Isolation and Characterization of Superdormant Spores of Bacillus Species

Abstract

Superdormant spores of Bacillus subtilis and Bacillus megaterium were isolated in 4 to 12% yields following germination with high nutrient levels that activated one or two germinant receptors. These superdormant spores did not germinate with the initial nutrients or those that stimulated other germinant receptors, and the superdormant spores' defect was not genetic. The superdormant spores did, however, germinate with Ca 2؉ -dipicolinic acid or dodecylamine. Although these superdormant spores did not germinate with high levels of nutrients that activated one or two nutrient germinant receptors, they germinated with nutrient mixtures that activated more receptors, and using high levels of nutrient mixtures activating more germinant receptors decreased superdormant spore yields. The use of moderate nutrient levels to isolate superdormant spores increased their yields; the resultant spores germinated poorly with the initial moderate nutrient concentrations, but they germinated well with high nutrient concentrations. These findings suggest that the levels of superdormant spores in populations depend on the germination conditions used, with fewer superdormant spores isolated when better germination conditions are used. These findings further suggest that superdormant spores require an increased signal for triggering spore germination compared to most spores in populations. One factor determining whether a spore is superdormant is its level of germinant receptors, since spore populations with higher levels of germinant receptors yielded lower levels of superdormant spores. A second important factor may be heat activation of spore populations, since yields of superdormant spores from non-heat-activated spore populations were higher than those from optimally activated spores. Spores of variousBacillus species are formed in sporulation and are metabolically dormant and very resistant to environmental stress factors (21, 37). While such spores can remain in this dormant, resistant state for long periods, they can return to life rapidly through the process of germination, during which the spore's dormancy and extreme resistance are lost (36). Spore germination has long been of intrinsic interest, and continues to attract applied interest, because (i) spores of a number of Bacillus species are major agents of food spoilage and food-borne disease and (ii) spores of Bacillus anthracis are a major bioterrorism agent. Since spores are much easier to kill after they have germinated, it would be advantageous to trigger germination of spores in foods or the environment and then readily inactivate the much less resistant germinated spores. However, this simple strategy has been largely nullified because germination of spore populations is heterogeneous, with some spores, often called superdormant spores, germinating extremely slowly and potentially coming back to life long after treatments are applied to inactivate germinated spores (8,9,16). The concern over superdormant spores in populations also affects decisions such as how lon...

Enhanced Inactivation of <i>Bacillus subtilis</i> Spores by Carbonation with Heating in the Presence of Monoglycerol-caprate

“…Previous studies have shown that carbonation with mild heating (<10 MPa, 20 _ 40℃) effectively inactivates vegetative bacterial cells and fungal cells (Watanabe et al, 2003). However, spores of various Bacillus species are metabolically dormant and are thus highly resistant to environmental stress factors (Ghosh and Setlow, 2009) including carbonation (Watanabe et al, 2003).…”

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

“…The t lag values were computationally obtained for more than 90% of spores imaged. The remaining spores were almost exclusively ones that did not germinate during the experiment, possibly because many of these spores were "superdormant" (6,7,34), and these spores were not analyzed.…”

“…The two other B. subtilis strains used were PY79 derivatives and were PY79 (wild type) and PB705 (32) (prkC) (obtained from J. Dworkin). Spores of B. subtilis strains were prepared on 2ϫ SG agar plates without antibiotics at 37°C, and spores were incubated, harvested, and cleaned as described previously (6,19). The B. megaterium strain used was QM B1551 (ATCC 12872) (originally obtained from H. S. Levinson).…”

The Ger Receptor Family from Sporulating Bacteria

“…Although germination is an all-or-nothing process for each individual spore, subpopulations of spores appear to have different germination thresholds (Ghosh and Setlow, 2009a). This might reflect a stochastic process due to the low expression levels of Ger receptor proteins.…”

“…This might reflect a stochastic process due to the low expression levels of Ger receptor proteins. Indeed, spores that can only germinate when multiple Ger receptors are activated simultaneously have been isolated in B. subtilis, B. megaterium and B. cereus (Ghosh and Setlow, 2009a;Ghosh and Setlow, 2009b). Future work should elucidate whether differences in Ger receptors levels could account for these superdormant spores.…”