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A Fouet

Publications and source records attributed to A Fouet.

32 records · Page 2Linked to original sources
PubMed1995

Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene.

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. The gene coding for the S-layer protein (sap) was cloned on two contiguous fragments in Escherichia coli, and the complete sequence of the structural gene was determined. The protein, Sap, is composed of 814 residues, including a classical prokaryotic 29-amino-acid signal peptide. The mature form has a calculated molecular mass of 83.7 kDa and a molecular mass of 94 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Sap possesses many charged residues, is weakly acidic, and contains only 0.9% methionine and no cysteine residues. The N-terminal region of Sap shares sequence similarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp alpha protein, and the Bacillus thuringiensis S-layer protein. Electron microscopy observations showed that this S-layer is not observed on B. anthracis cells in which sap has been deleted.

Amino Acid Sequence↗
PubMed1994

Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase.

The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N-terminal two-thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I. The suggested active-site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.

Amino Acid Sequence↗
PubMed1994

The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature.

The two Bacillus anthracis toxins are composed of three proteins, protective antigen, lethal factor, and edema factor. The structural genes for these three components are located on the virulence plasmid pXO1. We constructed transcriptional fusions between the regulatory region of each of these genes and lacZ. Each construct was then inserted as a single copy at the corresponding toxin gene locus on pXO1, resulting in three isogenic strains. Two environmental factors, bicarbonate and temperature, were found to induce beta-galactosidase synthesis in each recombinant strain. Furthermore, the transcription of the three toxin genes appears to be coordinately regulated.

Antigens, Bacterial↗
PubMed1992

Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion.

The production of protective antigen (PA), the common component of the two anthrax toxins, is influenced by the environment. In order to examine factors involved in its regulation, a transcriptional fusion between the promoter region of the PA gene (pag) and the lacZ gene was constructed and introduced into Bacillus anthracis Sterne. Activity of the pag promoter was followed by measuring beta-galactosidase activities under various growth and medium conditions. Expression from the pag promoter was observed throughout exponential-phase and was maximal in early stationary phase. The activity of the pag promoter was stimulated by the addition of glucose in the medium.

Antigens, Bacterial↗
PubMed1990

A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis.

Expression of the aconitase (citB) gene of Bacillus subtilis is subject to catabolite repression in cells grown in minimal media. In nutrient broth medium, citB expression is low in growing cells but is induced when cells enter sporulation. A 600-base-pair DNA fragment that extends from positions -400 through +200, relative to the transcription start site, was shown to include all of the cis-acting sequences necessary for catabolite repression and sporulation-associated regulation. This was demonstrated by fusing this DNA fragment to the Escherichia coli lacZ gene, integrating the fusion in the amyE locus of the B. subtilis chromosome, and measuring the regulation of expression of beta-galactosidase. By creating a series of deletions from either end of the 600-base-pair fragment, it was possible to define a target for catabolite repression; at least part of this target lies within the sequence between positions -84 and -68. DNA fragments that included positions -84 through +36, when carried on high-copy plasmids, caused derepression of aconitase synthesis, as if a negative regulator were being titrated. The same plasmids caused derepression of citrate synthase activity as well. Deletion of the sequence between positions -84 and -67 abolished this titration effect for both enzymes. Mutations that altered the target for catabolite repression also affected the inducibility of citB at the onset of sporulation, at least when sporulation was induced by the addition of decoyinine, an inhibitor of guanine nucleotide synthesis. When sporulation was induced by exhaustion of nutrient broth, there was no detectable difference in expression of citB-lacZ fusions whether or not they had the citB sequence from positions -84 to -67, suggesting that the mechanisms of regulation of citB in minimal medium and nutrient broth are different.

Aconitate Hydratase↗
PubMed1990

The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators.

The expression of the Bacillus subtilis sacPA operon is induced by sucrose. A DNA fragment containing the upstream region of this operon was cloned. This fragment contains a promoter from which the operon is expressed. This upstream region also contains a palindromic DNA sequence very similar to the transcriptional terminator which regulates the induction of the B. subtilis sacB gene. Of 37 nucleotides in a region partially overlapping the sacP palindromic sequence, 34 were identical to the corresponding region of the sacB gene. A similar motif is also present in the bgl operon of Escherichia coli. The sacT locus controlling sacPA expression had been identified by a single constitutive mutation sacT30 which mapped close to the sacPA operon. DNA fragments containing the sacT+ and sacT30 alleles were cloned and sequenced. The sacT gene product is very similar to the B. subtilis sacY and to the E. coli bglG gene products. The constitutive sacT30 mutation was identified. It corresponds to a Asp-96-to-Tyr missense mutation located in a highly conserved region in SacT and SacY. These results strongly suggest that sacT is a specific regulatory gene of the sacPA operon.

Amino Acid Sequence↗
PubMed1990

Multiple regulatory sites in the Bacillus subtilis citB promoter region.

The aconitase (citB) gene of Bacillus subtilis is repressed during growth in a medium that contains a rapidly metabolizable carbon source and a source of 2-ketoglutarate. It is derepressed when either of these nutrient sources becomes limiting. Repression by rapidly metabolizable carbon sources was shown previously to depend at least in part on a DNA sequence located 67 to 84 base pairs upstream of the start point of citB transcription. In the present work, this region and surrounding DNA were mutagenized to identify more precisely the target for carbon catabolite repression. Mutations in a symmetric sequence located between positions -73 and -59 led to constitutive transcription from the citB promoter in media that normally provoke catabolite repression. By gel mobility shift assays, it was shown that at least one protein in extracts of B. subtilis binds to the symmetric sequence and that DNA of constitutive mutants binds to this protein much less effectively. A second sequence located near position -45 was also implicated in this regulation. A second form of regulation of citB was also investigated. This gene is known to be derepressed when cells are induced to sporulate by exhaustion of a nutrient broth medium or limitation of guanine nucleotide synthesis. The mutations that led to constitutivity with respect to the carbon source had no effect on citB expression in nutrient broth medium, indicating that control by catabolite repression and control by components of nutrient broth (presumably amino acids) act by different mechanisms.

Aconitate Hydratase↗
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↗
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↗
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↗
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↗
PubMedDate not supplied

Molecular tools for the study of transcriptional regulation in Bacillus anthracis.

Bacillus anthracis produces two toxins composed of three proteins. Genetic tools were constructed to study the regulation of toxin synthesis. They included transcriptional fusions with various reporter genes, in replicative and integrative vectors. The reporter gene xylE, encoding catechol 2,3-dioxygenase, may be valuable for screening of strong promoters, as expression of the gene can be visualized directly and the studies of regulation in B. anthracis. Therefore, transcriptional fusions between a lacZ reporter gene and the toxin genes were constructed. Experiments with a multicopy plasmid in trans suggested that the transcriptional activator(s) of the toxin genes were not titrated. B. anthracis strains, which contain pXO1 carrying multiple copies of fusions, were analysed. Expression of the reporter gene was proportional to the fusion copy number. Indeed, single integration of a suicide plasmid can be distinguished from multiple integration according to the level of resistance to an appropriate antibiotic. Finally, recombination in B. anthracis was found to be very efficient (approximately 10(-2) recombinants per transconjugant cell.

Bacillus anthracis↗