Involvement of the spore coat in germination of Bacillus cereus T spores

Abstract

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65°C at a rate of 1°C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-M, cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight >12,000 were essential for fast germination.

Involvement of Calcium in Germination of Coat‐Modified Spores of Bacillus cereus T

“…Chemical treatment of the spores. In order to modify the coat, spores of the wild strain were chemically treated by the method described by Aronson and Fitz-James (1) which has subsequently been adopted by others (18,36). Briefly, 50 mg of clean spores were suspended in 50 ml of a freshly prepared solution of 5 mm DTT, 0.5% SDS and 0.1 M NaCl with the pH adjusted to 10.…”

“…There is abundant evidence in the literature that the coats may be involved in spore germination. Spores whose coats are modified by chemical treatments almost invariably show altered or reduced germinability (3,4,6,8,10,18,27,36). Some coatless or coat-defective mutant spores are also deficient in germination (2,5,8,14,17,22).…”

“…Evidences accumulated in recent years clearly indicate that the coats of bacterial spores play some important roles in germination (4). Spores whose coats were artificially removed by chemical treatments (3,6,8,17,18,27,36) or rendered defective by mutation (2,5,9,14,17,22) displayed altered or markedly reduced germinability. At present, we know very little why coat-defective spores cannot germinate normally.…”

Gene structure and precursor processing of a novel Bacillus subtilis spore coat protein

“…The proteinaceous coat layers of the bacterial spore (exclusive of the exosporium) are believed to have a protective function and to play some role in germination (Aronson and Fitz-James, 1976;Kutima and Foegeding, 1987). A cryptic cortex lytic enzyme (Brown et al, 1982;Foster and Johnstone, 1987) or proteases involved in the activation of this enzyme (Boschwitz etal., 1985) may be associated with an integral part of the coat.…”

Role of Calcium in Biphasic Germination of Bacillus cereus T Spores Sensitized to Lysozyme

“…The SDS-DTT-treated spores seem to be lacking in a significant portion of "functional" Ca2+ necessary for germination (21). Kutima and Foegeding (15) described that some components (Mr, > 12 kDa) located in the spore coat or the outer membrane were extracted by 0.5% SDS-100 mM DTT and were essential for rapid germination of coatdefective spores. Recently, Shyu and Foegeding (24,26) reported that a novel calcium-binding protein (Mr, 24 kDa) was purified from dormant spores of B. cereus T. From these results, it is considered that the SDS-DTT-treatment of B. cereus spores might remove functional Ca2+ and a certain component that specifically binds or traps Ca2+ , .…”