidied it up and uploaded it mainly for my own satisfaction. The work described was undertaken 45 years ago is unlikely to be of more than historical interest, if that, to contemporary readers. At this time I did some work on germination kinetics, which included a mathematical model of the process, whose development was stimulated by observations of differences in the kinetics of germination between l-alanine and inosine + alanine. The model showed that the differences arose depending on whether the initiation phase or the germination phase is rate limiting. I wrote the work up for publication but the draft was (justifiably) sent back for revision. It has always been a source of regret to me that I could not make the revisions and resubmit because I was moving jobs within Unilever. I have the original draft and I intend to produce an edited version that I will shortly upload as supplementary material to this paper on germination mutants.
