Search PubMed⌕ Search

Biomedical subjects

A Danchin

Publications and source records attributed to A Danchin.

At least 145 records · Page 8Linked to original sources
PubMed1989

A locus involved in kanamycin, chloramphenicol and L-serine resistance is located in the bglY-galU region of the Escherichia coli K12 chromosome.

Spontaneous mutants of Escherichia coli K12 displaying an increased level of the kanamycin resistance conferred by plasmid pGR71 were selected. Several mutants obtained in this way apparently carry large chromosomal deletions extending into galU and/or bglY (27 min). This positive selection of deletions allowed detection of a new locus located between galU and bglY. Deletions of this locus are responsible for increased resistance to kanamycin (Irk), decreased resistance to L-serine in minimal medium (Drs) and decreased resistance to chloramphenicol (Drc) when a cat gene is present in the bacteria.

Chloramphenicol Resistance↗
PubMed1989

Molecular characterization of two cya mutations, cya-854 and cyaR1.

The cya-854 mutation of Escherichia coli K-12 (E. Brickman, L. Soll, and J. Beckwith, J. Bacteriol. 116:582-587, 1973) has been shown to consist of a 200-base-pair deletion generating a new translation start that permits synthesis of the adenylate cyclase carboxy terminus. Contrary to expectations (C. Guidi-Rontani, A. Danchin, and A. Ullmann, J. Bacteriol. 148:753-761, 1981), cyaR1 results from the insertion of an IS1 element into the sequence coding for the catalytic center of the protein.

Adenylyl Cyclases↗
PubMed1988

Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis, a 45 kd secreted protein, is synthesized as a 1706 amino acid precursor. We have shown that this precursor is a bifunctional protein, carrying both adenylate cyclase and haemolytic activities. The 1250 carboxy-terminal amino acids of the precursor showed 25% similarity with Escherichia coli alpha-haemolysin (HlyA) and 22% similarity with Pasteurella haemolytica leucotoxin. Three open reading frames were identified downstream from the cyaA gene: cyaB, cyaD and cyaE, coding for polypeptides of 712, 440 and 474 amino acid residues, respectively. As for E. coli alpha-haemolysin, secretion of B.pertussis adenylate cyclase and haemolysin requires the expression of additional genes. The gene products of cyaB and cyaD are highly similar to HlyB and HlyD, known to be necessary for the transport of HlyA across the cell envelope and for its release into the external medium. Complementation and functional studies indicate that the B.pertussis adenylate cyclase-haemolysin bifunctional protein is secreted by a mechanism similar to that described for E.coli alpha-haemolysin, requiring, in addition to the cyaB and cyaD gene products, the presence of a third gene product specified by the cyaE gene.

Adenylate Cyclase Toxin↗
PubMed1988

Structural homology between virulence-associated bacterial adenylate cyclases.

The primary structure of the calmodulin-sensitive adenylate cyclase toxin from Bacillus anthracis has been determined from the corresponding nucleotide sequence and compared to that of the homologous toxin secreted by Bordetella pertussis. The cya gene of Bacillus anthracis encodes an 800 amino acid (aa) protein beginning with an N-terminal signal peptide. The central part of the B. anthracis adenylate cyclase includes a region of striking homology with the N-terminal part of the B. pertussis enzyme. In this region a particularly well conserved 24-aa peptide and two other less homologous peptides have been identified. These data corroborate the immunological relatedness of the two enzymes and suggest that the two prokaryotic calmodulin-sensitive adenylate cyclases originate from a common ancestor.

Adenylyl Cyclases↗
PubMed1988

Cloning and expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in Escherichia coli.

The adenylate cyclase gene of Bacillus anthracis, encoding the edema factor, a component of anthrax toxin, has been cloned and expressed in Escherichia coli. Clones were selected by their capacity to complement the cyclase deficiency (cya-) of an E. coli strain expressing the eukaryotic protein calmodulin, an essential activator of B. anthracis adenylate cyclase. The protein expressed in E. coli was shown to exhibit adenylate cyclase activity only in the presence of calmodulin. Experiments using a coupled in vitro transcription-translation system revealed that the protein synthesized from the cloned DNA fragment was enzymatically active, upon addition of calmodulin, and could be immunoprecipitated by antibodies directed against purified Bordetella pertussis adenylate cyclase toxin. This indicates that the two calmodulin-dependent adenylate cyclase toxins are immunologically related.

Animals↗
PubMed1988

Aspects of the regulation of adenylate cyclase synthesis in Escherichia coli K12.

In Escherichia coli K12 expression of the adenylate cyclase gene is subject to multiple controls. In order to gain understanding of the regulation of adenylate cyclase synthesis, operon and protein fusions were constructed by in vitro recombination either into bacteriophage lambda or low-copy-number plasmids, or directly on the chromosome at the cya locus. The fusions were used in physiological experiments as probes to study transcriptional and translational controls of cya expression. It was found that adenylate cyclase synthesis was insensitive to glucose effects. As already described by other workers, the CAP-cAMP complex had a moderate negative control on cya expression. In addition it was observed that concomitant with a severe slackening of growth rate, specific to the growth of cya strains in rich medium, cya expression was considerably enhanced. This increase of adenylate cyclase synthesis did not appear to be directly dependent on the presence of a functional cAMP receptor (CAP), and seemed to be controlled at the level of transcription. Finally, translation of the cya message was very weak when compared to cya transcription (the mRNA level was the same in protein and operon fusions.

Adenylyl Cyclases↗
PubMed1988

Phylogeny of metabolic pathways: O-acetylserine sulphydrylase A is homologous to the tryptophan synthase beta subunit.

The cysK gene of Escherichia coli K-12 encoding O-acetylserine sulphydrylase A, was cloned and its nucleotide sequence, together with that of the flanking regions, was determined. The deduced amino acid sequence of the carboxy-terminal moiety of O-acetylserine sulphydrylase A shows significant similarity to the amino acid sequence of tryptophan synthase beta chain from several organisms. This sequence similarity is likely to reflect the structural homologies of substrates shared by both enzymes. This may indicate that these proteins, although catalysing different reactions in different metabolic pathways, have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1988

The ptsH, ptsI, and crr genes of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: a complex operon with several modes of transcription.

The ptsH, ptsI, and crr genes, coding for three of the proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (HPr, enzyme I, and enzyme IIIGlc, respectively) have been studied by determination of their nucleotide sequence and analysis of their expression. The three genes constitute an operon, but analysis of the ptsH, ptsI, and crr transcripts by Northern (RNA) blotting revealed the existence of three major mRNA species. One encompassed the three cistrons, a second one the ptsH gene and part of the ptsI gene, and the third one only the distal gene crr. The short crr transcripts were initiated inside the ptsI open reading frame at points which were identified by S1 mapping. Expression of the genes was studied in vivo by using operon and protein fusions between the lacZ gene and the ptsH, ptsI, or crr gene on IncW low-copy-number plasmids. The present study showed that (i) the ptsH, ptsI, and crr genes exhibited high basal expression, (ii) transcription of the ptsH and ptsI genes was stimulated threefold by the cyclic AMP-cyclic AMP receptor protein complex and also by growth on glucose, but only in the presence of an active enzyme IIGlc, (iii) crr-specific expression was not sensitive to the complex or to growth on glucose, and (iv) under the growth conditions tested, the major part of crr transcription was initiated from internal promoters.

Amino Acid Sequence↗
PubMed1988

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli.

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.

Adenylate Cyclase Toxin↗
PubMed1988

Bordetella pertussis adenylate cyclase: the gene and the protein.

Using the adenylate cyclase-calmodulin interaction as a tool, the B. pertussis cya gene was cloned in a cya defective E. coli strain harbouring a plasmid which expressed high levels of calmodulin. The determination of the nucleotide sequence of the gene showed that adenylate cyclase is synthesized as a large precursor of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 400 amino acids whereas the 1300 amino acid carboxy-terminal part of the precursor is endowed with haemolytic activity. The catalytically active 43 kDa form of adenylate cyclase is organized in two domains: the N-terminal domain of 25 kDa harbors the catalytic site, and the 18 kDa C-terminal domain carries the main calmodulin-binding site. Immunological relatedness established between B. pertussis, B. anthracis and rat brain adenylate cyclases suggests a common evolutionary origin of a central domain of these calmodulin-stimulated enzymes. The secretion of the adenylate cyclase-haemolysin bifunctional protein (cyclolysin) requires the expression of three additional genes, contiguous to the cya gene. These four genes appear to form a single operon. The mechanism of secretion of the bifunctional protein should be similar to that described for E. coli alpha-haemolysin.

Adenylate Cyclase Toxin↗
PubMed1988

Structure and evolution of bacterial adenylate cyclase: comparison between Escherichia coli and Erwinia chrysanthemi.

The cya genes, coding for adenylate cyclase, from Escherichia coli and Erwinia chrysanthemi B374 are compared after determination of a 3632 bp long nucleotide sequence of the hemC-cya region of E. chrysanthemi, encompassing the whole cya gene. In spite of a large divergence between the two organisms, especially visible in non coding regions, the amino acid sequence of the proteins are very similar, except at the very distal carboxyl end. Codon usage is different in the two organisms, and E. chrysanthemi tends to restrict translation to codons ending in G or C. Conservation of the translation initiation start region (including the poor ribosome binding site GGCG, and the TTG start codon), suggests that a specific protein synthesis process controls adenylate cyclase expression. Finally a palindromic unit, of primary sequence differing from the E. coli counterpart, borders the gene in E. chrysanthemi.

Adenylyl Cyclases↗
PubMed1987

Structural and catalytic characteristics of Escherichia coli adenylate kinase.

The adk gene encoding adenylate kinase in Escherichia coli was cloned in pBR322. Adenylate kinase represented about 4% of total proteins in extracts of cells containing the pBR322:adk plasmid. This allowed preparation of more than 90% pure enzyme in a single-step purification procedure. Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the N-terminal sequence of the whole protein confirmed the primary structure of E. coli adenylate kinase predicted from the nucleotide sequence of the adk gene (Brune, M., Schumann, R., and Wittinghofer, F. (1985) Nucleic Acids Res. 13, 7139-7151). 2-Nitro-5-thiocyanatobenzoic acid reacted with the single cysteine residue of E. coli adenylate kinase. The cyanylated protein was cleaved upon exposure to alkaline pH, yielding two peptides corresponding to residues 1-76 and 77-214, respectively. A mixture of purified peptides tended to reassociate, recovering both catalytic activity and binding properties for adenine nucleotides. E. coli adenylate kinase has a broader specificity for nucleoside monophosphates than does the mammalian enzyme. In addition to 2'-dAMP, other nucleoside monophosphates such as 3'-dAMP, adenine-9-beta-D-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate.

Adenylate Kinase↗
PubMed1987

Cloning and characterization of the pH 2.5 acid phosphatase gene, appA: cyclic AMP mediated negative regulation.

The structural gene for an acid phosphatase coded for by the gene appA of Escherichia coli K12 was cloned from a cosmid library into pBR322 and the restriction map determined. Several appA deletion plasmids and a smaller appA+ plasmid were constructed by in vitro recombination techniques and tested for their ability to complement an appA1 mutation. The appA gene was localized within a 2.1 kb segment. Its orientation was determined by construction of a hybrid plasmid carrying an appA-lacZ fusion. beta-galactosidase synthesized from the appA promoter was negatively regulated by cyclic AMP.

Acid Phosphatase↗
PubMed1987

The structure of the promoter and amino terminal region of the pH 2.5 acid phosphatase structural gene (appA) of E. coli: a negative control of transcription mediated by cyclic AMP.

The regulatory and promoter regions of the gene for acid phosphatase of optimum pH 2.5 (appA) of Escherichia coli has been characterized by constructing appA-lacZ protein fusions. Monitoring of beta-galactosidase activity showed that the cloned fragment harbored a region that was responsible for a negative control of transcription mediated by the cAMP-cAMP receptor (CAP) complex. The nucleotide sequence of the segment was determined. A region containing a putative CAP binding site, overlapping the -10 region of the promoter was found. Deletion analysis around the promoter region, using appA-lacZ protein fusions substantiated the hypothesis for a role of the 'consensus' sequence in the negative control mediated by cyclic AMP. In addition, the coding sequence revealed the likely existence of a signal peptide similar to the one found for other exported proteins, upstream from the phosphatase protein sequence.

Acid Phosphatase↗
PubMed1987

Nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti.

The nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti has been determined. Its organization includes a short transcript with a promoter-like structure and two Rho-independent terminators about 10 bp downstream from the cloverleaf structure. The putative promoter shows good homology with the -45 and -35 region of the consensus (sigma 70) promoter sequence of Escherichia coli, but less with the -10 region. A discriminator-like sequence is present between the start of the transcript and the -10 region. The gene shows extensive homology to tRNA(leu)CAG genes from Gram-positive and Gram-negative bacteria. However, significant differences are encountered in the variable loop region. This is the first report of a tRNA sequence from R. meliloti.

Base Sequence↗