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

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 degrees C at a rate of 1 degree 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-Mr 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 greater than 12,000 were essential for fast germination.     

Involvement of calcium in germination of coat-modified spores of Bacillus cereus T.

The effect of calcium on germination of coat-modified Bacillus cereus T spores was investigated. Coat-modified spores produced either by chemical extraction (SDS-DTT-treated spores) or by mutagenesis (10LD mutant spores) were unable to germinate in response to inosine. While SDS-DTT-treated spores could germinate slowly in the presence of L-alanine, 10LD mutant spores could not germinate at all. The lost or reduced germinability of coat-modified spores was restored when exogenous Ca2+ was supplemented to the germination media. The calcium requirement of coat-modified spores for germination was fairly specific. The simultaneous presence of germinant with Ca2+ was also required for germination of coat-modified spores. The optimal recovery of germinability was observed in the presence of 1.0 mM of calcium acetate. The calcium requirement itself was remarkably diminished under the condition in which L-alanine and a certain purine nucleoside analog, adenosine or inosine, coexisted. The lost or diminished germinability observed in SDS-DTT-treated spores or 10LD mutant spores may be attributed to the loss of calcium associated with the spore integuments.     

Calmodulin antagonists inhibit germination of Bacillus cereus T spores

M. RACINE, J. DUMONT, C.P. CHAMPAGNE AND A. MORIN. 1991. The effects of lactose, ammonium and phosphate on the production of extracellular polysaccharide from Propionibacterium acidi-propionici VM-25 were studied in whey-based media. The polysaccharide was composed of a water-soluble fraction (15% w/w), a water-insoluble fraction (27% w/w) and ca 65% (w/w) of ash. Up to 15 g/l of polysaccharide was produced during growth on partially deproteinated whey, supplemented with lactose, NH₄ Cl and KH₂ PO₄, after incubation at pH 7.0 and 25.C for 90 h. The final viscosity of the medium remained under 20 centipoises at the end of the fermentation (100–140 h). The fermentation of whey enabled a reduction of the lactose content up to 50%. The polysaccharide-containing fractions were composed of glucose, galactose, mannose, rhamnose and fucose and had M, < 5800. The polysaccharide may have applications as a low viscosity stabilizing agent.     

Synthesis of Bacillus cereus spore coat protein

The major structural protein of Bacillus cereus spore coats was synthesized, commencing 1 to 2 h after the end of exponential growth, as a precursor with a mass of ca. 65,000 daltons. About 40% of this precursor, i.e. 26,000 daltons, was converted to spore coat monomers of 13,000 daltons each, perhaps as disulfide-linked dimers. The rate of conversion varied, being initially slow, most rapid at the time of morphogenesis of the coat layers, and then slow again late in sporulation, coincident with a decrease in intracellular protease activity. There was a second major spore coat polypeptide of about 26,000 daltons that was extractable from mature spores in variable amounts. This protein had a peptide profile and a reactivity with spore coat protein antibody that were very similar to those of the 13,000-dalton monomers. It is probably a disulfide-linked dimer that is not readily dissociated.     

Effect of phenolic compounds and oleuropein on the germination of Bacillus cereus T spores

The phenolic compounds extracted from olives with ethyl acetate inhibited germination and outgrowth of Bacillus cereus T spores. Purified oleuropein, a well-characterized component of olive extract, inhibited these processes also. The addition of oleuropein and olive extracts 3 or 5 min after germination began, immediately decreased the rate of change of phase bright to phase dark spores and delayed significantly outgrowth.     

Stimulation of germination of unactivated Bacillus cereus spores by ammonia

Inclusion of ammonia in germinant mixtures containing L-alanine and inosine stimulated germination of unactivated Bacillus cereus spores at rates equal to those obtained using heat-activated spores without ammonia. D-Alanine had little effect on germination of heat-activated spores, but severely inhibited germination of unactivated spores in the presence of ammonia. Ammonia did not replace the requirement for either L-alanine or inosine: all three compounds were required for rapid germination. Kinetic analysis suggested that the functions of ammonia and L-alanine were more closely related than the functions of ammonia and inosine. With rate-saturating concentrations of L-alanine and inosine, germination rates showed saturation kinetics for ammonia with a Km for NH4Cl of 5 mM. Comparisons of the effects of salts, amines and pH on germination rates suggested that NH4OH rather than NH+4 was the rate-limiting form of ammonia. In comparisons of various strains of B. cereus, stimulation of germination by ammonia occurred in all cases, although spores of most soil isolates germinated more rapidly than B. cereus T spores in the absence of ammonia.     

Inhibition of germination ofBacillus cereus T spores by phenylglyoxal

Phenylgloxal at a concentration of 0.6 mM inhibited germination of Bacillus cereus T spores as characterized by a decrease in absorbance, dipicolinic acid and loss in heat resistance in a chemically defined growth and sporulation medium. In a germination medium containing L-alanine and adenosine, phenylglyoxal inhibited decrease in absorbance and affected partial loss of viability. It is postulated that phenylglyoxal interacts with free amino groups of various enzymes or amino compounds present in the spore structure thereby causing the inhibition of germination.     

Studies on the bacterial spore coat. IX. The role of surface charge in germination of Bacillus megaterium spores

The surface charge of Bacillus megaterium QM B1551 spores was estimated to be negative, -0.2 ad -0.4 mueq/mg by colloidal titration using glycol chitosan (GCh) and methylglycol chitosan (MGCh), respectively, as positive colloids. MGCh, which reacts with all of the negatively charged groups including carboxylate, inhibited the second stage of the germination to result in semirefractile spores, but GCh, which reacts only with strong acidic groups such as phosphate, did not. The spores produced in a medium with limited phosphate had coats with low phosphate content and carried less negative charge, and they were induced to germinate with 0.4 mM KNO3, which is one-tenth of the minimum concentration required for the germination of the control spores. A similar increase in germinability was observed in spores incubated with calcium acetate. The results suggest that the role of the surface charge in germination is as follows. Strong acidic groups (such as phosphate) in the coat may block the action of ionic germinants and act as a barrier against the initiation of ionic germination. Positively charged compounds (such as calcium) may compensate for this blocking effect. Weak acidic groups (such as carboxylate) may be involved in the later stage of germination.     

A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)