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G Rapoport

Publications and source records attributed to G Rapoport.

At least 19 recordsLinked to original sources
PubMed1992

The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis.

Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of degradative enzymes in vivo.

Alleles↗
PubMed1992

Mutagenesis of the Bacillus subtilis "-12, -24" promoter of the levanase operon and evidence for the existence of an upstream activating sequence.

The levanase operon of Bacillus subtilis is controlled by RNA polymerase associated with sigma 54 factor and by the LevR activator that is homologous to the NifA/NtrC family of regulators. A "-12, -24" promoter is present at the appropriate distance from the transcription start site. The drastic down effect of base substitutions in the TGGCAC, TTGCA consensus sequence on the expression of the levanase operon confirmed the involvement of the "-12, -24" region in promoter function. Deletion derivatives of the upstream sequence of the operon promoter were constructed using translational levD'-'lacZ fusions and were integrated as single copies at the amyE locus of the B. subtilis chromosome. A cis-acting DNA sequence that is required for activation of the operon promoter by LevR was identified. This regulatory sequence is about 50 base-pairs long and is centered 125 base-pairs upstream from the transcription start site in a region containing a 16 base-pair palindromic structure. This region of dyad symmetry functions as a regulatory element when placed up to at least 600 base-pairs upstream from the "-12, -24" promoter, although the efficacy of activation is lowered. Thus, in common with most sigma 54-dependent promoters, an upstream activating sequence (UAS) is involved in the control of expression of the levanase operon. The isolation and characterization of eight mutations in the UAS region confirmed the importance of the palindromic structure in promoter activation. Moreover, the expression of the levanase operon was inhibited by placing the UAS in trans on a multicopy plasmid, probably through titration of the LevR polypeptide. In conclusion, the levanase promoter region can be divided into two regulatory sequences: the "-12, -24" promoter recognized by the sigma 54 RNA polymerase holoenzyme and the UAS, an inverted repeat sequence that is probably the LevR binding site.

Amino Acid Sequence↗
PubMed1992

Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase.

Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikström, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605 cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B. subtilis.

Amino Acid Sequence↗
PubMed1992

Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity.

The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B. subtilis SacY and the Escherichia coli BglG antiterminators. Expression of the sacT gene is shown to be constitutive. The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator. We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon. In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive. In this work, we show that this constitutivity is due to a fully active SacY antiterminator. In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination. It appears that the PTS exerts different effects on the sacB gene and the sacPA operon. The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity.

Bacillus subtilis↗
PubMed1992

Positive regulation in the gram-positive bacterium: Bacillus subtilis.

Temporally and environmentally regulated gene expression in prokaryotes occurs primarily at the level of transcription initiation. Two main modes of regulation have been described, including either the binding of a repressor that blocks transcription or the interaction of a positive regulator with the transcription complex, leading to transcription initiation. Several classes can be distinguished among positive regulators according to their mechanisms of action. This review describes the different types of positive regulators identified in Bacillus subtilis, a gram-positive bacterium. These include accessory regulatory polypeptides, classical positive regulators that bind to target sites located just upstream from the promoter, ambiactive regulators that can act both positively and negatively, antiterminators, two-component signal transduction systems, and positive regulators associated with specific secondary sigma factors.

Bacillus subtilis↗
PubMed1991

The Bacillus subtilis sigL gene encodes an equivalent of sigma 54 from gram-negative bacteria.

The levanase operon in Bacillus subtilis is expressed from a -12, -24 promoter and transcription is stimulated by the regulator LevR, which contains a domain homologous with the central domain of the NifA and NtrC family of regulators. We isolated mutants defective in the expression of the levanase operon. These strains contain mutations that define a gene, called sigL, located between cysB and sacB on the genetic map. The sigL gene was cloned and sequenced. It encodes a polypeptide containing 436 residues with a molecular weight of 49,644. The amino acid sequence of SigL is homologous with all sigma 54 factors from Gram-negative bacteria, including Rhizobium meliloti (32% identity) and Klebsiella pneumoniae (30% identity). B. subtilis sigL mutants have a pleiotropic phenotype: (i) the transcription of the levanase operon is strongly reduced and (ii) in minimal medium lacking ammonia, sigL mutants cannot grow when arginine, ornithine, isoleucine, or valine is the sole nitrogen source. These results indicate that the sigL gene encodes an equivalent of the sigma 54 factor in B. subtilis, to our knowledge, the first of this type to be identified in Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1991

The transcriptional regulator LevR of Bacillus subtilis has domains homologous to both sigma 54- and phosphotransferase system-dependent regulators.

The regulatory gene levR of the levanase operon of Bacillus subtilis was cloned and sequenced. It encodes a polypeptide of Mr 106,064 with two domains homologous to members of two families of bacterial activators. One domain in LevR is homologous with one region of bacterial regulators including SacT and SacY of B. subtilis and BglG from Escherichia coli. Another domain of LevR is homologous to one part of the central domain of NifA and NtrC, which control nitrogen assimilation in Gram-negative bacteria. The levanase promoter contains two regions almost identical to the -12, -24 consensus regions present in sigma 54-dependent promoters. The expression of the levanase operon in E. coli was strongly dependent on sigma 54. Taken together, these results suggest that the operon is expressed from a -12, -24 promoter regulated by a sigma 54-like-dependent system in B. subtilis.

Amino Acid Sequence↗
PubMed1991

Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity.

The cytA gene encoding the 28-kDa polypeptide of Bacillus thuringiensis subsp. israelensis crystals was disrupted in the 72-MDa resident plasmid by in vivo recombination, thus indicating that homologous recombination occurs in B. thuringiensis. The absence of the 28-kDa protein in B. thuringiensis did not affect the crystallization of the other toxic components of the parasporal body (68-, 125-, and 135-kDa polypeptides). The absence of the 28-kDa protein abolished the hemolytic activity of B. thuringiensis subsp. israelensis crystals. However, the mosquitocidal activity of the 28-kDa protein-free crystals did not differ significantly from that of the wild-type crystals when tested on Aedes aegypti and Culex pipiens larvae. The 28-kDa protein contributed slightly to the toxicity to Anopheles stephensi larvae. This indicates that the 28-kDa protein is not essential for mosquitocidal activity, at least against the three species tested.

Animals↗
PubMed1991

DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ.

Production of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis is regulated at the transcriptional level by a signal transduction pathway which includes the DegS-DegU two-component system and at least two additional regulatory genes, degQ and degR, encoding polypeptides of 46 and 60 amino acids, respectively. Expression of degQ was shown to be controlled by DegS-DegU. This expression is decreased in the presence of glucose and increased under any of the following conditions: growth with poor carbon sources, amino acid deprivation, phosphate starvation, and growth in the presence of decoyinine, a specific inhibitor of GMP synthetase. In addition, expression of degQ is shown to be positively regulated by the ComP-ComA two-component system. Separate targets for regulation of degQ gene expression by DegS-DegU and ComP-ComA were located by deletion analysis between positions -393 and -186 and between positions -78 and -40, respectively. Regulation of degQ expression by amino acid deprivation was shown to be dependent upon ComA. Regulation by phosphate starvation, catabolite repression, and decoyinine was independent of the two-component systems and shown to involve sequences downstream from position -78. The ComP-ComA and DegS-DegU two-component systems seem to be closely related, sharing several target genes in common, such as late competence genes, as well as the degQ regulatory gene. Sequence analysis of the degQ region revealed the beginning of an open reading frame directly downstream from degQ. Disruption of this gene, designated comQ, suggests that it also controls expression of degQ and is required for development of genetic competence.

Adenosine↗
PubMed1991

Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase.

The DegS-DegU protein kinase-response regulator pair controls the expression of genes encoding degradative enzymes as well as other cellular functions in Bacillus subtilis. Both proteins were purified. The DegS protein was autophosphorylated and shown to transfer its phosphate to the DegU protein. Phosphoryl transfer to the wild-type DegU protein present in crude extracts was shown by adding 32P-labeled DegS to the reaction mixture. Under similar conditions, the modified proteins encoded by the degU24 and degU31 alleles presented a stronger phosphorylation signal compared with that of the wild-type DegU protein. This may suggest an increased phosphorylation of these modified proteins, responsible for the hyperproduction of degradative enzymes observed in the degU24 and degU31 mutants. However, the degU32 allele, which also leads to hyperproduction of degradative enzymes, encodes a modified DegU response regulator which seems not to be phosphorylatable. The expression of the hyperproduction phenotype of the degU32 mutant is still dependent on the presence of a functional DegS protein. DegS may therefore induce a conformational change of the degU32-encoded response regulator enabling this protein to stimulate degradative enzyme synthesis. Two alleles, degU122 and degU146, both leading to deficiency of degradative enzyme synthesis, seem to encode phosphorylatable and nonphosphorylatable DegU proteins, respectively.

Bacillus subtilis↗
PubMed1990

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon.

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.

Amino Acid Sequence↗
PubMed1990

Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtilis by induction of chromosomal DNA amplification.

We have inserted a DNA fragment composed of (i) the promoter and the export signal of the Bacillus subtilis levansucrase gene; (ii) the sequence encoding the mature part of the Clostridium thermocellum endoglucanase A gene in a specific site of the B. subtilis chromosome. The insert was flanked by directly repeated pBR322 sequences of 3.9 kb. Plasmid pE194, which has a thermosensitive replication, was integrated adjacent to one of the repeats. When the integrated plasmid was allowed to replicate, the insert and one of the repeats was amplified up to a level of about 250 copies per chromosome. Endoglucanase A was efficiently synthesized in, and secreted from, cells containing the amplified structure, since the heterologous fusion protein was the major extracellular protein in a B. subtilis sacUh strain. The NH2-terminal sequence of the secreted protein revealed three different cleavage sites in the vicinity of the signal peptidase recognition sequence.

Amino Acid Sequence↗
PubMed1990

Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU.

The rates of synthesis of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis are controlled by a signal transduction pathway defined by at least four regulatory genes: degS, degU, degQ (formerly sacQ), and degR (formerly prtR). The DegS-DegU proteins show amino acid similarities with two-component procaryotic modulator-effector pairs such as NtrB-NtrC, CheA-CheY, and EnvZ-OmpR. By analogy with these systems, it is possible that DegS is a protein kinase which could catalyze the transfer of a phosphoryl moiety to DegU, which acts as a positive regulator. DegR and DegQ correspond to polypeptides of 60 and 46 amino acids, respectively, which also activate the synthesis of degradative enzymes. We show that the degS and degU genes are organized in an operon. The putative sigma A promoter of the operon was mapped upstream from degS. Mutations in degS and degU were characterized at the molecular level, and their effects on transformability and cell motility were studied. The expression of degQ was shown to be subject both to catabolite repression and DegS-DegU-mediated control, allowing an increase in the rate of synthesis of degQ under conditions of nitrogen starvation. These results are consistent with the hypothesis that this control system responds to an environmental signal such as limitations of nitrogen, carbon, or phosphate sources.

Amino Acid Sequence↗
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↗
PubMed1989

Nucleotide sequence and characterization of a new insertion element, IS240, from Bacillus thuringiensis israelensis.

The nucleotide sequence of two repeated sequences (RS) in opposite orientations flanking the 125-kDa toxin gene of Bacillus thuringiensis israelensis (C. Bourgouin et al., J. Bacteriol. 170, 3575-3583, 1988) is reported in this paper. The analysis of these sequences indicates that these two RS display characteristic features of bacterial insertion sequences (IS) and are therefore referred to as IS240. IS240 B is 865 bp long and has two perfect terminal-inverted repeats of 16 bp; IS240 A is 99% identical to IS240 B. A long open reading frame encoding a polypeptide of 235 amino acids spans almost the entire sequence of both IS240 elements. Both the sequence of the inverted repeats and the putative transposases are homologous to IS26 of Proteus vulgaris, IS15-delta of Salmonella panama, IS431 of Staphylococcus aureus, and ISS1 of Streptococcus lactis.

Amino Acid Sequence↗
PubMed1989

Expression of the MuIFN alpha 7 gene in Bacillus subtilis using the levansucrase system.

The mouse interferon alpha 7 gene, the signal sequence of which has been removed by oligonucleotide-directed mutagenesis, was introduced into a Bacillus subtilis secretion vector containing the promoter and the signal sequence of the B. subtilis levansucrase gene. Different B. subtilis strains were transformed with the fused levansucrase-interferon gene; their cell extracts and culture supernatants tested for antiviral activity and the IFN alpha 7 protein showed the presence of IFN alpha 7 only in the cell extracts. To promote IFN alpha 7 secretion, constructs were realized in order to restore the alpha helix conformation of the signal sequence of levansucrase and interferon protein junction. Our results suggest that factors other than the structure of the peptide around the cleavage site are involved in the secretion of IFN alpha 7 by B. subtilis.

Amino Acid Sequence↗