Microbes Environ. Vol. 19, No. 3, 199–202, 2004 http://wwwsoc.nii.ac.jp/jsme2/ Minireview The Role of Calcium in Bacterial Spore Germination YNTE P. DE VRIES1,2,3* 1 Wageningen Centre for Food Sciences, P.O. Box 557, 6700 AN, Wageningen, the Netherlands 2 Wageningen UR Laboratory of Food Microbiology, P.O. Box 8129, 6700 EV, Wageningen, the Netherlands 3 Agrotechnology and Food Innovations A&F, P.O. Box 17, 6700 AA, Wageningen, the Netherlands (Received May 22, 2004—Accepted June 30, 2004) The germination of bacterial endospores involves the following structural and physiological events: binding of nutrients to specific receptors located in the spore, the release of cations and calcium dipicolinate (CaDPA) from the core, degradation of the spore cortex, and the breakdown of small acid soluble proteins (SASP) protecting the DNA in the core. This minireview focuses specifically on the action of germination-specific lytic enzymes (GSLE) and the role of calcium ions in the degradation and collapse of the spore cortex. Several key questions for future research are discussed. Key words: endospore germination, calcium, dipicolinic acid, germination specific lytic enzymes Bacterial sporulation is a process triggered by starvation Spore germination and/or quorum sensing37,39). Bacterial spores are metaboli- In order to grow and proliferate, bacterial spores must cally dormant and resistant to many kinds of insults that are germinate (for recent reviews see references, 24 and 37). lethal to vegetative cells, such as heat, desiccation, pH ex- The dormant spore therefore monitors its environment for tremes, radiation and toxic chemicals36). The bacterial spore nutrients, and when conditions are favorable for growth, the has a unique structure43,45) (Fig. 1) that brings about its spore germinates and converts back to a vegetative cell that remarkable resistance and dormancy. A key feature is the can grow and divide. In addition to its intrinsic interest, dehydrated core that contains only 30–50% of water and an spore germination has attracted applied interest, because it enormous amount (10% of the total spore mass) of dipi- is through germination that spores ultimately cause food colinic acid (DPA), chelated with calcium ions as CaDPA. spoilage and poisoning37). Briefly, germination involves: 1) The process of sporulation, the spore structure, spore germi- binding of nutrients to specific receptors located at the inner nation and the environmental role of spores have recently (plasma) membrane of the spore; 2) the release of cations been excellently reviewed1,10,24,27,30,33,37,42). In this minireview, and CaDPA from the core by an unknown mechanism, re- I would like to focus on a specific area of spore germination sulting in partial rehydration of the core; 3) degradation of that has received little attention so far: the interaction of the cortex, enabling full rehydration of the core; 4) break- calcium ions with the spore cortex and their role in ger- down of small acid-soluble proteins (SASP) that protect the mination. DNA in the core and start up of metabolism resulting in a normal vegetative cell. Thus, spore germination involves the breakdown of the unique structure of the spore, and a key step is degradation of the cortex. The cortex is a thick * Corresponding author; E-mail: [email protected], Tel: 31– layer of modified peptidoglycan surrounding the core, 317–475109, Fax: 31–317–475347 which plays a key role in spore resistance and dormancy33). 200 VRIES proper moment is not completely clear. Paidhungat et al.29) hypothesized that partial rehydration of the core after CaDPA excretion results in a deformation of the cortex, and that SleB acts only on the cortex when it is in this deformed state. Furthermore, these authors have shown that CaDPA (extruded from the spore core during germination or added from outside as a germinant) seems to activate CwlJ in a more direct manner. Cortex collapse and calcium ions Additionally, CaDPA could help to initiate cortex degra- dation through structural modification of the peptidoglycan. Cortex peptidoglycan has an expanded structure, is electro- negative15) and loosely cross-linked2,31). The cortex structure is conserved amongst different species3). Loosely cross- linked peptidoglycan exhibits a significant change in vol- ume upon alteration of its ionic environment26,28). Nakatani et al.26) reported a decrease of 80% in volume of isolated cortex material from Bacillus megaterium in the presence of divalent cations. It is possible that the calcium from the Fig. 1. The structure of a bacterial spore, cross section. From Gould CaDPA that is excreted from the core during early germina- and Hurst14). tion causes a collapse of the expanded cortex structure, thereby helping to activate the GSLE and creating space to As explained below, calcium may play an important role in allow partial rehydration of the core. Thus, in addition to the the degradation of the cortex during germination. rehydrating core pushing the cortex to the outside29), the calcium excreted from the core as CaDPA could cause shrinking of the cortex, increasing the overall effect. Indeed, Cortex degradation 26) Nakatani et al. have shown that the volume of cortex The breakdown of the cortex is a later event in germina- peptidoglycan is reduced when CaDPA is present, indicat- tion and it involves the action of germination specific lytic ing that cortex peptidoglycan successfully competes with enzymes (GSLE)4) that specifically recognize muramic- DPA for Ca2-binding. Spores germinate in response to -lactam, the most distinguishing feature of cortex exogenous CaDPA, but not to calcium ions alone29). One peptidoglycan32). Research focusing on Bacillus subtilis has explanation could be that DPA chelates calcium ions revealed three enzymatic modifications of cortex material and thereby shuttles the positively charged calcium ions that occur upon germination2), while genetic analysis has from outside the spore through the coat into the cortex. yielded two enzymes, SleB and CwlJ, involved in cortex There, in addition to the direct effect on CwlJ, calcium from degradation. SleB was reported to be an amidase by the CaDPA can stimulate cortex collapse and degradation. Moriyama et al.25), while Boland et al.7) reported it to be a This theory of calcium binding to the cortex would lytic transglycosylase. SleB has been localized by immuno- explain the small lag that has been observed in calcium electron microscopy to the boundary region between the efflux as compared to DPA efflux at the beginning of spore’s cortex and coats25). CwlJ has been located in the germination11). Other supporting evidence is that the lysis of inner coats of the spore, close to the cortex5). CwlJ is, decoated spores by isolated and purified GSLE is optimal in like SleB, involved in cortex lysis, but its precise enzymatic the presence of 10 mM Ca2 or Mg2 44). More recently, activity has not been described. SleM, a GSLE from C. perfringens was shown to require While a GSLE from Clostridium perfringens has been divalent cations for its activity8), and Ca2 and Mg2 were shown to be activated upon germination by proteolytic suggested to be involved in cortex lytic enzyme activation cleavage23), both SleB and CwlJ are present in the spore during germination induced by high pressure17). Indeed, in a mature form, and how their activity is restricted to the the importance of cations in the germination of bacterial Calcium and Spore Germination 201 spores has since long been known11,12,13,20). Repeated observa- standing of spore germination is almost solely based on one tions are that B. cereus spores with a low DPA content ger- strain of B. subtilis, which has been propagated in the lab minate poorly and slowly in response to nutrient for many generations. Other species and wild isolates must germinants16,18,19,46), that DPA content is linked with Ca2 be more included in future research to validate the current content16,18), and that the germination of low-DPA spores in models and to provide much needed knowledge to the CaDPA is unaffected19). Moreover, the germination re- applied field. sponse of low-CaDPA spores in alanine is enhanced by the exogenous addition of calcium and accompanied by the up- take of these Ca2 ions18), while calcium channel blockers References 22) inhibit germination . Finally, acid-titrated “H”-spores, 1) Aronson, A. 2002. Sporulation and delta-endotoxin synthesis in which have been stripped from their cations, do not readily Bacillus thuringiensis. Cell. Mol. Life Sci. 59: 417–425. germinate. Their ability to germinate can be restored by the 2) Atrih, A. and S.J. Foster. 1999. The role of peptidoglycan addition of Ca2 ions6,34). These pieces of evidence suggest structure and structural dynamics during endospore dormancy and germination. Antonie van Leeuwenhoek 75: 299–307. a key role not only for DPA, but also for calcium ions in 3) Atrih, A. and S.J. Foster. 2001. Analysis of the role of bacterial germination. endospore cortex structure in resistance properties and demon- stration of its conservation amongst species. J. Appl. Microbiol. 91: 364–372. Key questions for future research 4) Atrih, A. and S.J. Foster. 2001. In vivo roles of the germination- specific lytic enzymes of Bacillus subtilis 168. Microbiology 147: The role of calcium ions in not only spore germination 2925–2932. but also spore dormancy deserves more attention. Key ques- 5) Bagyan, I. and P. Setlow. 2002. Localization of the cortex lytic tions for future research are: enzyme CwlJ in spores of Bacillus subtilis. J. Bacteriol. 184: 1219–1224. Is calcium required for the attainment of a dehydrated 6) Bender, G.R. and R.E. Marquis. 1985. Spore heat resistance and core? It has been shown that DPA-less spores have higher specific mineralization. Appl. Environ. Microbiol. 50: 1414– core water content then DPA-sufficient spores.