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A Danchin

Publications and source records attributed to A Danchin.

At least 19 recordsLinked to original sources
PubMed1996

Conformational transitions within the calmodulin-binding site of Bordetella pertussis adenylate cyclase studied by time-resolved fluorescence of Trp242 and circular dichroism.

The sequence situated around Trp242 in Bordetella pertussis adenylate cyclase, a bifunctional protein of 1706 amino acid residues, forms the core of the calmodulin-binding site. Peptides varying in size and in affinity for calmodulin, and preserving the same sequence around Trp242 were analyzed by time-resolved fluorescence spectroscopy. Their dynamic properties were compared to those of the catalytic domain of B. pertussis adenylate cyclase corresponding to the first 400 amino acid residues of the protein and in which the Trp69 residue was replaced by Phe. The heterogeneity of the fluorescence intensity decays of Trp242 is likely due to the existence of conformers in equilibrium as is suggested by the effect of trifluoroethanol both on the secondary structure content and the lifetime distributions. Binding to calmodulin leads to striking effects on the lifetime distribution profiles by selecting a major excited state population and therefore one major conformer. Trp242 still presents some degree of rotational freedom in the complexes. The reduction of rotational freedom is more important for the shorter peptides than for the longest one. A similar selection of one major conformer with the same lifetime was also observed for the Trp242 in the mutant protein when bound to calmodulin, as in the complexes with the peptides. We conclude that the site of interaction of B. pertussis adenylate cyclase with calmodulin has similar conformational flexibility as that evidenced in the isolated peptides. This property of the molecule allows a better adjustment of the enzyme upon interaction with calmodulin.

Adenylyl Cyclases↗
PubMed1996

Uneven distribution of GATC motifs in the Escherichia coli chromosome, its plasmids and its phages.

This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coli genome, compared to its distribution in phages and plasmids. At first sight the distribution of GATC words looks random. But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromasome), is used as a theoretical reference, strong biasesare observed. GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp. The last class is the only one present in E. coli parasites. It can be ascribed to the triggering sequences of the long-patch mismatch repair system. The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection. The other classes, which could be targets for a nucleic acid-binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs. Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions. In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions. In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift.

Bacteriophages↗
PubMed1996

CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

Amino Acid Sequence↗
PubMed1996

Comparative analysis of the cya locus in enterobacteria and related gram-negative facultative anaerobes.

Comparison of the cya loci (cya codes for adenylyl cyclase (AC)) from a variety of phylogenetically divergent facultative anaerobic Gram-negative bacteria reveals conserved sequence features. The entire locus structure in enterobacteria is preserved, including two major promoters (a conserved cya strong promoter, P2, and a divergent promoter for a heme biosynthetic operon, hemCD) present in the upstream region of the cya gene. The region between hemC and cya is much longer in Proteus mirabilis than in other enterobacteria, and lacks the P1 upstream cya promoter. In Aeromonas hydrophila the cya promoter (the strong P2 promoter in E coli) is preserved, including a putative GATC methylation site situated immediately downstream from the -10 box. Each cya frame analyzed uses TTG as the translation start codon and is preceded by an unusual ribosome binding site. This suggests that a lower translation efficiency of the cya transcript could be the result of some selection pressure. This has been substantiated by in vitro mutagenesis and by selection of up mutations which all map at the cya ribosome binding site. In enterobacteria the cyaY frame is the only conserved reading frame downstream of cya, with the orientation opposite to that of cya. This organization is not preserved in Aeromonas. Experiments involving fusions with the lacZ gene demonstrated that cyaY is expressed. Finally, comparison of the different polypeptide sequences of ACs permits discussion of important features of the catalytic and regulatory centers of the protein.

Adenylyl Cyclases↗
PubMed1996

Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis.

Bacillus subtilis can grow anaerobically in the presence of nitrate as a terminal electron acceptor. The two component regulatory proteins, ResD and ResE, and an anaerobic gene regulator, FNR, were previously shown to be indispensable for nitrate respiration in B. subtilis. Unlike Escherichia coli fnr, B. subtilis fnr transcription was shown to be highly induced by oxygen limitation. fnr is transcribed from its own promoter as well as from a promoter located upstream of narK, the first gene in the narK-fnr dicistronic operon. DNA fragments containing the narK promoter, the fnr promoter, and both of the promoters were used to construct three lacZ fusions to examine the transcriptional regulation of the narK-fnr operon. ResDE was found to be required for transcriptional activation of fnr from the fnr-specific promoter, and FNR was required for activation of narK-fnr transcription from the FNR-dependent narK operon promoter under anaerobiosis. In order to determine if the requirement for ResDE in nitrate respiration is solely to activate fnr transcription, fnr was placed under control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter, Pspac. The observed defect in anaerobic growth of a Pspac-fnr delta resDE mutant in the presence of IPTG indicated that resDE has an additional role in B. subtilis anaerobic gene regulation.

Anaerobiosis↗
PubMed1995

Anaerobic transcription activation in Bacillus subtilis: identification of distinct FNR-dependent and -independent regulatory mechanisms.

Bacillus subtilis is able to grow anaerobically using alternative electron acceptors, including nitrate or fumarate. We characterized an operon encoding the dissimilatory nitrate reductase subunits homologous to the Escherichia coli narGHJI operon and the narK gene encoding a protein with nitrite extrusion activity. Downstream from narK and co-transcribed with it a gene (fnr) encoding a protein homologous to E.coli FNR was found. Disruption of fnr abolished both nitrate and fumarate utilization as electron acceptors and anaerobic induction of narK. Four putative FNR binding sites were found in B.subtilis sequences. The consensus sequence, centred at position -41.5, is identical to the consensus for the DNA site for E.coli CAP. Bs-FNR contained a four cysteine residue cluster at its C-terminal end. This is in contrast to Ec-FNR, where a similar cluster is present at the N-terminal end. It is possible that oxygen modulates the activity of both activators by a similar mechanism involving iron. Unlike in E.coli, where fnr expression is weakly repressed by anaerobiosis, fnr gene expression in B.subtilis is strongly activated by anaerobiosis. We have identified in the narK-fnr intergenic region a promotor activated by anaerobiosis independently of FNR. Thus induction of genes involved in anaerobic respiration requires in B.subtilis at least two levels of regulation: activation of fnr transcription and activation of FNR to induce transcription of FNR-dependent promoters.

Amino Acid Sequence↗
PubMed1995

Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement.

Using the high efficiency of homologous gene recombination in Bacillus subtilis, a strategy for mutational analysis of the proton pumping aa3-600 quinol oxidase of this organism has been developed. The qox operon with the qoxA, qoxB, qoxC and qoxD genes, coding for the four subunits of this oxidase, was deleted and then replaced with mutated copies in which qoxC (subunit III) or qoxD (subunit IV) genes were deleted. The complete deletion of the qox operon caused disappearance of heme aa3-600 and a slight depression of the overall respiratory activity, compensated by alternative oxidase with no proton pumping activity. Deletion of qoxC probably resulted in a defective assembly of the aa3-600 quinol oxidase. The strain with deletion of qoxD gene expressed normal content of heme aa3-600 but exhibited a reduced respiratory activity and a significantly depressed proton pumping activity. These results show that subunit IV is critical for the activity of the proton pumping aa3-600 quinol oxidase.

Bacillus subtilis↗
PubMed1995

Analysis of a Bacillus subtilis genome fragment using a co-operative computer system prototype.

Analysis of the huge volume of data generated by large scale sequencing projects requires the construction of new, sophisticated computer systems. These systems should be able to manage the biological data as well as the results of their analysis. They should also help the user to choose the most appropriate methods, and to string them together in order to solve a global analysis task. In this paper we present the prototype of a software system providing an environment for the analysis of large-scale sequence data. As a first step toward this end, this environment has been put to the test within the Bacillus subtilis genome sequencing project. This system integrates both the descriptive knowledge of the entities involved (genes, regulatory signals and the like) and the methodological knowledge comprising an extensible set of analytical methods. A knowledge representation based on two existing object-oriented models is used to implement this integrated system. In addition, the present prototype provides a suitable user interface both for displaying simultaneously the results generated by several methods and for interacting with the objects. We present in this paper the analysis of a B. subtilis genome fragment, present in data libraries but not annotated. Annotation of the genes present in the fragment allowed us to combine the results of several methods used for predicting coding sequences, and to characterize it as comprising a cryptic phage, the skin element. Comparison between the annotation of the skin element and a standard region of the chromosome indicated that local features of the nucleotide sequence could discriminate between phage and non-phage DNA sequence.

Bacillus subtilis↗
PubMed1995

Detection of new genes in a bacterial genome using Markov models for three gene classes.

We further investigated the statistical features of the three classes of Escherichia coli genes that have been previously delineated by factorial correspondence analysis and dynamic clustering methods. A phased Markov model for a nucleotide sequence of each gene class was developed and employed for gene prediction using the GeneMark program. The protein-coding region prediction accuracy was determined for class-specific Markov models of different orders when the programs implementing these models were applied to gene sequences from the same or other classes. It is shown that at least two training sets and two program versions derived for different classes of E. coli genes are necessary in order to achieve a high accuracy of coding region prediction for uncharacterized sequences. Some annotated E. coli genes from Class I and Class III are shown to be spurious, whereas many open reading frames (ORFs) that have not been annotated in GenBank as genes are predicted to encode proteins. The amino acid sequences of the putative products of these ORFs initially did not show similarity to already known proteins. However, conserved regions have been identified in several of them by screening the latest entries in protein sequence databases and applying methods for motif search, while some other of these new genes have been identified in independent experiments.

Algorithms↗
PubMed1995

Cloning and sequence of the Bordetella bronchiseptica adenylate cyclase-hemolysin-encoding gene: comparison with the Bordetella pertussis gene.

The cyaA gene from Bordetella bronchiseptica (Bb), encoding the adenylate cyclase-hemolysin (AC-Hly), has been cloned and its complete nucleotide sequence has been determined. The deduced amino-acid sequence was compared to the AC-Hly from B. pertussis (Bp) and the main differences were found in the C-terminal repeat region of the molecule.

Adenylate Cyclase Toxin↗
PubMed1995

Cloning of the second adenylate cyclase gene (cya2) from Rhizobium meliloti F34: sequence similarity to eukaryotic cyclases.

A second adenylate cyclase (cya2) gene was isolated from a Rhizobium meliloti F34 gene bank. Complemented E. coli delta cya mutants were capable of utilizing a number of, but not all, carbon sources known to be regulated by cAMP. DNA hybridization studies showed cya2 to be unique to R. meliloti strains. The cya2 nucleotide sequence was determined and found to encode a protein of 363 amino acids. Residues were identified within the C-terminal domain which are conserved in both eukaryotic adenylate and guanylate cyclases, including a putative ATP binding site. Similar residues were also found in the prokaryotic R. meliloti Cya1 protein. A R. meliloti cya1/cya2 double mutant was constructed and characterized; however, cAMP production was still observed in this strain indicating the presence of a third cya gene.

Adenylyl Cyclases↗
PubMed1995

Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP.

The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The Escherichia coli DNA-binding protein H-NS is one of the first proteins to be synthesized after a nutritional upshift.

Using two-dimensional electrophoresis, the patterns of polypeptide expression of a wild type and an hns mutant of Escherichia coli were examined in exponential and late stationary phases. The same procedure was used for a kinetic study of proteins synthesized during the first 60 min following inoculation into fresh complete medium. The present study focusses on 35 polypeptides differentially synthesized according to the strain and/or the growth phase. One of the most striking and unexpected observations in this work was a strong and transient synthesis of H-NS in the wild-type strain just after the nutritional upshift.

Autoradiography↗
PubMed1995

SubtiList: a relational database for the Bacillus subtilis genome.

In the framework of the international collaborative project aiming to sequence the whole Bacillus subtilis chromosome, we have created a relational database for managing and analysing information associated with the molecular genetics of this bacterium: SubtiList. It allows recovery of non-redundant DNA sequences of the B. subtilis genome, as well as related information, i.e. genes, proteins, etc. A logical structure has been designed with appropriate links between the different objects, and a set of procedures has been implemented for data updating and management. The database is organized around a core constituted by all known contigs of B. subtilis, i.e. sets of non-redundant sequences created from original entries in the EMBL data library. A user-friendly interface has been developed to make the database easy to consult. Sequence analysis tools have been integrated into the database, such as a program for rapid similarity searching of protein data banks, and a powerful DNA pattern searching program. Thanks to the consistency of SubtiList, we have performed a codon usage analysis by Factorial Correspondence Analysis, and a study of the distribution of the isoelectric points of known proteins of B. subtilis. The SubtiList database is available through anonymous ftp (address 'ftp.pasteur.fr' or IP number 157.99.64.12, directory '/pub/GenomeDB/SubtiList').

Algorithms↗