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P Stragier

Publications and source records attributed to P Stragier.

50 records · Page 3Linked to original sources
PubMed1987

Molecular cloning, characterization, and chromosomal localization of dapF, the Escherichia coli gene for diaminopimelate epimerase.

The Escherichia coli dapF gene was isolated from a cosmid library as a result of screening for clones overproducing diaminopimelate epimerase. Insertional mutagenesis was performed on the cloned dapF gene with a mini-Mu transposon, leading to chloramphenicol resistance. One of these insertions was transferred onto the chromosome by a double-recombination event, allowing us to obtain a dapF mutant. This mutant accumulated large amounts of LL-diaminopimelate, confirming the blockage in the step catalyzed by the dapF product, but did not require meso-diaminopimelate for growth. The dapF gene was localized in the 85-min region of the E. coli chromosome between cya and uvrD.

Amino Acid Isomerases↗
PubMed1985

Two functional domains conserved in major and alternate bacterial sigma factors.

Sequences of the sigma factors of Escherichia coli and Bacillus subtilis were aligned with the sequences of two sigma-like proteins, HtpR, involved in the expression of heat-shock genes in E. coli, and SpoIIG, necessary for endospore formation in B. subtilis. An internal region is highly conserved in the four proteins and is proposed to be involved in binding of sigma factors to core RNA polymerase. The carboxy-terminal part of the four proteins presents the characteristic structure found in several prokaryotic DNA-binding proteins and is proposed to be involved in promoter recognition.

Amino Acid Sequence↗
PubMed1985

Expression in mammalian cells of the diaminopimelic acid decarboxylase of Escherichia coli permits cell growth in lysine-free medium.

The lysA gene of Escherichia coli encodes for a diaminopimelic acid decarboxylase (EC 4.1.1.20) which allows the conversion of diaminopimelic acid into lysine in bacteria. It has been cloned in an eukaryotic expression vector containing upstream the SV40 early promoting sequence, and downstream mouse alpha-globin maturating sequences. The recombinant plasmid pSB99 (4800 base pairs) has been introduced into several mammalian cell lines by cotransfection with a second selectable marker i.e. the polyoma-transforming DNA. Selection for morphologically transformed rat cells which contained the intact lysA sequences, allowed the determination of the concentration of diaminopimelic acid in the lysine-free medium that permitted cell growth. lysA-expressing clones were directly selected in a medium containing 10 mM diaminopimelic acid, after transfection with pSB99 alone. Southern blot analysis on selected clones have shown that they contain up to 30-50 integrated copies of the plasmid in tandem arrangement. Finally, we demonstrated that lysA-expressing clones incorporate a significant amount of radiolabelled [3H]diaminopimelic acid in acid-insoluble material. The recombinant plasmid can serve as a selectable marker, in growth medium in which lysine was replaced by its direct bacterial precursor.

Animals↗
PubMed1984

Nucleotide sequence and expression of the Escherichia coli dapB gene.

The Escherichia coli dapB gene encodes dihydrodipicolinate reductase. This enzyme is part of the diaminopimelate-lysine pathway, and its synthesis is repressed by lysine. The dapB gene was cloned into pBR322 from a transducing lambda bacteriophage, its complete nucleotide sequence established, and the transcriptional start localized. The DNA sequence predicts that the dapB gene codes for a 273-amino acid polypeptide, Mr 28,798. No attenuation-type sequence can be found between the mRNA start and the coding sequence. The dapB promoter signals appear to be weak as compared to RNA polymerase consensus sequences. Nevertheless an efficient in vivo synthesis of beta-galactosidase was obtained when the lac operon was inserted in vitro in the dapB gene, downstream of the dapB regulatory signals. Further studies were performed on an in-frame gene fusion constructed in vitro between the dapB and the lacZ genes. They indicated that repression by lysine is exerted on a DNA region restricted to a 153-base pair fragment with only 102 nontranscribed nucleotides. Finally, dapB gene expression showed a gene dosage effect which suggests that it is not controlled by an element present in limiting amounts in the cell.

Amino Acid Sequence↗
PubMed1984

Multiple regulatory signals in the control region of the Escherichia coli carAB operon.

The first reaction in pyrimidine and arginine biosynthesis in Escherichia coli is catalyzed by a single enzyme, carbamoyl-phosphate synthetase (EC 6.3.5.5), the product of the carAB operon. Expression of this operon is cumulatively repressed by arginine and pyrimidines. The nucleotide sequence of the carAB control region was determined and transcriptional starts were localized. Two adjacent promoters, 70 base pairs apart, appear to be used in vivo, the downstream one overlapping a typical arginine operator. The absence of any attenuation-like sequence excludes such a mechanism for pyrimidine-mediated repression. Various fragments of the carA promoter-proximal region were fused in vitro with the lacZ gene. Results obtained with these fusions indicate that (i) translation of the carA gene can be initiated in vivo without an AUG codon but very likely with an UUG or an AUU codon; (ii) the carAB downstream promoter is repressed by arginine; and (iii) the carAB upstream promoter is repressed by pyrimidines and subject to stringent control. When carried by a multicopy plasmid the carAB control region escapes repression by arginine and pyrimidines. The existence of a pyrimidine repressor, present in limiting amounts in the cell, is therefore postulated.

Amino Acid Sequence↗
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

Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. I. Identification of a lysR gene encoding an activator of the lysA gene.

The synthesis of diaminopimelate decarboxylase, which catalyzes the decarboxylation of diaminopimelate into lysine, is known to be repressed by lysine and induced by diaminopimelate in Escherichia coli K12. Until now only mutations in lysA, the structural gene for diaminopimelate decarboxylase, have been described that lead to a Lys- phenotype. A set of plasmids carrying adjacent inserts of the lysA region was constructed and employed to transform different Lys- mutants. The complementation pattern observed and the corresponding expression of the lysA gene show that in fact the Lys- phenotype can be obtained by mutations in two different and closely linked loci: one being the lysA structural gene, and the other called lysR. We propose that the lysR gene encodes a positive effector required for the full expression of the lysA gene. The synthesis of a hybrid lysA-lacZ protein constructed in vitro was observed to be decreased dramatically in lysR mutants. Moreover, all the regulatory features were lost, indicating that the LysR activator is necessary for the regulation of lysA expression. The gene order is thyA lysA lysR clockwise around 61 minutes on the chromosome, lysA being transcribed counter-clockwise.

Bacterial Proteins↗
PubMed1983

Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. II. Nucleotide sequence of the lysA gene and its regulatory region.

The complete nucleotide sequence of the lysA gene and its regulatory region was determined. At the 3' end of the lysA gene an open reading frame was revealed in the opposite direction and was identified as the galR coding region. Only six base-pairs are present between the two translational stops and thus both transcription units are overlapping in vivo. Translational gene fusions constructed in vitro with the beta-galactosidase gene were used to identify the lysA initiating ATG. The sequence encodes a 420 amino acid long peptide for a predicted molecular weight of 46,099. No attenuation-like sequence was found at the beginning of the lysA gene. A target of the LysR activator protein was localized on a 73 base-pair fragment found 48 base-pairs upstream from the lysA coding region. The presence of this DNA sequence on a multicopy plasmid led to a net decrease of lysA expression, indicating limiting amounts of active LysR protein in the cytoplasm.

Amino Acid Sequence↗
PubMed1983

Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. III. Nucleotide sequence and regulation of the lysR gene.

The complete nucleotide sequence of the lysR gene, which encodes the activatory protein required for lysA expression, has been determined. Bal31 deletions and translational fusions were used to localize the promoter region and the initiator ATG of the lysR gene which encodes a 311 amino acid polypeptide. Both lysA and lysR coding sequences were found to be divergent and separated by a very short intergenic region consisting of 121 base-pairs between the postulated ATGs of the two proteins. Transfer of the whole lysR gene on a plasmid carrying a lysR-lacZ fusion shows that lysR expression is autoregulated by a factor of 7. The same binding site (73 base-pairs fragment) could be involved in both effects of the LysR product, acting simultaneously as an operator for lysR expression and an initiator for lysA expression. The genetic organization of the whole region (4127 base-pairs) is given. A strikingly symmetrical pattern is observed with the four tightly packed galR, lysA, lysR and orfX (an unidentified open reading frame) genes, in a very unusual arrangement of both divergent and convergent overlapping transcription units.

Amino Acid Sequence↗
PubMed1983

Regulatory pattern of the Escherichia coli lysA gene: expression of chromosomal lysA-lacZ fusions.

The regulation of lysA which encodes the last enzyme for lysine biosynthesis in Escherichia coli, diaminopimelic acid-decarboxylase, was studied by using lysA-lacZ fusions. Our results indicate an absolute requirement for the LysR product for its activation, LysR protein present in a limiting amount which can be titrated by a multicopy plasmid carrying its target site and a negative regulatory role for the LysA protein itself which decreases lysA-lacZ expression 30-fold.

Bacterial Proteins↗
PubMed1982

Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal.

The asd gene of escherichia coli encodes aspartic semialdehyde dehydrogenase, an enzyme involved in lysine, threonine, and methionine biosynthesis; its synthesis is controlled by a multivalent repression mechanism. It was cloned in plasmid pBR322 and its complete nucleotide sequence determined. The sequence predicts a polypeptide chain of 367 amino acids, in good agreement with results obtained for the purified protein ( Biellmann et al., 1980a ). Our data indicate a Cys residue instead of a His residue, which was proposed after covalent labeling of the active center of the enzyme; this is more in line with the catalytic site of glyceraldehyde-3-phosphate dehydrogenase, an enzyme which carries out a similar reaction. The nucleotide sequence that precedes the translational start does not display any of the characteristic features of an attenuation signal. Hence the expression of the asd gene is probably not controlled in the same way as other multivalently repressed operons such as ilva and thr.

Aspartate-Semialdehyde Dehydrogenase↗
PubMedDate not supplied

The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis.

The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus. This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step. The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library. Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene. After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine. The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature. The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation. Possible additional roles of FtsA during sporulation are discussed.

Bacillus subtilis↗
PubMedDate not supplied

A developmental gene product of Bacillus subtilis homologous to the sigma factor of Escherichia coli.

Sporulation of Bacillus subtilis involves sequential morphological and biochemical changes and is regulated by specific genes (spo genes) estimated to occupy more than 30 loci. A mutation in any one of these genes blocks the sporulation process at the corresponding developmental stage. Despite intensive genetic studies, the nature and function of the spo gene products remain unknown. Vegetative B. subtilis RNA polymerase core enzyme may interact with several sigma factors and discriminate among different classes of promoters. During sporulation, new polypeptides are associated with the core enzyme which may have a central role in modifying its promoter recognition specificity. As a first step to understanding their function in the switch from vegetative to sporulation mode, several early sporulation genes have been cloned and analysed. Here we report the cloning and nucleotide sequence of the spoIIG gene of B. subtilis. This gene encodes a polypeptide with a predicted relative molecular mass of 27,652 which contains a 65-amino acid region highly homologous to an internal part of the Escherichia coli sigma factor.

Amino Acid Sequence↗