Triple fixation of Bacillus subtilis dormant spores.

A triple-fixation method with a sequential application of 5% glutaraldehyde, 1% osmium tetroxide, and 2% potassium permanganate gave superior preservation of the ultrastructure of Bacillus subtilis dormant spores with a thick spore coat.     

Transformation of germinated spores of Bacillus subtilis on agar plates

l-Alanine-germinated spores of Bacillus subtilis developed a competence on agar plates after 10 h of incubation. Addition of marker amino acid to the plates was required for the transformation.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Trypsinlike enzymes from dormant and germinated spores of Bacillus cereus T and their possible involvement in germination.

Trypsin-like enzymes were studied in dormant, activated, and germinated spores of Bacillus cereus T. Dormant spores contained two heat-labile enzyme activities. One was extractable with 2 M KCl and hydrolyzed azo-albumin. The second, a trypsinlike activity, was not extractable with 2 M KCl and hydrolyzed benzoyl-L-arginine-p-nitroanilide. Because of their heat instability, these two enzyme activities are probably not involved in the germination of heat-activated spores. Upon germination of heat-treated spores, a trypsinlike protease which was not detected in intact dormant spores was activated or exposed. This enzyme, when measured in intact germinated spores, hydrolyzed benzoyl-DL-arginine-p-nitroanilide but not azo-albumin and was inhibited in situ by sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid and Hg2+. There was a correlation between the inhibition of germination and enzymatic activity by sulfhydryl-blocking reagents. The enzyme was also inhibited by leupeptin, tosyl-L-lysine chromoethyl ketone, and tosyl-L-arginine methyl ester. Good correlation existed between the inhibition of germination and enzymatic activity by these agents. Electron micrographs showed that in the presence of trypsin inhibitors, the spores did not lose their cortex. The protein extracts of the inhibited spores formed a somewhat different electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the protein extracts of dormant or germinated spores.     

The structure of bacillomycin L, an antibiotic from Bacillus subtilis (author's transl)]

The structure of bacillomycin L, an antifungal agent isolated from the culture medium of a strain of Bacillus subtilis, has been investigated. The peptide moiety contains one mole each of D-aspartic acid, L-aspartic acid, L-glutamine, L-serine, D-serine, L-threonine, and D-tyrosine. The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid (46%), 3-amino-12-methyltetradecanoic acid (38%, 3-amino-14-methylpentadecanoic acid (11%), and two minor homologues. The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis. The molecular weight was determined by the thermoosmotic method; this showed that bacillomycin L has a monomeric structure which is given in Formula 1.     

Deoxyribonucleic acid hybridizations among some vibrio-like marine bacteria (author's transl)]

The genotypic relationships established by DNA/DNA hybridization in vitro confirm the results obtained by earlier phenotypic analyses of certain vibrio-like marine bacteria. These strains are closely related to the species Photobacterium fischeri, and do not belong to the genus Vibrio. The group of marine, vibrio-like bacteria that require Na+ for growth is genotypically very heterogeneous.     

Ultrastructural localization of dipicolinic acid in dormant spores of Bacillus subtilis by immunoelectron microscopy with colloidal gold particles.

The localization of dipicolinic acid in dormant spores of Bacillus subtilis was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were distributed mainly in the core regions of dormant spores and were not observed in those of germinated or autoclaved spores. This result clearly demonstrates that dipicolinic acid is localized in the cores of dormant spores.     

Permeability of dormant spores of Bacillus subtilis to malachite green and crystal violet

The permeability of dormant spores of Bacillus subtilis to malachite green (MG) and crystal violet (CV) was examined by using potassium trichloro(eta 2-ethylene)platinum(II) (KTPt) as an electron-opaque marker for the dyes. The spores were treated with the dyes and other substances at 30 degrees C for 30 min or at 80 degrees C for 5 min. When the spores were incubated in 50 mM-MG solution at 30 degrees C and in 50 mM-CV solution at 30 degrees C or 80 degrees C, many small electron-dense precipitates, which were chemical complexes of dyes and platinum, were seen, mainly around the boundary between the inner and outer coat regions. The spores treated under the above conditions were not stained. Treatment with 50 mM-MG alone or a mixture of 25 mM-oxalic acid and 50 mM-CV at 80 degrees C made the spores stainable and dye-TPt precipitates were observed mainly in the outer pericortex region. Pretreatment with 25 mM-oxalic acid and 5% (v/v) phenol at 80 degrees C followed by 50 mM-CV treatment at 30 degrees C gave the same results as above. It was considered from these results that the inner coat itself might function as the primary permeability barrier to MG and CV, and that a secondary barrier to the dyes might exist around the cortex region.