Quantitative Analysis of Spatial-Temporal Correlations during Germination of Spores of Bacillus Species

Abstract

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...

Summer meeting 2013 - when the sleepers wake: the germination of spores ofBacillusspecies

“…This likely precludes triggering of the germination of nearby dormant spores by CaDPA released from one germinating spore (Zhang et al . ). In contrast to CwlJ, CaDPA alone does not activate SleB, and its mechanism of activation in germination is not known.…”

“…The precise level of specific GRs in spores is almost certainly the major factor determining the rate of germination of spores of different populations, and also of different individuals in a single population (Zhang et al . , , , ) as discussed further below under SD spores.…”

Analysis of Metabolism in Dormant Spores of Bacillus Species by 31 P Nuclear Magnetic Resonance Analysis of Low-Molecular-Weight Compounds

“…This molecule triggers spore germination by activating the CLE CwlJ (2,26). While CaDPA released from a few spores that spontaneously germinate could cause germination of GR-less spores, CaDPA is effective only at rather high concentrations (14,26,27). Thus, we felt that using GR-less spores alone would reduce the germination of spores incubated at 37 and 50°C sufficiently such that more than enough dormant spores would remain after extended incubation to allow for analysis of small molecules in these spores.…”

Expression Level of Bacillus subtilis Germinant Receptors Determines the Average Rate but Not the Heterogeneity of Spore Germination

“…For microscopy experiments, spores were first heat activated for 30 min at 75°C and then cooled on ice for at least 15 min. The heat-activated spores were spread on 0.1% polylysine-coated glass dishes (18). Levels of GRs in spores on the polylysinecoated glass dishes were measured with epifluorescence microscopy as described previously (7).…”