Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

Abstract

b When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ϳ20% of spore DPA) from individual spores that takes place prior to the lag time, T lag , when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T 1 ; (ii) the period of slow DPA leakage (⌬T leakage ‫؍‬ T lag ؊ T 1 ) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T 1 times but increased values of ⌬T leakage ; (iv) upon germination with L-valine for 10 min followed by addition of D-alanine to block further germination, all germinated spores had T 1 times of less than 10 min, suggesting that T 1 is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T 1 and T lag times but not the amount of DPA released in ⌬T leakage ; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ⌬T leakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination. Spores of Bacillus species can remain dormant for long periods and are extremely resistant to a variety of environmental stresses. However, under appropriate conditions, normally upon the binding of specific nutrients to spores' nutrient germinant receptors (GRs), spores can rapidly return to life in the process of germination followed by outgrowth (1-3). Germination is important not only to spores but also to the food and medical product industries, since spores of a number of species are major agents of food spoilage and foodborne disease and must germinate to cause their deleterious effects (3). In contrast to the resistant dormant spores that are hard to kill, germinated spores have lost most dormant spore resistance properties and are killed relatively easily (2, 3). Consequently, there is much interest in the mechanism of spore germination, since preventing this process or promoting it efficiently could have significant applied microbiology applications.Germination of Bacillus subtilis spores can be triggered by L-alanine or L-valine or a combination of L-asparagine, D-glucose, D-fructose, and K ϩ (AGFK). These nutrient germinants trigger germination by binding to and in...

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

“…3 and Table 1). Notably, 13 to 16% of total spore CaDPA was released between T 1 and T lag , similar to results with nutrient germination of spores of Bacillus species (24). Given the good correspondence between CaDPA release and loss of DIC image intensity in spore germination, in further experiments we used DIC microscopy to monitor the germination of multiple individual C. difficile spores.…”

“…2a). In addition to T release , a number of other spore germination parameters have been previously described (23,24,40), including the following: T 1 , the time when CaDPA leakage starts; CaDPA leakage, the percent CaDPA released slowly between T 1 and T lag ; T lag , the time after T 1 when very rapid CaDPA begins; T release , the time when CaDPA release is complete; T lysis , the time when spore refractility becomes constant; and ⌬T release and ⌬T lysis , which are calculated as T release Ϫ T lag and T lysis Ϫ T release , respectively.…”

“…These bile salts were used at 0.5% (ϳ10 mM) with 19.6 mM glycine. Spore germination was also carried out at 37°C with 60 mM CaDPA made to pH 7.4 with Tris base and with 1.2 mM dodecylamine at 42°C in 25 mM K-HEPES buffer (pH 7.4) as described previously (24).…”

“…Measurement of commitment of C. difficile spores to germinate. A notable feature of B. subtilis spores is their irreversible commitment to germination, followed by some slow CaDPA leakage after a short exposure to germinant, with commitment taking place at approximately T 1 (22,24,50). Since C. difficile spores also showed a T 1 time followed by some slow CaDPA leakage, we examined if C. difficile spores also showed commitment after exposure to taurocholate and glycine for various periods, followed by germinant removal, extensive rinsing, and further incubation in buffer (Fig.…”

“…The process of an individual spore's germination has been divided into four phases according to a spore's optical intensity in differential interference contrast (DIC) or phase-contrast microscopy, with the different phases ending at times T 1 , T lag , T release , and T lysis (23,24). T 1 is the time when slow CaDPA leakage begins after germinant addition and is probably coincident with the time of commitment, T lag is the time after the start of slow CaDPA leakage when the initiation of very rapid CaDPA release begins, and T release is the time for completion of rapid CaDPA release; following T release , there is a further small decline in spore refractility due to the hydrolysis of the spore cortex and spore core swelling, and the time when spore refractility becomes constant is termed T lysis .…”