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Biomedical subjects

J Szulmajster

Publications and source records attributed to J Szulmajster.

At least 19 recordsLinked to original sources
PubMed1990

Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity.

The two genes of Bacillus sphaericus 1953M coding for the 51.4-kDa and 41.9-kDa proteins are both required for the expression of the active larvicidal toxin in Escherichia coli. The minimal size of the active peptide of the 41.9-kDa toxin was defined by in vitro deletion analysis of the gene and found to consist of 338 amino acids (38.3 kDa). N-terminal deletions past the Ile18 residue and C-terminal deletions past the His352 residue result in the loss of toxic activity and rapid degradation of such modified toxins by host proteases. The minimal active 38.3-kDa peptide produced in E. coli seems to mimick the stable processed form of the toxin found in larval midguts. However, it still requires the action of the synergistic 51.4-kDa protein for the larvicidal activity.

Amino Acid Sequence↗
PubMed1990

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression.

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.

Aedes↗
PubMed1989

On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis.

The 3.6 kb HindIII DNA fragment of B. sphaericus 1593M chromosomal DNA bears two genes encoding two polypeptides of 41.9 kDa (protein "42") and 51.4 kDa (protein "51"). DNA fragments carrying only one of these two genes when expressed in E. coli yield products that are inactive towards Culex larvae. The larvicidal activity is recovered when Triton X-100 treated E. coli cells containing each one of the two genes are incubated together. In E. coli these two polypeptides are acting synergistically. The protein "51" appears to be involved in the maturation of protein "42" for expression of the larvicidal activity. In B. subtilis however the toxicity is expressed by cells carrying only the gene coding for protein "42". There is no need of the "51" gene product for the maturation of the "42" polypeptide, suggesting that the maturation is most likely accomplished by host enzymes.

Animals↗
PubMed1988

Protein folding.

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Amino Acid Isomerases↗
PubMed1987

Purification and characterization of the larvicidal toxin of Bacillus sphaericus 1593M.

We present here a procedure for purifying the larvicidal toxin from sporulating cells of Bacillus sphaericus 1593M and describe some of the biochemical and biophysical properties of this toxin. The procedure involves solubilization of the cell-wall/membrane bound toxin by sonication of cells followed by repeated rounds of freezing and thawing at 50 degrees C. Further purification involved Sephadex G-100 and DEAE Sephacel chromatography. We show by Sephadex G-100 chromatography that at pH 7.5 the smallest active form of the toxin has an Mr of 38,000 and that this toxin can reversibly aggregate to molecular forms of a size higher than 2 X 10(5) Mr. By shifting the pH from 7.5 to 8.5 only the aggregated forms can be observed.

Bacillus↗
PubMed1987

Expression of the larvicidal gene of Bacillus sphaericus 1593M in the cyanobacterium Anacystis nidulans R2.

A 3.6 kb HindIII DNA fragment from Bacillus sphaericus 1593M was cloned and expressed in Escherichia coli and B. subtilis using pHV33 as shuttle vector and in the cyanobacterium Anacystis nidulans R2 with pUC303 as shuttle vector. The level of toxin activity of the respective recombinant plasmids pGsp04 and pGsp12 against Culex mosquito larvae was found to be the same in Escherichia coli and in the cyanobacterium.

Animals↗
PubMed1985

Bacillus subtilis sigma factor sigma 29 is the product of the sporulation-essential gene spoIIG.

Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31. This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation. spoIIG-specific RNA is present in B. subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not. A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B. subtilis strain with a mutation at the spoIIG locus (spoIIG41). The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31.

Antibodies, Monoclonal↗
PubMed1984

Biocide gene(s) and biocidal activity in different strains of Bacillus sphaericus. Expression of the gene(s) in E. coli maxicells.

The recently cloned biocidal determinant of the highly toxic strain of B. sphaericus 1593M (Ganesan et al. 1983) was used as probe to investigate homologous sequences in different toxic and non-toxic strains of B. sphaericus. It was found that the potent strains we have analysed are characterised by the presence of DNA sequences (6.6, 6.4, 5.8, 1.6, 1.3 and 0.6 Kb) not found in the non-toxic strains. These results further show that one of the two weakly toxic strains analysed presents a hybridisation pattern completely different from that observed with the highly potent strains of B. sphaericus. When the DNA of the two non-toxic strains was analysed, SSII-I failed to hybridise to the probe and Rem4 exhibited mainly one hybridisable sequence of 2.3 Kb not detectable in the toxic strains. No region of homology to the probe was found in the DNA of two strains of B. thuringiensis (var. berliner and var. israeliensis) analysed. By dot blot hybridisation experiments it was estimated that the larvicidal determinant might be present in about one to three copies per genome. With the use of E. coli maxicells we have shown further that the toxin gene(s) encoded four polypeptides with molecular weights of 21, 19, 15 and 12 Kd. The significance of these findings is discussed.

Animals↗
PubMed1984

Nucleotide sequence of the spo0B gene of Bacillus subtilis and regulation of its expression.

The spo0B gene is one of the genes involved in initiation of sporulation of Bacillus subtilis. This gene, previously cloned into the pHV33 shuttle vector, is expressed in Escherichia coli and B. subtilis. We have determined the sequence of 1118 base pairs (bp) of the DNA insert carrying the spo0B gene. The promoter sequence of this gene shows the canonical T-A-T-A-A-T region at 10 bp from the transcriptional start (-10 region) but an unusual sequence, T-T-T-T-C-T-, in the -35 region. The nucleotide sequence shows an open reading frame encoding a 192-amino-acid polypeptide of Mr 22,542, which is close to the molecular weight of the spo0B product synthesized in E. coli minicells. To investigate the regulation of the spo0B gene under a variety of physiological conditions, we constructed an in-frame fusion between the spo0B promoter proximal region and the lacZ gene of E. coli. This hybrid gene was subsequently integrated into the B. subtilis chromosome, and the beta-galactosidase activity was measured. It was found that the spo0B gene is preferentially expressed during exponential growth; it is not induced by exhaustion of the growth medium nor repressed by glucose.

Bacillus subtilis↗
PubMed1983

Cloning and expression in Escherichia coli of a DNA fragment from Bacillus sphaericus coding for biocidal activity against mosquito larvae.

A 3.7 kb DNA fragment from Bacillus sphaericus 1593 was cloned and expressed in E. coli using the plasmid pHV33. The level of larvicidal activity of the hybrid plasmid, pGsp03, against two species of mosquito larvae was comparable to that found with sporulating cell suspensions of B. sphaericus. A limited restriction cleavage map of the cloned insert is given.

Animals↗
PubMed1979

Characterization of a thermosensitive sporulation mutant of Bacillus subtilis affected in the structural gene of an intracellular protease.

A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 degrees C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-15R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 degrees C it synthesizes a 'cryptic' protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30 degrees C). The intracellular proteases from the wild type and from ts-15 grown at 30 degrees C and 42 degrees C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30 degrees C was 64000--65000 in the absence of sodium dodecylsulfate and 31000--32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42 degrees C showed the same molecular weight of 32000--34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.

Bacillus subtilis↗
PubMed1977

Specific alteration of the 30S ribosomal subunits of Bacillus subtilis during sporulation.

Active 30S ribosomal subunits were isolated from vegetative and sporulating cells of Bacillus subtilis. Both subunits were able to function in polyuridylic acid of phage phie messenger ribonucleic acid-dependent protein synthesis in vitro. The sporulation 30S subunits were highly active in polyuridylic acid-dependent polyphenylalanine synthesis but showed a reduced activity in the presence of natural messenger ribonucleic acid as compared with their vegetative counter-parts. The reduced activity was independent of the source of 50S particles and initiation factors (vegetative or sporulation). The alteration of the 30S sporulation subunits appears to be related to the sporulation process, since the same subunits isolated from stationary-phase cells of an asporogenic mutant did not show any impairment in protein synthesis in vitro.

Bacillus subtilis↗
PubMed1976

Transcription from complementary deoxyribonucleic acid strands in various sporogenic and asporogenic mutants of Bacillus subtilis. Hybridization-competition studies on ribonucleic acid synthesized in vivo by a thermosensitive sporulation mutant (ts-4).

The present paper describes an investigation, at the transcription level, in a thermosensitive sporulation mutant of Bacillus subtilis (ts-4) grown at the permissive (30 degrees C) or restrictive (42 degrees C) temperature where sporulation capacity is respectively expressed or arrested. These studies were carried out by analysing the ribonucleic acid from vegetative and stationary phase cells (t3 cells) grown under both conditions, by hybridization-competition experiments.

Bacillus subtilis↗