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A Danchin

Publications and source records attributed to A Danchin.

At least 73 records · Page 4Linked to original sources
PubMed1997

Comparison between the Escherichia coli and Bacillus subtilis genomes suggests that a major function of polynucleotide phosphorylase is to synthesize CDP.

Genome comparison permits identification of chromosome regions conserved during evolution. Bacillus subtilis and Escherichia coli are so distant that there exists very few conserved landmarks in their genome organisation. Analysis of the conserved cmk rpsA cluster pinpointed the importance of cytosine nucleotide metabolism. In these bacteria, mRNA turnover provides an efficient means to fulfil the need for CDP as a precursor of DNA synthesis. The cmk rpsA operon is responsible for CDP synthesis. This function is self-explained in the case of the cmk gene (which codes for cytidylate kinase). The case of rpsA, that codes for ribosomal protein S1, is more subtle. It is suggested here that S1 is a RNA-binding protein helping polynucleotide phosphorylase (PNPase, known to be phylogenetically related to S1) to degrade mRNA, or helper molecule involved in other RNase activities. This provides an explanation for the elusive function of PNPase, which generates nucleoside diphosphates (not monophosphates) when degrading RNA. This also accounts for the discovery that the B. subtilis comR gene product is PNPase. This article briefly discusses the availability of cytosine nucleotides in eukaryotes, and suggests that they are derived from phospholipids turnover. Finally, the GC content of genomes is discussed in this new light.

Bacillus subtilis↗
PubMed1997

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca.

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.

Adenosine↗
PubMed1997

The Bacillus subtilis genome from gerBC (311 degrees) to licR (334 degrees).

As part of the international project to sequence the Bacillus subtilis genome, the DNA region located between gerBC (311 degrees) and licR (334 degrees) was assigned to the institut Pasteur. In this paper, the cloning and sequencing of 176 kb of DNA and the analysis of the sequence of the entire 271 kb region (6.5% of the B. subtilis chromosome) is described; 273 putative coding sequences were identified. Although the complete genome sequences of seven other organisms (five bacteria, one archaeon and the yeast Saccharomyces cerevisiae) are available in public database, 65 genes from this region of the B. subtilis chromosome encode proteins without significant similarities to other known protein sequences. Among the 208 other genes, 115 have paralogues in the currently known B. subtilis DNA sequences and the products of 178 genes were found to display similarities to protein sequences from public databases for which a function is known. Classification of these genes shows a high proportion of them to be involved in the adaptation to various growth conditions (non-essential cell wall constituents, catabolic and bioenergetic pathways); a small number of the genes are essential or encode anabolic enzymes.

Amino Acid Sequence↗
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

In vivo positive effects of exogenous pyrophosphate on Escherichia coli cell growth and stationary phase survival.

We have studied the effect of exogenous pyrophosphate on growing cells of Escherichia coli. In the presence of 10 mM of pyrophosphate, the entry into the stationary phase was delayed and thus a significant increase in the growth yield was observed (25 to 35%) when the bacteria were grown in glucose minimal medium. Furthermore, the synthesis of 52 polypeptides was affected, as demonstrated by two-dimensional electrophoresis. Among the 22 proteins identified by comparison with the E. coli gene-protein index and/or by microsequencing procedures, 15 were involved either in catabolic or anabolic pathways of the intermediary metabolism or in stress responses. Subsequent physiological experiments enabled us to conclude that pyrophosphate exerted a direct or indirect effect on bacterial growth by (1) conferring upon cells a better capacity to use carbon sources and (2) inducing biosynthetic processes. Finally, we show that exogeneous pyrophosphate enhanced the stationary phase survival of E. coli cells.

Autoradiography↗
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↗