ABSTRACT
Protein complexes present different degrees of stability. We have previously described a glycoprotein from Bacillus thuringiensis that appeared as a multimer unable to be dissociated by the usual SDS-containing sample buffers of pH 6.8. In order to dissociate the complex, a SDS-containing sample buffer of pH 9 was described. In the present report three additional protein complexes with different degrees of stability and the effect of that dissociating sample buffer are described. The study of SDS critical micellar concentration values as a function of pH explains the improvement of dissociating properties at pH 9.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Sodium Dodecyl Sulfate , Bacterial Proteins/chemistry , Buffers , MicellesABSTRACT
Two glycoproteins (205 and 72 kDa) were found in Bacillus thuringiensis sporangia. They were predominantly localized in the exosporium and/or the spore coat, although a small proportion was also found in membranes. A method for the dissociation of hydrophobic aggregates that resist the usual conditions of SDS-PAGE is described. Using this method we established that the 205 kDa glycoprotein is a multimer of the 72 kDa one. Deglycosylation of the 205 kDa and 72 kDa glycoproteins with trifluoromethanesulfonic acid yielded a 54 kDa polypeptide in both cases. At least three species of oligosaccharides were O-glycosidically linked to serines of the 54 kDa polypeptide chain. One of the oligosaccharides had N-acetylgalactosamine at the reducing end, rhamnose and a component not yet identified.
Subject(s)
Bacillus thuringiensis/chemistry , Bacterial Proteins/chemistry , Glycoproteins/chemistry , Oligosaccharides/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , Chromatography, Paper , Electrophoresis, Polyacrylamide Gel , Glucose/metabolism , Glycoproteins/biosynthesis , Glycoproteins/isolation & purification , Hydrogen-Ion Concentration , Pronase/metabolism , Protein Conformation , Serine/metabolism , Spores, Bacterial , Time FactorsABSTRACT
Bacitracin induced one protein (bacitracin-induced protein [BIP]) in Bacillus thuringiensis and two proteins (BIP1 and BIP2) in Bacillus subtilis that were localized in the membrane. Divalent cations acted as cofactors for induction in all three cases. Growth was initially inhibited by the antibiotic, but following induction of proteins growth resumed. B. subtilis cells possessing BIPs were able to duplicate at a normal rate in the presence of bacitracin. The amount of B. subtilis BIPs diminished markedly after a few divisions in the absence of the antibiotic and the organism simultaneously reverted to the susceptible state. Induction of the proteins did not take place after the fourth or fifth hour of the stationary phase. The B. thuringiensis BIP was also induced by vancomycin. Bacitracin did not induce the synthesis of specific proteins in susceptible (Micrococcus lysodeikticus) or outer membrane-possessing resistant bacteria (Escherichia coli).
Subject(s)
Bacillus subtilis/metabolism , Bacillus thuringiensis/metabolism , Bacitracin/pharmacology , Bacterial Proteins/biosynthesis , Bacillus subtilis/drug effects , Bacillus thuringiensis/drug effects , Drug Resistance, Microbial , Electrophoresis, Polyacrylamide Gel , Escherichia coli/drug effects , Escherichia coli/metabolism , Micrococcus/drug effects , Micrococcus/metabolism , Sulfur RadioisotopesABSTRACT
Crystal serine-proteases of B. thuringiensis subsp. israelensis were able to process the 28,000-dalton protein during crystal solubilization. On the other hand, solubilized crystal proteins were degraded during the larvicidal bioassay by the action of serine-proteases liberated by mosquito larvae into the medium, with loss of toxicity. However, proteins in intact crystals were protected from the action of these proteases. This resistance to degradation of crystals partly explains the observation that they are more toxic than solubilized crystal proteins.
Subject(s)
Bacillus thuringiensis/pathogenicity , Bacterial Proteins/metabolism , Bacterial Toxins/antagonists & inhibitors , Culex/enzymology , Endotoxins , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/antagonists & inhibitors , Bacterial Toxins/metabolism , Hemolysin Proteins , Larva , Peptide Hydrolases/metabolismABSTRACT
The effect of bacitracin on Bacillus thuringiensis is described. When added after the end of the exponential phase, it produces a marked increase in the size of spores and parasporal crystals. The cultures to which the antibiotic was added are able to produce more crystal proteins than non-treated cultures. The protein composition of crystals from bacitracin-treated cultures is the same as that of crystals purified from control cultures.
Subject(s)
Bacillus thuringiensis/metabolism , Bacitracin/pharmacology , Bacterial Proteins/biosynthesis , Bacterial Toxins , Endotoxins , Bacillus thuringiensis/growth & development , Bacillus thuringiensis/physiology , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Hemolysin Proteins , Mannose/metabolism , Molecular Weight , Spores, Bacterial/drug effects , Spores, Bacterial/metabolism , Time FactorsSubject(s)
Geobacillus stearothermophilus/metabolism , Protein Biosynthesis , Ribosomal Proteins/pharmacology , Ribosomes/metabolism , Bacteriophages , Cell Cycle , Hypertonic Solutions , Peptide Initiation Factors/pharmacology , Poly U/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Ribosomes/drug effectsABSTRACT
The appearance of a protein (association factor I) in ribosomes from Bacillus stearothermophilus at stationary phase of growth is described. Association factor I is present on 30S subunits and 30S-50S ribosomal couples, but not on 50S subunits. This protein is responsible for the low levels of polyphenylalanine synthesis shown by stationary phase ribosomes. Association factor I is able to bind to free 30S-50S ribonsomal couples but not to polysomes, and exerts its effect by inhibiting the initiation step of protein synthesis. Ribonsomes preincubated with association factor I have a decreased ability for polypeptide synthesis directed page mRNA or poly(U).
Subject(s)
Bacterial Proteins/pharmacology , Geobacillus stearothermophilus/physiology , Peptide Biosynthesis , Peptide Chain Initiation, Translational/drug effects , Peptides , Ribosomal Proteins/pharmacology , Cell Cycle , Coliphages , Phenylalanine/analogs & derivatives , Phenylalanine/biosynthesis , Poly U/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Ribosomes/metabolismSubject(s)
Bacterial Proteins/pharmacology , Peptide Biosynthesis , Ribosomes/drug effects , Depression, Chemical , Geobacillus stearothermophilus/metabolism , Kinetics , Magnesium/pharmacology , Peptide Chain Initiation, Translational/drug effects , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructureABSTRACT
When putrescine is added to polyamine starved cultures of an E. coli strain difficient in the biosynthesis of putrescine, the protein synthesis is enhanced almost immediately and the ribosomal pattern changes concomitantly, increasing the ratio 70S monomer/ribosomal subparticles. Studies with cell-free systems derived from polyamine starved or unstarved bacteria show that the translation of synthetic and natural mRNAs is several fold higher in system prepared from cells grown in the presence of polyamines. This effect depends on the ribosomal particles and more specifically on the 30S subunit. The results on association of ribosomal subunits strongly suggest that polyamines are involved in this reaction occurring in vivo.
Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli/metabolism , Putrescine/metabolism , Ribosomal Proteins/biosynthesis , Animals , Polyamines/metabolism , RNA/biosynthesisABSTRACT
Chromatography through Bio-Gel columns is a simple and rapid method for the purification of different ribosomal particles. This mild procedure, easily adapted to many purposes with good yields, can substitute the conventional sucrose gradient centrifugations, especially when the integrity of labile particles or complexes must be preserved.
Subject(s)
Polyribosomes/analysis , Cell Fractionation , Chromatography, Gel/methods , Escherichia coli/ultrastructureABSTRACT
Chromatography through Bio-Gel columns is a simple and rapid method for the purification of different ribosomal particles. This mild procedure, easily adapted to many purposes with good yields, can substitute the conventional sucrose gradient centrifugations, especially when the integrity of labile particles or complexes must be preserved.
ABSTRACT
Chromatography through Bio-Gel columns is a simple and rapid method for the purification of different ribosomal particles. This mild procedure, easily adapted to many purposes with good yields, can substitute the conventional sucrose gradient centrifugations, especially when the integrity of labile particles or complexes must be preserved.
ABSTRACT
The association of ribosomal subparticles induced by several associating agents has been studied under different conditions. The following observations were made: 1. Spermidine was able to produce the association of subunits, and the concentration and temperature curves of this reaction were similar to those obtained with association factor. The product formed in the latter case was more stable. 2. The association at low Mg2+ concentration was higher with association factor than with polyamines. 3. The temperature-dependent binding of spermidine to 30-S subunits formed an active complex, which was converted into the 30S-50S couples by the addition of 50-S subparticles at low temperature. A similar behaviour has been previously shown for the complete association factor and its low molecular weight fraction. 4. The same unstable form of 30S-50S couples has been obtained either with spermidine or with the low molecular weight component (AFII) of the association factor. In both cases the protein fraction AFI was able to complete the reaction by stabilizing the subunit couple. 5. After glutaraldehyde fixation the products of the reactions with spermidine or association factor behaved in a similar way when they were submitted to long sucrose-gradient centrifugations. 6. The analysis of association factor preparations has shown that they contain spermidine as well as spermine. The polyamine levels in association factor could account for part of the total associating activity.
Subject(s)
Bacterial Proteins/metabolism , Geobacillus stearothermophilus/metabolism , Ribosomes/metabolism , Spermidine/pharmacology , Binding Sites , Centrifugation, Density Gradient , Geobacillus stearothermophilus/drug effects , Kinetics , Magnesium/pharmacology , Ribosomes/drug effects , Ribosomes/ultrastructure , TemperatureSubject(s)
Bacillus/cytology , Bacterial Proteins/pharmacology , Ribosomes/metabolism , Bacterial Proteins/isolation & purification , Centrifugation, Density Gradient , Glutaral , Hot Temperature , Hydrostatic Pressure , Magnesium/pharmacology , Phosphates , Phosphorus Isotopes , Polynucleotides , Protein Binding , Protein Biosynthesis , RNA, Messenger , Ribosomes/analysis , Ribosomes/drug effects , TemperatureSubject(s)
Bacterial Proteins/biosynthesis , Ribosomes/metabolism , Anti-Bacterial Agents/pharmacology , Cell Division , Centrifugation, Density Gradient , Escherichia coli/cytology , Escherichia coli/metabolism , Kinetics , Magnesium , Methionine , Models, Biological , Peptide Chain Termination, Translational , Peptides , Phosphorus Radioisotopes , Polyribosomes/metabolism , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribonucleosides/pharmacology , Ribosomes/drug effects , Species Specificity , Spectrophotometry, Ultraviolet , Spermidine/pharmacology , Time Factors , UltracentrifugationABSTRACT
The isolation of a new factor, which can cause the in vitro association of 30S and 50S ribosomal subunits at low Mg(++) concentration, is described. The association factor is eluted together with the dissociation protein when ribosomes of Bacillus stearothermophilus are washed with salt solutions of high concentration. The association activity is heat-stable, whereas dissociation factor is inactivated after 10 min at 80 degrees C. This treatment allows the separation of both factors. Several properties rule out the possibility that uncharged, amino-acyl-, or peptidyl-tRNA are responsible for the association process described in this report. Digestion with trypsin shows that the association factor contains at least two components, one of which is a protein.
