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.
Aim To analyse the effect of wet heat treatment on nutrient and non‐nutrient germination of individual spores of Clostridium perfringens. Methods and Results Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat‐treated spores of Cl. perfringens with various germinants. When incubated in water at 90–100°C for 10–30 min, more than 90% of spores were inactivated but 50–80% retained their Ca2+‐dipicolinic acid (CaDPA). The wet heat‐treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640–1680 cm−1 (amide I) and 1230–1340 cm−1 (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat‐treated spores that retained CaDPA germinated with KCl or l‐asparagine, but wet heat treatment increased values of Tlag, ΔTrelease and ΔTlys, during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl. perfringens spores lacking the essential cortex‐lytic enzyme (CLE), SleC, exhibited longer Tlag and ΔTrelease values during KCl germination than wild‐type spores and germinated poorly with CaDPA. Wet heat‐treated wild‐type spores germinating with CaDPA or dodecylamine exhibited increased Tlag, ΔTrelease and ΔTlys values, as did wet heat‐treated sleC spores germinating with dodecylamine. Conclusions (i) Some proteins important in Cl. perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat. Significance and Impact of the Study This study provides information on the germination of individual Cl. perfringens spores and improves the understanding of effects of wet heat treatment on spores.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.