Monitoring of Commitment, Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

Abstract

bShort exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ϳ7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (T release ) greater than the time of germinant removal (T b ) increased, while the time T lag ؊ T b , where T lag represents the time when rapid DPA release began, was decreased but rapid DPA release times (⌬T release ‫؍‬ T release ؊ T lag ) were increased; Factors affecting average T release values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants.

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA 2mob Operon in Foodborne Strains of Bacillus subtilis

“…Under laboratory conditions, spores of B. subtilis are often heat activated (HA) for 30 min at 70°C to increase their germination responses to nutrients (51)(52)(53). In this study, germination (which, under a phase-contrast microscope, is reflected by the phase-bright to phase-dark transition of spores) in response to L-alanine and AGFK was assessed for HA and non-HA spores of various B. subtilis strains that exhibit low to extremely high heat resistance.…”

Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

“…In most experiments, germination of multiple individual spores was monitored by PC microscopy as described previously (11,(20)(21)(22). Changes in wild-type spores' PC image intensities at least through T release of germination are almost certainly due to changes in spores' CaDPA content, and this is also true for analyses of spore germination by DIC microscopy (see below) (9)(10)(11).…”

“…In experiments measuring spores' commitment to germinate, spores were germinated with 10 mM L-valine for 10 min at various temperatures, and then the L-valine was replaced with 10 mM D-alanine in 25 mM K-HEPES buffer (pH 7.4) as described previously (20) and incubation was continued at 37°C. Previous work has shown that D-alanine addition to spores germinating with L-valine blocks further commitment but allows completion of the germination of spores that had become committed prior to D-alanine addition (7).…”

Investigating germination and outgrowth of bacterial spores at several scales

“…Successive stages of the germination and outgrowth of bacterial spores, observed under phase-contrast microscopy and methods applied for monitoring. Commitment to germination can be followed by the release of CaDPA (Zhang, Liang, Yi, Setlow, & Li, 2014). Spores remain phase-bright.…”

“…Spore germination can be monitored by titration in the culture medium of CaDPA released by spores (5. Zhang et al, 2014). Bioluminescence methods consist in observing bioluminescence of lux-containing engineered spores (6.…”