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G Rapoport

Publications and source records attributed to G Rapoport.

At least 73 records · Page 4Linked to original sources
PubMed1988

A Bacillus thuringiensis subsp. israelensis gene encoding a 125-kilodalton larvicidal polypeptide is associated with inverted repeat sequences.

A gene encoding a 125-kilodalton (kDa) mosquitocidal delta-endotoxin was cloned from the 72-MDa resident plasmid of Bacillus thuringiensis subsp. israelensis. This gene is similar in its 3' region to the gene encoding the 135-kDa protein previously cloned (C. Bourgouin, A. Klier, and G. Rapoport, Mol. Gen. Genet. 205:390-397, 1986). Escherichia coli recombinant clones harboring the 125-kDa gene were toxic to larvae of the three mosquito species Aedes aegypti, Anopheles stephensi, and Culex pipiens. In addition, the B. thuringiensis subsp. israelensis DNA fragment carrying the 125-kDa protein gene contains two sets of inverted repeat sequences, identified either by the S1 nuclease method or by electron microscopic observation. The structural organization of inverted repeat sequences and of the 125-kDa gene was analyzed and suggests that this B. thuringiensis subsp. israelensis delta-endotoxin gene is located within a transposable element.

Aedes↗
PubMed1987

Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase.

The structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli. The cloned gene was mapped by PBS1 transduction near the sacL locus on the B. subtilis chromosome, between leuA and aroD. Expression of the enzyme was demonstrated both in B. subtilis and in E. coli. The presence of sacC allowed E. coli to grow on sucrose as the sole carbon source. The complete nucleotide sequence of sacC was determined. It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli. The apparent molecular weight of purified levanase is 73 kDa. The sacC gene product was characterized in an in vitro system and in a minicell-producing strain of E. coli, confirming the existence of a precursor form of levanase of about 75 kDa. Comparison of the predicted aminoacid sequence of levanase with those of the two other known beta-D-fructofuranosidases of B. subtilis indicated a homology with sucrase, but not with levansucrase. A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.

Amino Acid Sequence↗
PubMed1987

Bacillus subtilis sucrose-specific enzyme II of the phosphotransferase system: expression in Escherichia coli and homology to enzymes II from enteric bacteria.

Sucrose is transported into Bacillus subtilis cells by way of a phosphotransferase system, which consists of a specific enzyme II, a nonspecific enzyme I, and a histidine-containing phosphocarrier protein. Mutations in the sacP locus abolish the specific transport of sucrose. The B. subtilis sacP gene was cloned and expressed in Escherichia coli, and transformed cells could transport and phosphorylate sucrose. This indicates that the sacP gene product is enzyme II of the sucrose phosphotransferase system of B. subtilis. The nucleotide sequence of the sacP gene was determined and was found to overlap with the sacA gene at the tetranucleotide ATGA, which may allow a translational coupling between sacP and sacA. The two genes are therefore probably organized in an operon structure with the promoter located 5' to sacP gene. The deduced amino acid sequence gave a Mr of 48,945 for the sucrose-specific enzyme II polypeptide. The amino acid sequence was compared to that of three other known enteric bacterial enzymes II (beta-glucoside-specific enzyme II, mannitol-specific enzyme II, and glucose-specific enzyme II). Homology was found with beta-glucoside enzyme II, and well conserved regions were identified through the comparison of the proteins.

Amino Acid Sequence↗
PubMed1987

Distinct control sites located upstream from the levansucrase gene of Bacillus subtilis.

The sacR regulatory region, which modulates the expression of sacB, the structural gene for levansucrase, was separated into two parts: an upstream region which carries a constitutive promoter and a downstream region which carries a palindromic structure. Three types of fusions were constructed in which the aphA3 gene coding for kanamycin resistance of Streptococcus faecalis was placed downstream from different deleted sacR regions. Other fusions were constructed by inserting a promoter from phage SPO1 upstream from the sacB gene and part of the sacA region. A third kind of fusion was constructed in which the palindromic structure was flanked by a heterologous promoter and a heterologous structural gene. After introduction of these fusions into the chromosomal DNA of mutants affected in sacB regulation, it was possible to reveal different targets for the regulatory genes sacU, sacQ and sacS: the sacU and sacQ genes act on a region located near or just upstream from the promoter, and the sacS gene, which is involved in the induction process, acts on the palindromic structure.

Bacillus subtilis↗
PubMed1987

Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis.

The sacQ gene from Bacillus licheniformis was cloned and expressed in Bacillus subtilis. Deletion analysis shows that it encodes a 46-amino-acid polypeptide homologous to the B. subtilis sacQ gene product. The polypeptide, when it is overexpressed, activates the expression of a number of target genes in B. subtilis, all encoding secreted enzymes: alkaline protease, levansucrase, beta-glucanase(s), xylanase, and alpha-amylase. The maximum stimulations measured for alkaline protease and levansucrase were by a factor of 70 and 50, respectively, when the sacQ gene from B. licheniformis was present on a multicopy plasmid in B. subtilis. The sacQ genes from B. subtilis and B. licheniformis, cloned in the same multicopy plasmid, were compared under the same conditions. The sacQ gene from B. licheniformis was more efficient than the sacQ gene from B. subtilis in producing the hypersecretion phenotype. The sacQ structural genes from B. subtilis and B. licheniformis were placed under the control of the same inducible promoter. Hypersecretion was specifically obtained under conditions of full induction of the promoter. The target site of levansucrase regulation by sacQ was identified as a 440-base-pair fragment located in the 5' noncoding region of sacB, suggesting transcriptional control.

Amino Acid Sequence↗
PubMed1987

Bacillus larval toxin crystal protein.

During sporulation some bacteria produce parasporal inclusions which are toxic for insect larvae. The proteins responsible for this toxicity have been characterized and their genes have been cloned. Using genetic engineering methods, it is now possible to establish new bacterial strains and to introduce these genes into plant genomes.

Animals↗
PubMed1986

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis.

The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichia-coli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 130 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.

Aedes↗
PubMed1986

Nucleotide sequence of the sucrase gene of Bacillus subtilis.

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.

Amino Acid Sequence↗
PubMed1985

Recent aspects of genetic manipulation in Bacillus thuringiensis.

The conjugative plasmid pAM beta 1 was transferred from Streptococcus faecalis to several strains of Bacillus thuringiensis by a filter-mating process. From a transconjugant clone of B. thuringiensis a hybrid plasmid resulting from an in vivo insertion into pAM beta 1 of a 3 Md DNA sequence was isolated. This 3 Md DNA molecule (Th sequence) is related to several host plasmids found in different serotypes of B. thuringiensis. A reciprocal conjugation-like process involving the transfer of pAM beta 1 from B. thuringiensis to S. faecalis was also demonstrated. The comparison of the restriction maps of the crystal genes from plasmid and chromosomal origins of different serotypes, six of which having been cloned in E. coli, revealed the existence of two classes of genes which are very similar in the map corresponding to the N-terminal part of the protein, and which differ essentially in the 3' region. The presence of the transposon-like Th sequence was found in several cases associated with the crystal gene in the same host plasmid, and a model for their structural organization is proposed.

Bacillus thuringiensis↗
PubMed1985

Cloning and expression in Escherichia coli of the regulatory sacU gene from Bacillus subtilis.

The regulatory wild-type locus sacU, which has a pleiotropic effect in Bacillus subtilis, notably on the synthesis of secreted proteins, was obtained from a colony bank of Escherichia coli harboring recombinant cosmids representative of the B. subtilis genome. It was shown that the sacU gene is located on a 2.4-kilobase KpnI-EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. The wild-type phenotype was recovered after transformation of SacU-, SacUh, and SacU- Rec- strains with the recombinant cosmid, indicating that the sacU locus has been cloned in totality. The sacU gene was expressed in a minicell-producing E. coli strain, and it was shown that it coded for a 46-kilodalton protein. In addition to the hypersecretion of proteins, SacUh mutants were characterized by the presence of a 46-kilodalton protein in the membrane fraction in higher amounts than were found in the wild-type strain. These mutants were also devoid of a 36-kilodalton polypeptide corresponding to the flagellin subunit. Analysis of the mRNA content of a secreted protein (levansucrase) in SacU- and SacUh mutants strongly suggested that the pleiotropic action of the sacU gene on the synthesis of levansucrase is exerted at a posttranscriptional level in B. subtilis cells and is probably correlated with the mechanism of secretion of exoenzymes.

Bacillus subtilis↗
PubMed1984

Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis.

Expression of the cloned levansucrase gene (sacB) was demonstrated in E. coli minicells by assay of the enzyme in crude extracts, SDS-polyacrylamide gel electrophoresis and immunoblotting. The existence of a precursor form of the enzyme of MW 53000 was also demonstrated and confirmed by the DNA sequence corresponding to the NH2 terminal region of the protein.

Amino Acid Sequence↗
PubMed1983

In vitro transcription of the cloned chromosomal crystal gene from Bacillus thuringiensis.

We have determined the conditions required for in vitro transcription of the cloned chromosomal crystal gene from Bacillus thuringiensis using either the homologous vegetative RNA polymerase or a sporulation specific form of this enzyme. The gene is actively transcribed by the latter enzyme (form II) but not by the vegetative one. Evidence for a specific recognition between the form II enzyme and the promotor site of the crystal gene was obtained by binding experiments. They showed that the binding is increased by the presence of some additional factors, which change the specificity of the vegetative core-enzyme. The sequence of the promoter has been determined and the start-point of the transcription deduced. Two hexanucleotide sequences, TACAAT and CCTACG, centered at - 10 and - 35 bp are present, but are somewhat different from the consensus sequences previously described in other bacilli.

Bacillus thuringiensis↗
PubMed1982

Cloning and expression of the crystal protein genes from Bacillus thuringiensis strain berliner 1715.

From a clone bank of the entire genome of Bacillus thuringiensis, one clone that contains a plasmid ( pBT 15-88) harboring a sporulation gene was identified by molecular hybridization. This gene, identified as the crystal protein gene, occurs both on a large host plasmid DNA and in the chromosomal DNA in B. thuringiensis strain berliner 1715. The inserted sequence of pBT 15-88, which corresponds to the chromosomal sequence, was not expressed in Escherichia coli. In B. thuringiensis (kurstaki), the crystal gene was found only on a large host plasmid while in B. thuringiensis ( dendrolimus ), it is only on the chromosomal DNA. The plasmid crystal gene was cloned by ligation of a 14-kb BamHI fragment of a host plasmid DNA of 42 megadaltons from strain berliner 1715 into the BamHI site of the bifunctional vector pHV33 . In E. coli and in sporulating B. subtilis the plasmid pBT 42-1 coded for a polypeptide, detected by antibodies against the crystal protein, with the same electrophoretic mobility as the crystal protein of B. thuringiensis. The crystal gene was not expressed in vegetative cells of B. subtilis, suggesting that the control at the transcriptional level is the same in B. subtilis and in B. thuringiensis. Protein extracts from the clones harboring the hybrid plasmid are toxic for the larvae of Pierris brassicae and the protein antigen forms cytoplasmic inclusion bodies in E. coli and B. subtilis, which are visible under the light microscope.

Bacillus thuringiensis↗
PubMed1982

Cloning and expression in Escherichia coli of the sucrase gene from Bacillus subtilis.

A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank of E. coli harboring recombinant cosmids representative of the B. subtilis genome. It was shown that the sacA gene is located in a 2kb EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for in B. subtilis and E. coli. Complementation of a sacA mutation was observed in Rec+ and REc- strains of B. subtilis. Expression of sucrase was also demonstrated in E. coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic in E. coli since the presence of the recombinant plasmids did not allow the transport of [U14C] sucrose and the growth of the cells. It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport of B. subtilis.

Bacillus subtilis↗
PubMed1979

Structure of cloned ribosomal DNA cistrons from Bacillus thuringiensis.

A library of B. thuringiensis DNA has been prepared by using the plasmid pBR322 as a cloning vehicle and E. coli as a host cell. By screening this collection with specific probes, 17 clones were identified whose hybrid plasmids contain rRNA genes of B. thuringiensis. Several of these plasmids have been mapped with restriction endonucleases and by DNA-RNA hybridization. By using maps of overlapping fragments, we have been able to establish an overall map of the ribosomal gene cluster.

Bacillus thuringiensis↗
PubMed1979

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA).poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid. Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such at thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec- recipient strain of B. subtilis was used.

Bacillus subtilis↗
PubMed1979

On the nature of tetracycline resistance in Bacillus subtilis mediated by the plasmid pT 127.

The nature of tetracycline resistance was studied in a strain of Bacillus subtilis carrying the plasmid pT 127 in comparison with the parental strain. The resistance has been shown to be inducible in both strains upon exposure to subinhibitory concentrations of tetracycline. No modification of the protein-synthesizing activity of the ribosomes or intracellular inactivation of the antibiotic was observed in both strains. Accumulation of labeled tetracycline in B. subtilis was found to be particularly low in the wild-type strain, compared to other bacterial species, with concentration gradients of only 2 to 3 fold. From the kinetics obtained it is likely that the permeation of the antibiotic does not correspond to an active process in B. subtilis. A fairly good correlation was established between the level of resistance obtained after induction or by the presence of the plasmid pT 127 and a decrease in the binding capacity of the cell for the antibiotic.

Bacillus subtilis↗