Role of menaquinone in inactivation and activation of the Bacillus cereus forespore respiratory system.

The respiratory systems of the Bacillus cereus mother cell, forespore, and dormant and germinated spore were studied. The results indicated that the electron transfer capacity during sporulation, dormancy, and germination is related to the menaquinone levels in the membrane. During the maturation stages of sporulation (stages III to VI), forespore NADH oxidase activity underwent inactivation concomitant with a sevenfold decrease in the content of menaquinone and without major changes in the content of cytochromes and segment transfer activities. During the same period, NADH oxidase and menaquinone levels in the mother cell compartment steadily decreased to about 50% at the end of stage VI. Dormant spore membranes contained high levels of NADH dehydrogenase and cytochromes, but in the presence of NADH, they exhibited very low levels of O2 uptake and cytochrome reduction. Addition of menadione to dormant spore membranes restored NADH-dependent respiration and cytochrome reduction. During early germination, NADH-dependent respiration and cytochrome reduction were restored simultaneously with a fourfold increase in the menaquinone content; during germination, no significant changes in cytochrome levels or segment electron transfer activities of the respiratory system took place.     

Respiratory system of vegetative and sporulating Bacillus cereus.

The composition and organization of the Bacillus cereus respiratory system were studied. The abolition of NADH-dependent respiration in vegetative and sporulating cell membranes by near-UV light (360 nm) indicated that electrons reduce oxygen only through a quinone-cytochrome pathway. Difference spectroscopy demonstrated the presence of cytochromes b555, c548, aa3, b562, and a2. This composition and studies with respiratory inhibitors suggested that cytochromes are organized in at least two branches, one being highly sensitive to cyanide.     

Protein composition of cell and forespore membranes of Bacillus subtilis

A sporulation mutant of Bacillus subtilis 168 was isolated and characterized. The mutant, designated SB-23, releases viable forespores at the end of the developmental period. Forespores were isolated on linear Renografin gradients and used as a source of forespore membranes. The protein composition of forespore membranes was found to differ from the protein composition of vegetative cell membranes by discgel electrophoresis. The results are discussed in relationship to morphological and physiological differentiation during bacterial sporulation.     

A forespore checkpoint for mother cell gene expression during development in B. subtilis

Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the bofA and/or bofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.     

Condensation of the forespore nucleoid early in sporulation of Bacillus species.

Fluorescence microscopic examination coupled with digital videoimage analysis of 4',6-diamidino-2-phenylindole-stained sporulating cells of Bacillus megaterium or Bacillus subtilis revealed a striking condensation of the forespore nucleoid. While both mother cell and forespore compartments had equal amounts of DNA, the forespore nucleoid became greater than 2-fold more condensed than the mother cell nucleoid. The condensation of the forespore nucleoid began after only the first hour of sporulation, 2 to 3 h before expression of most forespore-specific genes including those for small, acid-soluble spore proteins, and was abolished in spo0 mutants but not in spoII or spoIII mutants. It is possible that this striking condensation of forespore DNA plays some role in modulating gene expression during sporulation.     

Dihydrodipicolinate reductases from Bacillus cereus and Bacillus megaterium.

Dyhydrodipicolinate reductases were purified 100-fold from crude extracts of B. cereus and B. megaterium and their properties were compared with those of the reductase from B. subtilis. The molecular weights of the reductases of B. cereus and B. megaterium were fount to be 155,000 and 150,000, respectively. These reductases were shown to be free of flavin, unlike the B. subtilis enzyme, which contains flavin. Both NADPH and NADH acted as coenzymes for these two reductases. NADPH being three or four times more effective than NADH. The Km values for NADPH and dihydrodipicolinate were 8 micrometer and 62 micrometer, respectively, with B. cereus reductase, and 13 micrometer and 59 micrometer with B. megaterium reductase. The pH optima of the enzymes from B. cereus and B. megaterium were pH 7.4 and 7.2, respectively. The reductases were inhibited by dipicolinate noncompetitively with respect to dihydrodipicolinate and the Ki values were 85 micrometer and 140 micrometer, respectively. Lysine and diaminopimelate were not inhibitory. The properties of the reductases from B. cereus and B. megaterium were similar, but they differed considerably from those of the B. subtilis enzyme. However, all three Bacillus reductases were markedly inhibited by dipicolinate, unlike the enzyme from E. coli.     

Membrane-bound nucleotidase of Bacillus cereus.

A membrane-bound nucleotidase of Bacillus cereus T was solubilized by digestion with trypsin and subsequently purified more than 300-fold. The purified nucleotidase was most active on ribonucleoside 5'-monophosphates and was slightly less active (40 to 60%) on deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates. In addition to hydrolytic activity, the nucleotidase preparation possessed phosphotransferase activity by which phosphate is transferred from a phosphate donor to the 5' position of nucleosides.     

Mechanisms of Bacillus cereus enteropathy.

Three strains of Bacillus cereus isolated from sausages (Salami and Trekker, RANBAC, Ranchi) produced enterotoxin which caused vascular permeability in skin and haemorrhage in the ligated ileal loops of rabbits. Histopathological studies revealed haemorrhage and congestion in submucosa, mononuclear cell infiltration in lamina propria and submucosa and villous atrophy. Histochemical studies ruled out the effect on mitochondrial enzymes of intestinal epithelial cells. Purified enterotoxin given intradermally to rabbits caused severe necrotic reaction at the site of injection and death within 4 hr. Histopathological changes observed in liver included congestion of portal veins and sinusoids, vacuolar degeneration of hepatocytes, and hyperplasia of bile ducts. These suggested that B. cereus enterotoxin affected the capillaries of blood vessels locally and also systemically resulting into release of proteinaceous exudates and red blood cells.     

Studies on the ATPase of Bacillus cereus.

The membrane ATPase (EC 3.6.1.3) of Bacillus cereus was solubilized by a 'shock-wash' process and purified. The non-specific phosphatase contaminant was separated by glycerol density gradient centrifugation. The optimum temperature was 39.5 degrees C and the pH optimum at 7.5. On SDS-polyacrylamide gel electrophoresis two classes of subunits were observed in equal proportions with molecular weights of 70 K and 83 K. The effect of various compounds on the enzymatic activity was studied. The enzyme was insensitive to NaN3, oligomycin and to divalent cations, but was inhibited by citrate and oxalate.     

Physical map of the Bacillus cereus chromosome.

A physical map of the Bacillus cereus chromosome has been constructed by aligning 11 NotI fragments, ranging in size from 200 to 1,300 kilobases. The size of the chromosome is about 5.7 megabases. This is the first Bacillus genome of which a complete physical map has been described.     

Protein synthesizing systems from spores and vegetative cells of Bacillus cereus

A system of polyphenylalanine synthesis was optimized for a comparison of the polymerizing activities of ribosomes from spores and vegetative cells of Bacillus cereus T. Ribosomes of both types react similarly, showing a magnesium optimum of about 6 mM and spermidine optima of about 5 mM and 4 mM for vegetative and spore ribosomes, respectively. These lead to optimum mono- to multivalent cation rations of 9 and 10 respectively at 100 mM ammonium ion. A comparison of the response of these ribosomes to suboptimal concentrations of magnesium and spermidine show that they differ qualitatively from each other, suggesting that they possess different structure, macromolecular or ionic components.Abbreviations DFP diisopropylfluorophosphate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

The forespore line of gene expression in Bacillus subtilis

Endospore formation by Bacillus subtilis involves three differentiating cell types, the predivisional cell, the mother cell, and the forespore. Here we report the program of gene expression in the forespore, which is governed by the RNA polymerase sigma factors sigma(F) and sigma(G) and the DNA-binding proteins RsfA and SpoVT. The sigma(F) factor turns on about 48 genes, including the gene for RsfA, which represses a gene in the sigma(F) regulon, and the gene for sigma(G). The sigma(G) factor newly activates 81 genes, including the gene for SpoVT, which turns on (in nine cases) or stimulates (in 11 cases) the expression of 20 genes that had been turned on by sigma(G) and represses the expression of 27 others. The forespore line of gene expression consists of many genes that contribute to morphogenesis and to the resistance and germination properties of the spore but few that have metabolic functions. Comparative genomics reveals a core of genes in the sigma(F) and sigma(G) regulons that are widely conserved among endospore-forming species but are absent from closely related, but non-spore-forming Listeria spp. Two such partially conserved genes (ykoU and ykoV), which are members of the sigma(G) regulon, are shown to confer dry-heat resistance to dormant spores. The ykoV gene product, a homolog of the non-homologous end-joining protein Ku, is shown to associate with the nucleoid during germination. Extending earlier work on gene expression in the predivisional cell and the mother cell, we present an integrated overview of the entire program of sporulation gene expression.     

Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group

A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42 degrees C, and no reduction in activity was observed even after 24 h of growth at 42 degrees C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22 degrees C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.     

Toxin-Producing Ability among Bacillus spp. Outside the Bacillus cereus Group

A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42°C, and no reduction in activity was observed even after 24 h of growth at 42°C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22°C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.