Biomedical subjects

A Danchin

Publications and source records attributed to A Danchin.

At least 19 recordsLinked to original sources

PubMed1992

Positive regulation of the expression of the Escherichia coli pts operon. Identification of the regulatory regions.

The pts operon of Escherichia coli is composed of the ptsH, ptsI and crr genes coding for three proteins central to the phosphoenolpyruvate dependent phosphotransferase system (PTS), the HPr, enzyme I and EIIIGlc proteins, respectively. We previously showed that transcription from the promoter region located upstream from the pts operon is regulated by two control circuits, which can occur independently from each other. Transcription of the pts operon is (1) stimulated by the CAP-cAMP complex and (2) enhanced during growth on glucose, a PTS substrate. The DNA regions involved in regulation of the expression of the pts operon have been identified. Two promoters, P0 and P1, separated by 100 bp are located upstream from the pts operon. In these promoter regions, we identified two sequences showing similarity with the consensus of CAP-binding sites, CAPa located near P0 and CAPb located in the -35 region of P1. In vivo experiments showed that binding of CAP-cAMP at the CAPa site stimulates transcription from the P0 promoter. The binding sites of CAP-cAMP and/or RNA-polymerase on a DNA fragment containing both P0 and P1 promoters as well as both CAPa and CAPb sites were examined by the technique of DNase I footprinting. These in vitro experiments suggested that CAP-cAMP binding at the CAPb site might also play a role in regulation of the pts operon expression. In addition, we showed that the DNA region carrying the CAPa site is important for regulation by glucose. We finally propose that the expression of the pts operon is controlled by two alternative positive regulatory mechanisms, which are designed to allow activation of the pts operon under a great variety of growth conditions.

Amino Acid Sequence↗

PubMed1992

From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity.

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.

Adenylyl Cyclases↗

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

The role of histidine 63 in the catalytic mechanism of Bordetella pertussis adenylate cyclase.

Of the 9 histidines located in the catalytic domain of Bordetella pertussis adenylate cyclase, three (His63, His106, and His298) were found to be conserved in the adenylate cyclase of Bacillus anthracis, another calmodulin-dependent enzyme. Substitution of His63 with Arg, Glu, Gln, or Val decreased the catalytic efficiency of adenylate cyclase between 2 and 3 orders of magnitude and altered the kinetic properties of the enzyme. These effects varied in relation to the nature of the substituting residue, pH, and direction of the reaction, i.e. ATP cyclization (forward) or ATP synthesis (reverse). Arg was the best substituent for His63 as catalyst in the forward reaction, with shift of the optimum pH to the alkaline side, whereas Glu was the best substituent for His63 in the reverse reaction, with shift of the optimum pH to the acidic side. Diethyl pyrocarbonate, which had a deleterious effect on wild-type adenylate cyclase was ineffective on His63 mutants. From these results we conclude that His63 is involved in the reaction mechanism of adenylate cyclase, which requires a general acid/base catalyst, most probably as an intermediate in a charge-relay system.

Adenylyl Cyclases↗

PubMed1992

Mutations in bglY, the structural gene for the DNA-binding protein H1 of Escherichia coli, increase the expression of the kanamycin resistance gene carried by plasmid pGR71.

bglY mutants of Escherichia coli K12 which show higher levels of kanamycin resistance (Kmr) in the presence of plasmid pGR71 have been previously described. In this work, we show that this increased resistance to an aminoglycoside antibiotic is not due either to low drug uptake or to alteration of its target, the ribosome. The copy number of plasmid pGR71 is not modified. The fact that increased antibiotic resistance is observed with only some of the Kmr determinants used in this study suggests a specific role for the bglY gene product. Moreover, for one such determinant, a higher level of resistance was observed when it was inserted in the chromosome but not when harbored by a plasmid. This discrepancy can be explained by the twin transcriptional-loop model, which proposes that transcription can lead to local variation in topology. A kan-lacZ fusion was constructed from the Kmr gene of plasmid pGR71 and inserted into a low copy number vector. Assay of beta-galactosidase in wild-type and mutant strains showed that expression of the antibiotic resistance gene was directly affected by H1 protein, the bglY gene product.

Aminoglycosides↗

PubMed1991

Evidence for horizontal gene transfer in Escherichia coli speciation.

After extracting more than 780 identified Escherichia coli genes from available data libraries, we investigated the codon usage of the corresponding coding sequences and extended the study of gene classes, thus obtained, to the nature and intensity of short nucleotide sequence selection, related to constraints operating at the nucleotide level. Using Factorial Correspondence Analysis we found that three classes ought to be included in order to match all data now available. The first two classes, as known, encompass genes expressed either continuously at a high level, or at a low level and/or rarely; the third class consists of genes corresponding to surface elements of the cell, genes coming from mobile elements as well as genes resulting in a high fidelity of DNA replication. This suggests that bacterial strains cultivated in the laboratory have been fixed by specific use of antimutator genes that are horizontally exchanged.

Amino Acids↗

PubMed1991

Fructose catabolism in Xanthomonas campestris pv. campestris. Sequence of the PTS operon, characterization of the fructose-specific enzymes.

In Xanthomonas campestris pv. campestris, fructose is transported and phosphorylated into fructose 1-phosphate through a phosphoenolpyruvate-dependent phosphotransferase system. The nucleotide sequence of the fruA gene encoding the phosphotransferase system permease specific of fructose (EIIFru) was determined. The fructose 1-phosphate produced by the phosphotransferase system is phosphorylated into fructose 1,6-bisphosphate by a 1-phosphofructokinase. This enzyme was characterized and the corresponding gene (fruK) was sequenced. Sequence comparisons revealed that FruK is a member of a new family of ATP-binding proteins composed of sugar (or sugar-phosphate) kinases. In phosphotransferase system-deficient strains, fructose can still be transported by an unidentified permease. The intracellular fructose is then phosphorylated by a multimeric fructokinase of 135 kDa specific for fructose and inhibited by fructose, fructose 1,6-bisphosphate, and mannose. Several other enzymes of fructose metabolism were assayed and a potential pathway for fructose catabolism is presented.

Amino Acid Sequence↗

PubMed1991

Isolation and characterization of catalytic and calmodulin-binding domains of Bordetella pertussis adenylate cyclase.

A truncated Bordetella pertussis cya gene product was expressed in Escherichia coli and purified by affinity chromatography on calmodulin-agarose. Trypsin cleavage of the 432-residue recombinant protein (Mr = 46,659) generated two fragments of 28 kDa and 19 kDa. These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin-binding properties. The 28-kDa peptide, corresponding to the N-terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein. The 19-kDa peptide, corresponding to the C-terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl-calmodulin. T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure. The two fragments reassociated into a highly active calmodulin-dependent species. Reassociation was enhanced by calmodulin itself, which 'trapped' the two complementary peptides into a stable, native-like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.

Adenylyl Cyclases↗

PubMed1991

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.

Adenosine Triphosphate↗

PubMed1991

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Fructose was shown to be phosphorylated by a specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose EII. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.

Amino Acid Sequence↗

PubMed1991

Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12.

In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.

Amino Acid Sequence↗

PubMed1991

A pyruvate-stimulated adenylate cyclase has a sequence related to the fes/fps oncogenes and to eukaryotic cyclases.

The pyruvate-stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450 microM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C-terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N-terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.

Adenylyl Cyclases↗

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↗