Structure of the Bacillus brevis metalloprotease gene

Primary structure of DNA fragment of 2355 b.p., encoding metalloprotease gene of Bac. brevis, had been determined. Open reading frame for a protein with size of 528 amino acid residues was found in this sequence. The encoding protein is homologous to metalloproteases of Bac. stearothermophilus, Bac. cereus, Bac. subtilis and Bac. amyloliquefaciens. The structure of Bac. brevis metalloprotease gene reveals that this enzyme is synthesised as pre-pro-protease with signal peptide and pro-region, which are cut during its synthesis. The proposed size of mature protease is 304 amino acid residues. The residues, essential for catalysis, binding of Zn ion and Ca ions were found on the basis of Bacilli metalloproteases structures comparison.     

Specific phages used for differentiation of the entomopathogenic bacterium Bacillus thuringiensis]

The sensitivity to specific phages and morphological, physiological, and antigenic properties were compared among several strains of Bacillus thuringiensis isolated from insects inhabiting various geographical zones. All 43 cultures assigned to Bac. thuringiensis var. sotto and 198 among 170 cultures classed as Bac. thuringiensis var. dendrolimus were found to belong to Bac. thuringiensis var. dendrolimus. None of these cultures was resistant to its specific phage. The same was true of 22 studied cultures of Bac. thuringiensis var. galleriae. Only two among studied 45 cultures of Bac. thuringiensis var. thuringiensis were resistant to phages specific for this variety. Therefore, the abundance of variants resistant to specific phages in natural conditions differs among the varieties of Bac. thuringiensis. In most cases, cultures of the same variety of Bac. thuringiensis isolated from various insects inhabiting different geographical zones are identical by their sensitivity to specific phages and by other important characteristics.     

Oxidative burst elicited by Bacillus mycoides isolate Bac J, a biological control agent, occurs independently of hypersensitive cell death in sugar beet

Response of sugar beet cultivars C40 and USH11 to syringe infiltration of live and dead Bacillus mycoides isolate Bac J, a biological control agent, and virulent and avirulent isolates of Erwinia carotovora pv. betavasculorum was measured by monitoring systemic acquired resistance control of Cercospora beticola, specific activity of chitinase and beta-glucanase, the oxidative burst, and hypersensitive cell death at the infiltration site. Priming sugar beet with B. mycoides Bac J (1 x 10(8) cells/ml) and avirulent isolates of E. carotovora pv. betavasculorum (1 x 10(6) cells/ml) reduced C. beticola symptoms by nearly 70% on distal, untreated leaves. Systemic resistance responses elicited by live B. mycoides Bac J and avirulent E. carotovora pv. betavasculorum isolates, measured by assays for chitinase and beta-glucanase, were statistically equivalent, and biphasic hydrogen peroxide production was observed. Although similar in timing, the second hydrogen peroxide burst was twofold lower for B. mycoides Bac J than for avirulent E. carotovora pv. betavasculorum. Hypersensitive cell death was elicited by avirulent E. carotovora pv. betavasculorum but not B. mycoides Bac J. An oxidative burst was elicited by spray-applied B. mycoides Bac J under both light and green light conditions, indicating that the signal produced by B. mycoides Bac J was not reliant on the stomata for entry into sugar beet. A working model for signal delivery and systemic resistance induction by B. mycoides Bac J in sugar beet is proposed.     

Cloning in Bacillus subtilis cells of the Bacillus mesentericus genes that determine the enzyme synthesis of tryptophan metabolism

In this work, we tried to clone some Bacillus mesentericus genes coding for tryptophan synthesis in Bacillus subtilis cells. We succeeded in obtaining two identical plasmids carrying some genes of Bac. mesentericus and complementing the trpC2 and trpF mutations of Bac. subtilis. The cloned genes of Bac. mesentericus completely replaced the functions of trpC2 and trpF genes.     

Extracellular nuclease of Bacillus mesentericus]

Bacillus mesentericus is found to secrete three type of nucleases: alkaline ribonuclease (EC 2.7.7.17), acidic ribonuclease (EC 2.7.7.17) and Ca2+-activated exonucleease (EC 3.1.4.7). These nucleases are purified and characterized. They are similar to those from Bac. subtilis in main biochemical and physico-chemical properties and in their chromatographical behaviour. Studying physiological functions of Bac. mesentericus extracellular nucleases, it is shown that bacteria, which are capable to produce extracellular nucleases, utilize exogenous RNAs and a bit worse, DNAs as a single and additional source of nitrogen or phosphorus. In view of this it is believed that extracellular nucleases participate in bacteria nutrition.     

Taxonomy of denitrifying bacteria in soddy podzolic soil

The taxonomic composition of denitrifying bacteria in soddy podzolic soil was studied by the succession analysis method. This method revealed a significant variation in the taxonomic composition of denitrifying microorganisms in the course of succession. In contrast to succession analysis, the single microbiological analysis of soil samples reflected only the late stage of succession and thus led to an underestimation of the major members of succession. Myxobacteria were found to be the most active denitrifiers at the early stages of succession, whereas bacilli dominated at its late stages. The bacilli were represented by three facultatively anaerobic species, Bacillus cereus, Bac. circulans, and Bac. polymyxa.     

Effect of EcoRI restrictase on the transforming DNA of different bacilli]

The transforming activity of DNA from Bacillus subtilis 168, Bac. subtilis W23, Bac. subtilis NRS and Bac. aterrimus after the EcoRI restrictase treatment was studied. The auxotrophic strains of Bac. subtilis 168 were used as recipients in bacterial transformation. The transforming activity for different markers decreased up to 0.001--6 per cent from the initial level for different Bacilli DNAs. In some cases the sensitivity of the same marker from different Bacilli differed in more than 50 times. Bac. subtilis and Bac. aterrimus have demonstrated the maximal differences. Such differences can be used for the identification of close related Bacilli.     

Restriction-modification systems in Bacillus strains related to Bacillus subtilis

127 strains of bacilli sensitive to different phages of Bacillus subtilis were isolated from the soil of Moscow and its country-side. In 6 strains, restriction and modification systems were discovered which differed from these previously described for Bac. subtilis BsuR system. Two strains has identical restriction-modification systems, and one strain possessed two different systems. Using DNA from all 6 strains, it was possible to transform competent cells of Bac. subtilis RUB834. Two of these 6 strains could serve as recipients in transformation and transfection experiments.     

Dynamic of the volume and structure of the soil bacterial complex in the presence of azobenzene

Azobenzene exerted no significant effect on the dynamics and the species composition of the saprophytic soil bacterial complex, which remained almost the same as in the control and was characterized by the predominance of Curtobacterium sp., Arthrobacter globiformis, and Bacillus megaterium in all stages of succession. Some heterotrophic bacteria were found to be able to accumulate azobenzene. Bac. cereus and Bac. polymyxa degraded azobenzene during their cultivation in nutrient media.     

The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is widespread among bacteria where it mediates carbohydrate uptake and often serves in carbon control. Here we present cloning and analysis of the monocistronic ptsI gene of Corynebacterium glutamicum R, which encodes PTS Enzyme I (EI). EI catalyzes the first reaction of PTS and the reported ptsI was shown to complement the corresponding defect in Escherichia coli. The deduced 59.2-kDa EI of 564 amino acids shares more than 50% homology with EIs from Bacillus stearothermophilus, Bacillus subtilis, and Lactobacillus sake. Chromosomal inactivation of ptsI demonstrated that EI plays an indispensable role in PTS of C. glutamicum R and this system represents a dominant sugar uptake system. Cellobiose was only transported and utilized in adaptive mutants of C. glutamicum R. Cellobiose transport was also found to be PTS-dependent and repressed by PTS sugar glucose.     

Taxonomic composition of denitrifying bacteria in soddy podzolic soil

The taxonomic composition of denitrifying bacteria in soddy podzolic soil was studied by the succession analysis method. This method revealed a significant variation in the taxonomic composition of denitrifying microorganisms in the course of succession. In contrast to succession analysis, the single microbiological analysis of soil samples reflected only the late stage of succession and thus led to an underestimation of the major members of succession. Myxobacteria were found to be the most active denitrifiers at the early stages of succession, whereas bacilli dominated at its late stages. The bacilli were represented by three facultatively anaerobic species:Bacillus cereus, Bac. circulons, andBac. polymyxa.     

Effect of amino acids on exoprotease synthesis by Bacillus thuringiensis

The influence of certain L-amino acids and their mixtures on the synthesis of exoprotease from Bacillus thuringiensis was studied. Physiological experiments showed that the mixture of 20 amino acids added to the artificial medium repressed the synthesis of exoprotease. Among the compounds studied there are both the compounds which stimulate the synthesis of exoprotease (glutamic and aspartic acids, glycine), and the compounds which repress the synthesis of the enzyme (proline, tryptophane, tyrosine, asparagine, serine, cystein). None of the amino acids caused a change in the exoprotease activity. It has been assumed that the repression of the protease synthesis in the presence of the amino acids is accomplished by ammonium ions, which are formed when using the amino acids of Bac. thuringiensis. The glutamine synthetase activity of cells was determined during the growth of Bac. thuringiensis both on a medium containing triptone and after the addition of certain amino acids to the cell suspension. The correlation between the influence of different amino acids on the synthesis of exoprotease and the glutamine synthetase activity was demonstrated.     

Antibiotic formation in plasmid and plasmid-free strains]

Antibiotic activity of three plasmid and three plasmid-free strains of Bacillus pumilus was studied. The antibiotic activity was found in one plasmid and two plasmid-free strains. It was supposed that both the chromosomic and extrachromosomic genes could participate in regulation of the antibiotic production in Bac. pumilus. It was shown that the antibiotics produced by Bac. pumilus had a very narrow spectrum and inhibited multiplication only of several grampositive bacteria.     

A superfamily of voltage-gated sodium channels in bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.     

Instability of the auxotrophic markers in Bacillus thuringiensis

Auxotrophic mutants of Bacillus thuringiensis have been isolated and compared with the mutants of Bacillus subtilis. It has been found that after spore formation and a prolonged storage, the considerable part of cells in a population of some strains display a genetically unstable additional requirement for arginine with high frequency. The correlation between tetracycline sensitivity and unstable requirement for arginine and other growth factors has been found. The possible reasons for instability of genetic markers in Bac. thuringiensis are discussed.     

Using a combined transcription-translation system for determining the role of cryptic plasmids of Bacillus thuringiensis

The possibility of entomocyde crystal protein synthesis was studied using a heterological cell-free system with Bacillus thuringiensis plasmid DNA as template. The high level of template activity is usual for Bac. thuringiensis plasmid DNA. Immunochemical studies of the in vitro synthesized polypeptides showed that Bac. thuringiensis plasmid DNA does not direct crystal protein synthesis.     

Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

Effects of Phosphatidylinositol on Bacterial Protoplasts

Effects of phospholipids on the bacterial protoplasts or membranes were investigated. Phosphatidylinositol (PI) has, most of all, active bursting action on the protoplasts of Bacillus megaterium which was found to contain a very small amount of inositol, about 0.006% of dry cell weight. This action of PI was less active on the spheroplasts or protoplasts of Escherichia coli or Bacillus subtilis than Bac. megaterium. The bursting action of PI was dependent on temperature, but not on pH or osmotic pressure(concentration of sucrose). This action of PI on the protoplasts of Bac. megaterium was more marked when the incubation was carried out in phosphate buffer than in Tris buffer. High concentration of Mg ion inhibited this PI action in the phosphate buffer, but accerelated that in the Tris buffer.

Phospholipids, especially PI, elevated the activity of succinate dehydrogenase of membrane fraction of Bac. megaterium, but sodium laurylsulfate (SLS) inhibited this enzyme.

These actions of PI were compared with those of other phospholipids and detergenic Substances.     

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Characterization of the cellulolytic activity of a Bacillus isolate

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.