Search PubMed⌕ Search

Biomedical subjects

A Danchin

Publications and source records attributed to A Danchin.

At least 127 records · Page 7Linked to original sources
PubMed1991

Escherichia coli molecular genetic map (1500 kbp): update II.

The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987) and modified by others. This second update adds a further 500 kbp, increasing the amount of the E. coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base. The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene. The location of rare-cutting sites such as AvrII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file. The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences. There appears to be a significant counter-selection against several such sequences in E. coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E. coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.

Base Sequence↗
PubMed1991

Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases.

The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.

Adenylyl Cyclases↗
PubMed1991

Positive regulation of the pts operon of Escherichia coli: genetic evidence for a signal transduction mechanism.

The pts operon of Escherichia coli is composed of the genes ptsH, ptsI, and crr, which code for three proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS): the HPr, enzyme I (EI), and EIIIGlc proteins, respectively. These three genes are organized in a complex operon in which the major part of expression of the distal gene, crr, is initiated from a promoter region within ptsI. Expression from the promoter region of the ptsH and ptsI genes has been studied in vivo by using gene fusions with lacZ. Transcription from this promoter region is under the positive control of catabolite activator protein (CAP)-cyclic AMP (cAMP) and is also enhanced during growth in the presence of glucose (a PTS substrate). This report describes a genetic characterization of the mechanism by which growth on glucose causes transcriptional stimulation of the pts operon. This regulation is dependent on transport through the glucose-specific permease of the PTS, EIIGlc. Our results strongly suggest that transcriptional regulation of the pts operon is the consequence of an increase in the level of unphosphorylated EIIGlc which is produced during glucose transport. Furthermore, overproduction of EIIGlc in the absence of transport was found to stimulate expression of the pts operon. We also observed that CAP-cAMP could cause stimulation independently of the EIIGlc and that glucose could activate in the absence of cAMP in a strain overproducing EIIGlc. Our results indicate that glucose acts like an environmental signal through a mechanism of signal transduction. A sequence similarity between the C terminus of EIIGlc and the consensus of transmitter modules of the sensor proteins defined by E. C. Kofoid and J. S. Parkinson (Proc. Natl. Acad. Sci. USA 85:4981-4985, 1988) suggests that EIIGlc might have properties in common with the sensors of the two-component systems.

Escherichia coli↗
PubMed1991

A gene encoding a tyrosine tRNA synthetase is located near sacS in Bacillus subtilis.

Within the frame of an attempt to sequence the whole Bacillus subtilis genome, a region of 5.5 kbp of the B. subtilis chromosome near the sacS locus has been sequenced. It contains five complete coding sequences, including the sequence of sacY, three unknown CDS and a sequence coding for a tyrosine tRNA synthetase. That the corresponding CDS encodes a functional synthetase has been demonstrated by complementation of an Escherichia coli mutant possessing a thermosensitive tRNA synthetase. Insertion of a kanamycin resistance cassette in the B. subtilis chromosome at the corresponding locus resulted, however, in no apparent phenotype, demonstrating that this synthetase is dispensable. Finally phylogenetic relationships between known tyrosine and tryptophan tRNA synthetases are discussed.

Amino Acid Sequence↗
PubMed1990

Binding of 3'-anthraniloyl-2'-deoxy-ATP to calmodulin-activated adenylate cyclase from Bordetella pertussis and Bacillus anthracis.

3'-Anthraniloyl-2'-deoxyadenosine 5'-triphosphate (Ant-dATP), a fluorescent analogue of ATP, was tested as a probe for the nucleotide-binding site of calmodulin (CaM)-activated adenylate cyclases from Bordetella pertussis (BPCYA47) and Bacillus anthracis (BACYA62). Ant-dATP competitively inhibited both bacterial enzymes expressed in Escherichia coli (ki approximately 10 microM). Binding of the analogue to adenylate cyclase was monitored by equilibrium dialysis and by an increase in its fluorescence emission at 420 nm upon excitation at 330 nm. Whereas the fluorescence of Ant-dATP was little influenced by divalent cations, CaM, or adenylate cyclase alone, the Ca2+.CaM.cyclase complex increased up to 4 times the quantum yield of Ant-dATP. Binding of the analogue to the catalytic site of BPCYA47 and BACYA62 was specific as shown by its displacement with ATP or 3'-dATP. Our results substantiate the role of CaM in favoring substrate binding to CaM-activated enzymes.

Adenosine Triphosphate↗
PubMed1990

Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli.

A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.

Adenylyl Cyclases↗
PubMed1990

Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon.

A series of isogenic strains harboring known deletions in the pts operon of Escherichia coli have been constructed by reverse genetics. Strains bearing deletions for the whole pts operon failed to grow on maltose or on carbon sources of the same class. In these strains the total cAMP synthesis was significantly lower than in a strain deleted only for the crr gene. This indicated that enzyme I or phosphorylated histidine-containing phosphotransferase protein in addition to its role in phosphorylating enzyme IIIGlc, is involved in adenylate cyclase (AC) activation or cAMP excretion. It was further shown that deletions in the pts operon do not affect synthesis of AC.

Adenylyl Cyclases↗
PubMed1990

Mutations in the bglY gene increase the frequency of spontaneous deletions in Escherichia coli K-12.

A strong mutator effect has been observed in Escherichia coli K-12 strains mutated in the bglY gene (27 min). The frequency of point mutations is not modified in bglY mutant strains. In contrast, a strong increase in spontaneous generation of large deletions has been observed in these strains, both for chromosomal markers (10-fold increase of galETK-chlA deletions and 100-fold increase of ptsI-cysK deletions) and for plasmid DNA (100-fold increase of large deletions in the region located upstream of the chloramphenicol-resistance gene in plasmid pGR71). bglY mutations are recessive and can be complemented by a DNA fragment of 900 base pairs assumed to contain the entire bglY wild-type gene. This mutator effect is recA-independent.

Chromosome Deletion↗
PubMed1990

Characterization of Escherichia coli adenylate cyclase mutants with modified regulation.

In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein. In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymeIII-glucose, a component of the phosphotransferase system. It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background. Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background. For three mutants the mutation was identified as a single point mutation. This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.

Adenylyl Cyclases↗
PubMed1990

Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome.

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.

Chromosome Inversion↗
PubMed1990

A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity.

A DNA fragment from Xanthomonas campestris pv. campestris that partially restored the carbohydrate fermentation pattern of a cya crp Escherichia coli strain was cloned and expressed in E. coli. The nucleotide sequence of this fragment revealed the presence of a 700-base-pair open reading frame that coded for a protein highly similar to the catabolite activation factor (CAP) of E. coli (accordingly named CLP for CAP-like protein). An X. campestris pv. campestris clp mutant was constructed by reverse genetics. This strain was not affected in the utilization of various carbon sources but had strongly reduced pathogenicity. Production of xanthan gum, pigment, and extracellular enzymes was either increased or decreased, suggesting that CLP plays a role in the regulation of phytopathogenicity.

Amino Acid Sequence↗
PubMed1990

Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases.

A gene from Rhizobium meliloti coding for an adenylate cyclase was sequenced, and the deduced protein sequence was compared with those of other known adenylate cyclases. No similarity could be detected with the procaryotic counterparts. However, striking similarity was found with the catalytic region of Saccharomyces cerevisiae adenylate cyclase, the cytoplasmic domains of bovine adenylate cyclase, and two mammalian guanylate cyclases. The gene was fused to the enteric beta-galactosidase, and the chimeric protein was purified by affinity chromatography. This fusion protein was found to direct the synthesis of cyclic AMP in vitro. This activity was strongly inhibited by the presence of GTP, but no cyclic GMP synthesis could be detected in conditions permitting cyclic AMP synthesis.

Adenylyl Cyclases↗
PubMed1990

Escherichia coli molecular genetic map (1000 kbp): update I.

The sequenced genes from Escherichia coli that are available in the EMBL library (release 21) have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987). One thousand kbp of sequenced DNA are incorporated in this update; this is equivalent to 23% of the total genome. The accuracy of the map is assessed, and it is corrected and updated where appropriate. A significant number of sites were missing from the original map, mainly involving two of the eight enzymes used by Kohara et al. (1987), ie. PvuII and EcoRV. The nucleotide environment of such missing sites was examined and, using an Artificial Intelligence approach, it appears that the site for these enzymes is sensitive to context effects. Several genes of known position on the E. coli chromosome could not be placed on the restriction map; this suggests that additional gaps are likely to exist on the restriction map, in addition to the original seven identified by Kohara et al. We have also obtained information about the probable direction of transcription of chromosomal genes with respect to the map. Most genes are transcribed in the same direction as the replication forks, particularly around oriC at 84 min.

Artificial Intelligence↗
PubMed1989

Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli.

Cloning of higher eukaryotic genes has seldom been performed by complementation of a defective prokaryotic function. This is especially true in the case of functions that are normally absent from the prokaryotic host. We demonstrate here that it is possible to identify by complementation the cDNA from mouse brain, which encodes calmodulin (CaM) synthesis, in spite of the fact that the recipient strain, Escherichia coli, does not normally harbour a CaM function. A three-component cloning procedure in which a gene product requiring CaM for activity, adenylate cyclase from the pathogen Bordetella pertussis, was used to screen a cDNA library for cAMP synthesis in E. coli. The nucleotide sequence of the corresponding cDNA is also reported.

Adenylyl Cyclases↗
PubMed1989

Identification of a common domain in calmodulin-activated eukaryotic and bacterial adenylate cyclases.

Bordetella pertussis and Bacillus anthracis, two taxonomically distinct bacteria, secrete adenylate cyclase toxins that are activated by the eukaryotic protein calmodulin. The two enzymes contain a well-conserved stretch of 24 amino acid residues [Escuyer et al. (1988) Gene 71, 293-298]. Antibodies have been obtained against two synthetic heptadecapeptides, covering part of the conserved sequences. The anti-peptide antibodies specifically reacted in Western blots with the rat brain adenylate cyclase as well as with the two bacterial enzymes. Anti-rat brain adenylate cyclase serum contained antibodies that were retained by the immobilized peptides, and the affinity-purified antibodies yielded the same recognition pattern of the eukaryotic enzyme as did the unfractionated serum. These results indicate that the eukaryotic adenylate cyclase contains an epitope closely related to that specified by the conserved bacterial sequence. The synthetic peptides and the bacterial adenylate cyclases appeared to compete for ATP (KD of the ATP-peptide complex ca. 0.2 mM), suggesting that the conserved sequence may be part of the substrate binding site in these two enzymes.

Adenylyl Cyclases↗
PubMed1989

Antisense expression at the ptsH-ptsI locus of Escherichia coli.

A Mud(Ap, lac) prophage has been shown to be inserted into the ptsH gene of E. coli. The insertion is likely to have generated an operon fusion revealing antisense transcription at this locus. This suggests that the ORF previously identified, overlapping with ptsH ORF in the opposite orientation, might be functional. A model of regulation for the ptsH-ptsI-crr operon transcription is presented which accounts for antisense transcription.

Cloning, Molecular↗
PubMed1989

Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.

Adenylyl Cyclases↗