Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

Abstract

Aim: To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results: Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12 h and with temporal resolutions of ≤30 s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca‐dipicolinic acid (CaDPA) (Tlag and Trelease times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔTrelease times (Trelease−Tlag) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Conclusions: Since (i) ΔTrelease times were essentially identical in GR‐dependent germination of SD and dormant spores; (ii) rates of GR‐independent germination of SD and dormant spores were identical; (iii) large increases in Tlag times were the major difference in the GR‐dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter Tlag times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. Significance and Impact of the Study: This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.

Levels of Germination Proteins in Dormant and Superdormant Spores of Bacillus subtilis

“…Not surprisingly, increased average GR levels decrease the T lag , and lower average GR levels are associated with a longer T lag , with SD B. subtilis spores having T lag s of hours (25,40). The latter effect of GR levels on the T lag is certainly consistent with the low levels of GRs activated by the germinant used for SD spore isolation being responsible for these SD spores' extremely slow germination with this germinant and the extremely long T lag for such a germination (41). However, there appear to be variables, in addition to GR levels, that play roles in germination heterogeneity and, thus, presumably spore superdormancy.…”

“…However, there is some evidence that GRs can act cooperatively (11,39), so perhaps a linear relationship between GR levels and rates of spore germination is not to be expected. It is also notable that different SD spore isolates have been found to give rather different quantitative results when germination via GRs not selected against in SD spore isolation is measured (7,41; this work). These results have varied from large decreases in germination via unselected GRs to only small decreases, even though all these SD spores germinated extremely poorly via the GRs selected against in SD spore isolation.…”

“…Work with spores of several Bacillus species as well as Clostridium perfringens spores has shown that the major variable in the kinetics of spore germination with nutrients is the time between the mixing of the spores and germinant and the initiation of a rapid release of CaDPA, termed T lag (14,25,36,37,40,41). Values of T lag can vary between a few minutes to many hours for individuals in the same spore population.…”

“…Unfortunately, the simple strategy described above cannot generally be applied, since the germination of populations of spores of both Bacillus and Clostridium species is quite heterogeneous (7,8,10,13,14,28,30,36,37,40,41). Thus, while the great majority of a spore population may germinate within 30 to 60 min after the addition of nutrient germinants, others may not germinate for many hours or even days.…”

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

“…It is not known precisely what causes the variability in the T lag times for individual spores in populations, although factors that affect the commitment times of spore populations and T lag times of individual spores include spore heat activation, germinant concentrations, and GR levels (15,19,21). Spores that have higher GR levels germinate faster than spores with lower GR numbers, and spores with extremely low GR levels germinate extremely slowly and are termed superdormant (22,23).…”