Regulation of protoxin synthesis in Bacillus thuringiensis.

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.     

Intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki and their role in protoxin activation

The intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki were studied to identify the endogenous proteases involved in the activation of protoxin. The proteases obtained with 30% ammonium sulfate saturation were analysed by both gelatin zymography and azocasein hydrolysis. Three proteases with molecular mass 92 kDa, 78 kDa and 69 kDa were identified on gelatin gel and their gelatinolytic activity was inhibited by ethylenediamine tetraacetic acid. Significantly, 1,10-phenanthroline caused an inhibition of the azocasein hydrolytic activity by 98% and ethylenediamine tetraacetic acid by 28%. The three proteases were heat-stable at 65 °C, while the 69-kDa protease was active up to 75 °C. Intracellular protease-deficient mutants (ethyl methanesulfonate mutagenesis) could not generate the active toxin suggesting the existence of a specific enzyme affecting the conversion of protoxin to toxin.     

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B . subtilis . After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.     

Identification and purification of the 69-kDa intracellular protease involved in the proteolytic processing of the crystal delta-endotoxin of Bacillus thuringiensis subsp. tenebrionis

The dynamics of appearance of intracellular proteases in relation to the synthesis of crystal delta-endotoxin was studied to identify the native intracellular protease(s) involved in the proteolytic processing of the 73-kDa protoxin of Bacillus thuringiensis subsp. tenebrionis. In vitro proteolytic activation of the 73-kDa protoxin indicated the possible role of 69-kDa protease in the proteolytic processing of 73-kDa protoxin. The purified 69-kDa protease was able to cause the proteolytic activation of the 73-kDa protoxin to 68-kDa toxin and this conversion was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline.     

Nitrite loss and spoilage microflora development in chub-packed luncheon meat

Residual nitrite was lost from chub-packed luncheon meat during storage through both chemical breakdown and microbial consumption. The relative importance of these mechanisms in this pasteurized product was determined by the speed of development of the spoilage microflora, which is influenced by storage conditions. The nitrite half-life due to chemical loss was 13 d at 25°C and 36 d at 10°C. When microbial growth occurred these half-lives were reduced to 2.6 d and 21 d, respectively. Qualitative differences in the microflora that developed at these two temperatures (denitrifying Bacillus spp. at 25°C and non-denitrifying Streptococcus spp. at 10°C) account for the large temperature effect. Growth of Streptococcus spp. increased the rate of chemical nitrite loss in chubs by reducing the pH value. Nitrite did not inhibit the aerobic growth of either Bacillus or Streptococcus species associated with spoilage but did inhibit the anaerobic growth of Bacillus spp. This bacteriostatic effect of residual nitrite in anaerobic conditions will decrease during storage as nitrite level falls and oxygen penetrates the chub pack. Nitrite-mediated bacteriostasis does not obviate the need for refrigerated storage but does afford a real, if ephemeral, safeguard against spoilage occurring during short periods of temperature abuse.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Climatic influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil

Bacillus weihenstephanensis strains are psychrotolerant and grow from below 7°C to 38°C. Closely related mesophilic Bacillus cereus strains can grow from above 7°C to 46°C. We classified 1060 B. cereus group isolates from different soil samples with respect to their psychrotolerant and mesophilic genotypes by polymerase chain reaction (PCR) targeting of specific 16S rDNA and cold shock protein A gene signatures. In parallel, growth tests at 7°C were carried out to determine the thermal phenotype. The geographic distribution of psychrotolerant and mesophilic isolates was found to depend significantly on the prevalent annual average temperature. In one tropical, one temperate and two alpine habitats, the proportion of psychrotolerant cspA genotypes was found to be 0%, 45% and 86% and 98%, respectively, with the corresponding annual average temperatures being 28°C, 7°C, 4°C and 1°C. In the tropical habitat, only the mesophilic B. cereus was found, characterized by correspondence of thermal genotype and phenotype. In the alpine habitat, almost only the psychrotolerant B. weihenstephanensis was isolated. In the temperate habitat, mesophilic B. cereus and psychrotolerant B. weihenstephanensis as well as 'intermediate thermal types' occurred, the latter having opposite thermal genotypes and phenotypes or opposing sets of thermal DNA signatures, characterized by the coexistence of mesophilic and psychrotolerant 16S rDNA operon copies within a single isolate. Both sugar utilization and DNA fingerprinting patterns revealed a high, probably non-clonal microsite diversity within the population of the temperate habitat. We interpret our observations in terms of a temperature-dependent selection regime, acting on recombining B. cereus / B. weihenstephanensis populations in soil.     

Effect of temperature in the regulation of DNA synthesis in synchronous cultures of Chlorella

The influence of temperature on DNA replication of Chlorella pyrenoidosa grown at 30 °C was studied in synchronous cultures. Final DNA content and cell counts/ml were 30 to 40% less than controls after a 4 h exposure to 15 °C, but reached normal levels after being kept temporarily at 10 °C. The rates of DNA synthesis at these two temperatures were, respectively, one-sixth and one-tenth of the rate at 30 °C, but during the 15 °C treatment the cells lost the ability to enter a further replication cycle more rapidly than at 10 °C. This loss of capacity to initiate further replication cycles was prevented by inhibiting protein synthesis with cycloheximide.     

Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7‐selective peptide – protoxin II

Protoxin II is biologically active peptide containing the inhibitory cystine knot motif. A synthetic version of the toxin was generated with standard Fmoc solid phase peptide synthesis. If N‐methylmorpholine was used as a base during synthesis of the linear protoxin II, it was found that a significant amount of racemization (approximately 50%) was observed during the process of cysteine residue coupling. This racemization could be suppressed by substituting N‐methylmorpholine with 2,4,6‐collidine. The crude linear toxin was then air oxidized and purified. Electrophysiological assessment of the synthesized protoxin II confirmed its previously described interactions with voltage‐gated sodium channels. Eight other naturally occurring inhibitory knot peptides were also synthesized using this same methodology. The inhibitory potencies of these synthesized toxins on Nav1.7 and Nav1.2 channels are summarized. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Changes in protein synthesis during stratification and dormancy release in embryos of sugar maple (Acer saccharum)

Protein synthesis in dormant embryos of sugar maple ( Acer saccharum ) was investigated in seeds stratified at 4°C or incubated at 15°C. Seeds stratified at 4°C germinated after 27 days; seeds incubated at 15°C failed to germinate. Stratification increased the embryo's capacity for protein synthesis by day 11 as measured by in vivo incorporation of [³⁵S]-methionine into purified protein. At 4°C protein synthesis in the embryonic axis rose in a linear fashion prior to germination, whereas in cotyledons it increased until day 20 and then declined. Analysis of radiolabelled proteins by two-dimensional gel electrophoresis revealed that the levels of specific proteins were altered by temperature, primarily in the cotyledons. Several proteins were expressed in the cotyledons at 15°C but were absent in unstratified embryos and in embryos stratified at 4°C. That is, the expression of these proteins was repressed during stratification and release from dormancy. Levels of other proteins in the cotyledons declined at 4°C during stratification. We suggest that one or more of these proteins may be associated with the inhibition of growth of the embryonic axis imposed by the cotyledons.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light.

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Correlation between alkaline activation of Bacillus thuringiensis var. kurstaki spores and crystal production

The spores of crystal-forming (Cry⁺) and non-crystal-forming (Cry^-) strains of Bacillus thuringiensis var. kurstaki and Bacillus cereus were tested for the ability to be activated by 0.1 m K₂CO₃ (pH 10). Only the spores of crystal-forming strains could be activated, and this phenotype was independent of whether crystals were present with the spores in the activation solution. The spores of a B. thuringiensis var. kurstaki strain that is temperature sensitive for protoxin accumulation could be activated by the alkaline solution when produced at the permissive temperature, whereas spores produced at the nonpermissive temperature were not activated. The results indicate that protoxin in the spore coat is responsible for the alkaline-activation phenotype and may serve an ecological function for the organism.     

A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition

We have analysed the function of a gene of Bacillus subtilis , the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.     

Gene expression during leaf development in Lolium temulentum: Patterns of protein synthesis in response to heat-shock and cold-shock

At an optimal growth temperature of 20°C, expending 4th leaves of Lolium temulentum L. synthesised a broad spectrum of polypeptides which altered with the maturity of the leaf tissue. Elevation of the temperature to 35°C, or above, induced synthesis of heat-shock proteins (hsp), and all parts of the 4th leaf were capable of this response. The threshold temperature for induction of hsp synthesis was little affected by the growth temperature (5 or 20°C). In contrast, a sudden 15–18°C decrease in temperature did not result in a marked alteration of protein synthesis patterns. It is concluded that in this species adaptation to rapid temperature reduction is not mediated by stress protein synthesis.     

Nutrition-dependent modulations of protein synthesis in Candida albicans during germ-tube formation or maintenance of the yeast form in N-acetyl glucosamine media

Abstract Stationary phase, yeast-form cells of Candida albicans grown in glucose-yeast extract medium were shifted to N -acetylglucosamine (GlcNAc) and/or glucose medium, and the pattern of protein synthesized under conditions of a progressive decrease in the rate of total protein synthesis was analyzed by SDS-PAGE and autoradiography.
Marked temporal modulations in the rate of synthesis of some cytoplasmic proteins were detected both in cells forming germ-tubes (at 37°C) and in yeast cells (at 28°C). The major modulated components showed molecular weights of 63, 53, 48 and 34 kDa. These products could not be qualified as heat-shock or heat-stroke proteins, because analogous modulations were observed on shifting cells from 28°C to 37°C or from 28°C to 28°C. However, no marked modulations in the synthesis of specific proteins were detected when amino acids were added to the medium fostering germ-tube formation under conditions of unimpaired overall rate of protein synthesis.
It is suggested that the modulations observed in cells incubated in GlcNAc-glucose medium could represent a response to a nutritional stress.