Chemical and Morphological Studies of Bacterial Spore Formation : III. The Effect of 8-Azaguanine on Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.     

Adenine metabolism of Ehrlich mouse ascites cells in proliferating and resting phase of tumor growth.

The incorporation of 14C from [U-14C]adenine into the pools of purine nucleotides, nucleosides and bases in Ehrlich mouse ascites cells (EMAC1) during the proliferating and resting phases of tumor growth was compared. In the proliferating phase the total 14C incorporation into purine pools is much faster than in the resting phase. The ATP turnover as well as the purine breakdown to hypoxanthine and uric acid are increased in the proliferating phase. That corresponds to previous findings on higher nucleotide pool sizes and higher ATP yield and ATP-consuming processes in this growth period.     

Expression of a small RNA, BS203 RNA, from the yocI-yocJ intergenic region of Bacillus subtilis genome

We isolated and characterized a novel small RNA from Bacillus subtilis. We termed this molecule BS203 RNA from the length of its mature form (203 nt) and located the corresponding gene at the yocI-yocJ intergenic region on the B. subtilis genome. Northern blotting revealed that it is transcribed in vegetative growing cells and that the amount of BS203 RNA decreased in the middle of the vegetative phase. A computer-aided prediction of the BS203 RNA secondary structure revealed three characteristic stem-loop structures. Despite active expression during the vegetative phase, growth of the knockout mutant was not affected by depletion of BS203 RNA. A phylogenetic comparison of the sequence of the BS203 RNA with other Bacillus species including B. cereus and B. halodurans C-125, or Clostridium perfringens suggests that the sequence is unique to Bacillus subtilis.     

Modelling the number of viable vegetative cells of Bacillus cereus passing through the stomach

Aims:  Model the number of viable vegetative cells of B. cereus surviving the gastric passage after experiments in simulated gastric conditions.
Materials and Methods:  The inactivation of stationary and exponential phase vegetative cells of twelve different strains of Bacillus cereus , both mesophilic and psychrotrophic strains isolated from food and faeces from healthy and ill individuals, in simulated gastric conditions was determined using decimal reduction times at low pH ( D _pH). Subsequently inactivation rates were calculated. Inclusion of the inactivation rates into models describing the course of the gastric pH after the consumption of meal of solid food and the transfer of food from the stomach to the small intestine resulted in numbers of viable Bacillus cereus vegetative cells able to pass the stomach.
Conclusions:  According to the model, 3–26% of the ingested vegetative cells from Bacillus cereus may survive the gastric passage, dependent on the growth phase of the vegetative cells, the type of strains, and the age of the consumer.
Significance and Impact of the Study:  Vegetative cells of Bacillus cereus may be involved in the onset of diarrhoeal disease to a greater extent than expected since up to 26% of the ingested cells survive simulated gastric conditions.     

Effect of Purine Analogs and Their Interactions with Benzyladenine on Bud Burst and Shoot Growth in Apple Bud Explants

The purine analogs 8-azaadenine, 8-azaguanine, 4-aminopyrazolo [3,4-d] pyrimidine, 2-fluoroadenine and 6-methylpurine inhibited bud burst in non-dormant vegetative axillary bud explants obtained from apple (Malus domestica Borkh.) seedlings. The reversal of inhibition was attempted by incorporating increasing concentrations of benzyladenine (BA) in the culture medium. The inhibition caused by 8-azaadenine and 8-azaguanine was reduced especially when BA was added to the media at equimolar ratios along with the inhibitors. The inhibition due to 4-aminopyrazolo [3,4-d] pyridine was reduced only with equimolar BA, BA was ineffective or only minimally effective in reducing inhibition due to 2-fluoroadenine and 6-methypurine. Short pretreatment with 8-azaadenine and 8-azaguinine failed to cause any inhibition when explants were subsequently transferred to BA -containing medium. The effectiveness of 2-fluoroadenine and 6-methylpurine was maintained or only slightly reduced, indicating their long-term inhibitory action.     

Characteristics of Bacillus cereus dissociants]

The autoregulation of the phenotypic (populational) variability of the Bacillus cereus strain 504 was studied. The isolated colonial morphotypes of this bacterium were found to differ in their growth characteristics and the synthesis of extracellular proteases. The phenotypic variabilities of vegetative proliferating cells and those germinated from endospores and cystlike refractory cells were different. Bacterial variants also differed in the production of the d1 and d2 factors (the autoinducers of dormancy and autolysis, respectively) and sensitivity to them. The possible role of these factors in the dissociation of microorganisms is discussed.     

Comparative studies on sporulation-promotive actions of cyclic AMP,theophylline and caffeine in Saccharomyces cerevisiae

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylling did significantly when added to a presporulation medium containing glucose Caffeine inhibited the vegetative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Exogenous nucleosides stimulate RNA synthesis in resting cells of a culture of scarlet rose

RNA synthesis in response to exogenous nucleoside precursors was studied in a suspension culture of rose cells. Exponentially growing and resting cells were prelabeled with [3H] uridine, an excess of unlabeled uridine added, and subsequent isotopic incorporation into nuclear and ribosomal fractions measured. The data were compared to control values in cells continuously labeled in the absence of unlabeled uridine. Addition of uridine to the growing culture reduced the further uptake, and incorporation of [3H] uridine into RNA. In contrast, in resting cells, the addition of uridine (or, purine nucleosides) enhanced the apparent utilization of [3H] uridine in RNA synthesis by 2- to 4-fold.     

Hormonal control of enzyme synthesis: on the mode of action of gibberellic Acid and abscisin in aleurone layers of barley

Gibberellic acid (GA) enhances the synthesis of α-amylase and ribonuclease in isolated aleurone layers and this process is inhibited by abscisin. Removal of gibberellic acid in mid-course of α-amylase production results in a slowing down of α-amylase synthesis, suggesting a continued requirement of GA for enzyme synthesis. This is paralleled by a continuous requirement for RNA synthesis. Addition of 6-methylpurine or 8-azaguanine in mid-course results in an inhibition of α-amylase synthesis within 3 to 4 hours. However, actinomycin D added in mid-course is almost without effect. This is not due to its failure to enter the cells, because it does inhibit ¹⁴C-uridine incorporation at this stage. Addition of abscisin to aleurone layers which are synthesizing α-amylase results in an inhibition of this synthesis within 2 to 3 hours. Cycloheximide on the other hand inhibits enzyme synthesis immediately upon its addition. These data are consistent with the hypothesis that the expression of the GA effect requires the synthesis of enzyme-specific RNA molecules. The similarity in the kinetics of inhibition between abscisin on the one hand and 8-azaguanine or 6-methylpurine on the other suggests that abscisin may exert its action by inhibiting the synthesis of these enzyme-specific RNA molecules or by preventing their incorporation into an active enzyme-synthesising unit.     

Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus

Carvacrol, a natural plant constituent occurring in oregano and thyme, was investigated for its bactericidal effect towards the food-borne pathogen Bacillus cereus . Carvacrol showed a dose-related growth inhibition of B. cereus . At concentrations of 0·75 mmol l⁻¹ and above, total inhibition of the growth was observed. Below this concentration, carvacrol extended the lag-phase, reduced the specific growth rate and reduced the maximum population density. Incubation for 40 min in the presence of 0·75–3 mmol l⁻¹ carvacrol decreased the number of viable cells of B. cereus exponentially. Spores were found to be approximately 2·3-fold less sensitive to carvacrol than vegetative cells. Bacillus cereus cells showed reduced susceptibility towards carvacrol at pH 7·0 compared with different values between pH 4·5 and 8·5. The culture and exposure temperatures had a significant influence on the survival of vegetative cells. The highest death rate of cells was observed at an exposure temperature of 30 °C. Membrane fluidity was found to be an important factor influencing the bactericidal activity of carvacrol.     

Spores of microorganisms

Preexisting¹⁴C-DAP in vegetative cells ofBacillus cereus is not incorporated into the spores, but is released into the medium after sporogenesis is complete. Exogenous¹⁴C-DAP added to the medium before sporulation is incorporated intensively into the sporangia and practically all of it is taken up by the spores. During sporogenesis, two periods of increased incorporation of¹⁴C into hot TCA-precipitate of cells are found after adding¹⁴C-DAP— one before formation of the spores, when¹⁴C-lysine formed by decarboxylation is incorporated together with¹⁴C-DAP, and one during the “whitening” phase, when any¹⁴C-lysine is no longer incorporated. The incorporation of exogenous¹⁴C-lysine into the sporangial proteins is also markedly elevated during the presporulation phase and at the outset of sporogenesis.     

Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens.

The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined. Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium. The rate of RNA synthesis decreased as sporulation progressed. Deoxyadenosine increased uptake of [14C]uracil and [14C]thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml. Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation. The rate of protein breakdown during vegetative growth was 1%/h. During sporulation this rate increased to 4.7%/h. When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h. If added at 3 h the rate decreased to 2.1%/h. The role of proteases in this process is discussed.     

Azaguanine: A Theoretical Study of Its Tautomerism and Protonation in the Gas Phase and Aqueous Solution

The tautomerism and protonation of 8-azaguanine (azaG) have been studied by means of ab initio methods, both in the gas phase and in aqueous solution. An elimination procedure to choose the most stable tautomeric forms, based on AM1 and HF/6–31G* energies, has been applied. Tautomers azaG(1,9), azaG(1,7) and azaG(9,15) have been selected and their energies calculated at MP2/6–311++G**//HF/6–31G* level. Self-consistent reaction field IPCM, based on polarizable continuum model (PCM), has been applied to study the solvent effects. The stability order in the gas phase is azaG(1,7) = azag(1,9) > azaG(9,15), whereas in solution the order becomes azaG(1,7) > azaG(1,9) > azaG(9,15), the latter being just 0.76 kcal/mol over azaG(1,7). The calculations of proton affinities allowed to unambiguously determine the preferred sites of protonation of these species.     

A Role for Purines and Pyrimidines of Ribonucleic Acid in the Induction of Two-dimensional Growth in the Gametophytes of the Fern, Asplenium nidus

The inhibition of two-dimensional growth in the gametophytesof Asplenium nidus induced by purine and pyrimidine analoguesand the reversal of inhibition by natural purine and pyrimidinebases and their derivatives have been studied. Adenine and guanineand their ribosides and ribotides were more effective than cytosine,uracil, thymine, and their derivatives in preventing the inhibitiondue to 8-azaadenine and 8-azaguanine. Likewise, the inhibitoryeffects of 2-thiocytosine, 2-thiouracil,6-azauracil, and 5-fluorouracilwere overcome by the pyrimidines and their derivatives, butnot usually by the purines.Combinations of two purine analoguesor two pyrimidine analogues or one purine analogue and one pyrimidineanalogue inhibited growth more effectively than single compounds.The combined inhibitions were maximally reversed when both naturalbases or their derivatives were added to the medium. It is concludedthat there is a requirement for both purines and pyrimidinesof ribonucleic acid in the induction of two-dimensional growthin the gametophytes of Asplenium nidus.     

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium. Methods and RESULTS: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h). CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.     

Blood glycoprotein from antarctic fish. Possible conformational origin of antifreeze activity

Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared to be a tetramer of molecular weight 95 000. The other enzyme, adenosine phosphorylase, was specific for adenosine and deoxyadenosine. The molecular weight of the native enzyme was 153 000 +/- 10% and the molecular weight of the subunits was 25 500 +/- 5%. This indicates a hexameric structure. The adenosine phosphorylase was inactivated by 10(-3) M p-chloromercuribenzoate and protected against this inactivation by phosphate, adenosine and ribose 1-phosphate.     

Location of Bacterial Polysaccharide During Various Phases of Growth

Polysaccharide-containing material was located by histochemical staining on ultrathin sections of Bacillus cereus and several clostridial species during various phases of growth. These components were located along the cell wall in young vegetative cells and along the developing cortex in sporulating cells. In B. cereus any carbohydrate-containing material present along the forespore membrane was too small to be detected by direct staining. However, in three clostridial species such material was detected at all stages of forespore development.     

Production and characterization of monoclonal antibodies against vegetative cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Comparative studies on sporulation-promotive actions on cyclic AMP, theophylline and caffeine in Saccharomyces cerevisiae.

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylline did significantly when added to a presporulation medium containing glucose. Caffeine inhibited the vegatative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.