The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore's DPA once rapid DPA release had begun (⌬T release ) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ⌬T release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination >15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T lag , between mixing spores with nutrient germinants and the beginning of ⌬T release . (iv) A number of factors decrease spores' T lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.Spores of various Bacillus species are metabolically dormant and can survive for years in this state (30). However, spores constantly sense their environment, and if appropriate small molecules termed germinants are present, spores can rapidly return to life in the process of germination followed by outgrowth (25,29,30). The germinants that most likely trigger spore germination in the environment are low-molecularweight nutrient molecules, the identities of which are strain and species specific, including amino acids, sugars, and purine nucleosides. Metabolism of these nutrient germinants is not needed for the triggering of spore germination. Rather, these germinants are recognized by germinant receptors (GRs) located in the spore's inner membrane that recognize their cognate germinants in a stereospecific manner (17,24,25,29). Spores have a number of such GRs, with three functional GRs in Bacillus subtilis spores and even more in Bacillus anthracis, Bacillus cereus, and Bacillus megaterium spores (6,29,30).Binding of nutrient germinants to some single GRs is sufficient to trigger spore germination, for example the triggering of B...
Bacteria of Bacillus species sporulate upon starvation, and the resultant dormant spores germinate when the environment appears likely to allow the resumption of vegetative growth. Normally, the rates of germination of individual spores in populations are very heterogeneous, and the current work has investigated whether spore-to-spore communication enhances the synchronicity of germination. In order to do this work, time-lapse optical images of thousands of individual spores were captured during germination, and an image analysis algorithm was developed to do the following: (i) measure the positions and germination rates of many thousands of individual spores and (ii) compute pairwise correlations of their germination. This analysis showed that an individual spore's germination rate was dependent on its distance from other spores, especially at short distances. Thus, spores that were within a few micrometers exhibited an increased synchronicity in germination, suggesting that there is a mechanism for short-range communication between such spores during germination. However, two molecules known to be germinants that are released during germination, L-alanine and the 1:1 chelate of Ca 2؉ and dipicolinic acid, did not mediate spore-to-spore communication during germination.Bacteria of Bacillus species form spores in order to survive adverse environmental conditions (18, 28). These spores are metabolically dormant and highly resistant to most antibacterial agents. However, the dormant spores continually monitor their environment and can resume vegetative growth once the environment becomes suitable. The first step in a spore's return to growth is germination, which can be initiated by some specific nutrient germinant molecules, including amino acids, nucleosides, and sugars (16,27). A key early step in germination is the release of the spores' large pool (ϳ20% of the dry weight of spores' central region or core) of dipicolinic acid (DPA) that is chelated to divalent metal ions, predominantly Ca 2ϩ (Ca-DPA) (12,25,27). Recent studies with individual spores have shown that once rapid release of Ca-DPA during germination has begun, it is completed in only a few minutes (4,13,22,27,35). However, there are generally long lag times (t lag ) between the time of addition of a nutrient germinant to the initiation of rapid Ca-DPA release for individual spores, and t lag values vary greatly from spore to spore even in genetically identical populations germinating under the same conditions (4, 35). In contrast, the time needed for release of ϳ90% of a spore's Ca-DPA during germination is relatively constant at several minutes.The precise kinetics of spore germination are of great interest to the food and medical products industries, because once spores have germinated they are much easier to kill than the more resistant dormant spores. Unfortunately, the rates of germination of individual spores are extremely heterogeneous due to the variations in t lag (4,11,13,35). Some of the factors that influence t lag values have recently...
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