Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination. Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore’s germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca2+ chelated to DPA (Ca‐DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination. Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca‐DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores. Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.

Aims:  To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Methods and Results:  Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (Tlag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔTrelease) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average Tlag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The Tlag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average Tlag and ΔTrelease values in dodecylamine germination that does not utilize GRs. Conclusions:  Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical. Significance and Impact of the Study:  This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.

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.

Significance and Impact of the Study: This work shows that with Bacillus subtilis spore populations in which approximately 95% of individual spores were killed by several oxidizing agents, >95% of the spores in these populations germinated with nutrients, albeit slowly. This is important, as assay of an early germination event, release of dipicolinic acid, has been suggested as a rapid assay for spore viability and would give false-positive readings for the level of the killing of oxidizing agent-treated spore populations. Analysis of the germination kinetics of multiple individual untreated or oxidizing agenttreated spores also provides new information on proteins damaged by oxidizing agent treatment, and at least some of which are in spores' inner membrane. AbstractMore than 95% of individuals in populations of Bacillus subtilis spores killed approximately 95% by hydrogen peroxide or hypochlorite germinated with a nutrient, although the germination of the treated spores was slower than that of untreated spores. The slow germination of individual oxidizing agent-treated spores was due to: (i) 3-to 5-fold longer lag times (T lag ) between germinant addition and initiation of fast release of spores' large dipicolinic acid (DPA) depot (ii) 2-to 10-fold longer times (DT release ) for rapid DPA release, once this process had been initiated; and (iii) 3-to 7-fold longer times needed for lysis of spores' peptidoglycan cortex. These results indicate that effects of oxidizing agent treatment on subsequent spore germination are on: (i) nutrient germinant receptors in spores' inner membrane (ii) components of the DPA release process, possibly SpoVA proteins also in spores' inner membrane, or the cortex-lytic enzyme CwlJ; and (iii) the cortex-lytic enzyme SleB, also largely in spores' inner membrane. This study further indicates that rapid assays of spore viability based on measurement of DPA release in spore germination can give false-positive readings.

Aims:  To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results:  Bacillus megaterium was sporulated with no added MnCl2 and up to 1 mmol l−1 MnCl2. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H2O2. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions:  Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study:  The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.