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A Danchin

Publications and source records attributed to A Danchin.

At least 55 records · Page 3Linked to original sources
PubMed1999

Imagene: an integrated computer environment for sequence annotation and analysis.

MOTIVATION: To be fully and efficiently exploited, data coming from sequencing projects together with specific sequence analysis tools need to be integrated within reliable data management systems. Systems designed to manage genome data and analysis tend to give a greater importance either to the data storage or to the methodological aspect, but lack a complete integration of both components. RESULTS: This paper presents a co-operative computer environment (called Imagenetrade mark) dedicated to genomic sequence analysis and annotation. Imagene has been developed by using an object-based model. Thanks to this representation, the user can directly manipulate familiar data objects through icons or lists. Imagene also incorporates a solving engine in order to manage analysis tasks. A global task is solved by successive divisions into smaller sub-tasks. During program execution, these sub-tasks are graphically displayed to the user and may be further re-started at any point after task completion. In this sense, Imagene is more transparent to the user than a traditional menu-driven package. Imagene also provides a user interface to display, on the same screen, the results produced by several tasks, together with the capability to annotate these results easily. In its current form, Imagene has been designed particularly for use in microbial sequencing projects. AVAILABILITY: Imagene best runs on SGI (Irix 6.3 or higher) workstations. It is distributed free of charge on a CD-ROM, but requires some Ilog licensed software to run. Some modules also require separate license agreements. Please contact the authors for specific academic conditions and other Unix platforms. CONTACT: imagene home page: http://wwwabi.snv.jussieu.fr/imagene

Bacillus subtilis↗
PubMed1999

Analysis of long repeats in bacterial genomes reveals alternative evolutionary mechanisms in Bacillus subtilis and other competent prokaryotes.

Prokaryotic genomes seem to be optimized toward compactness and have therefore been thought to lack long redundant DNA sequences. However, we identified a large number of long strict repeats in eight prokaryotic complete genomes and found that their density is negatively correlated with genome size. A detailed analysis of the long repeats present in the genome of Bacillus subtilis revealed a very strict constraint on the spatial distribution of repeats in this genome. We interpret this as the hallmark of selection processes leading to the addition of new genetic information. Such addition is independent of insertion sequences and relies on the nonspecific DNA uptake by the competent cell and its subsequent integration in the chromosome in a circular form through a Campbell-like mechanism. Similar patterns are found in other competent genomes of Gram-negative bacteria and Archaea, suggesting a similar evolutionary mechanism. The correlation of the spatial distribution of repeats and the absence of insertion sequences in a genome may indicate, in the framework of our model, that mechanisms aiming at their avoidance/elimination have been developed.

Antigenic Variation↗
PubMed1999

Detecting and analyzing DNA sequencing errors: toward a higher quality of the Bacillus subtilis genome sequence.

During the determination of a DNA sequence, the introduction of artifactual frameshifts and/or in-frame stop codons in putative genes can lead to misprediction of gene products. Detection of such errors with a method based on protein similarity matching is only possible when related sequences are available in databases. Here, we present a method to detect frameshift errors in DNA sequences that is based on the intrinsic properties of the coding sequences. It combines the results of two analyses, the search for translational initiation/termination sites and the prediction of coding regions. This method was used to screen the complete Bacillus subtilis genome sequence and the regions flanking putative errors were resequenced for verification. This procedure allowed us to correct the sequence and to analyze in detail the nature of the errors. Interestingly, in several cases in-frame termination codons or frameshifts were not sequencing errors but confirmed to be present in the chromosome, indicating that the genes are either nonfunctional (pseudogenes) or subject to regulatory processes such as programmed translational frameshifts. The method can be used for checking the quality of the sequences produced by any prokaryotic genome sequencing project.

Bacillus subtilis↗
PubMed1999

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis.

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.

Acetate Kinase↗
PubMed1999

Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon.

Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of fliA and fliC, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the flhDC master operon restored, at least in part, motility in crp and hns mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates flhDC expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the flhDC promoter was decreased in the crp strain and increased in the hns mutant. In contrast, the activity of a transcriptional fusion encompassing the entire flhDC regulatory region extending to the ATG translational start codon was strongly reduced in both hns and crp mutants. These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in a crp mutant by activation-deficient CAP mutated proteins and characterization of cfs, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates flhDC transcription by binding to its promoter and interacting with RNA polymerase.

Bacterial Proteins↗
PubMed1999

Immunochemical analysis of UMP kinase from Escherichia coli.

Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole E. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.

Animals↗
PubMed1998

Oligonucleotide bias in Bacillus subtilis: general trends and taxonomic comparisons.

We present a general analysis of oligonucleotide usage in the complete genome of Bacillus subtilis . Several datasets were built in order to assign various biological contexts to the biased use of words and to reveal local asymmetries in word usage that may be coupled with replication, the control of gene expression and the restriction/modification system. This analysis was complemented by cross-comparisons with the complete genomes of Escherichia coli , Haemophilus influenzae and Methanococcus jannaschii . We have observed a large number of biased oligonucleotides for words of size up to 8, throughout the datasets and species, indicating that such long strict words play an important role as biological signals. We speculate that some of them are involved in interactions with DNA and/or RNA polymerases. An extensive analysis of palindrome abundances and distributions provides the surprising result that prophage-like elements embedded in the genome exhibit a smaller avoidance of restriction sites. This may reinforce a recently proposed hypothesis of a selfish gene phenomena in the transfer of restriction/modification systems in bacteria.

Bacillus subtilis↗
PubMed1998

Global analysis of genomic texts: the distribution of AGCT tetranucleotides in the Escherichia coli and Bacillus subtilis genomes predicts translational frameshifting and ribosomal hopping in several genes.

Present availability of the genomic text of bacteria allows assignment of biological known functions to many genes (typically, half of the genome's gene content). It is now time to try and predict new unexpected functions, using inductive procedures that allow correlating the content of the genomic text to possible biological functions. We show here that analysis of the genomes of Escherichia coli and Bacillus subtilis for the distribution of AGCT motifs predicts that genes exist for which the mRNA molecule can be translated as several different proteins synthesized after ribosomal frameshifting or hopping. Among these genes we found that several coded for the same function in E. coli and B. subtilis. We analyzed in depth the situation of the infB gene (experimentally known to specify synthesis of several proteins differing in their translation starts), the aceF/pdhC gene, the eno gene, and the rplI gene. In addition, genes specific to E. coli were also studied: ompA, ompFand tolA (predicting epigenetic variation that could help escape infection by phages or colicins).

Acetyltransferases↗
PubMed1998

On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi, a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats. An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli, purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.

Amino Acid Sequence↗
PubMed1998

Characterization of polyamine synthesis pathway in Bacillus subtilis 168.

The ubiquitous polyamines fulfil a variety of functions in all three kingdoms of life. However, little is known about the biosynthesis of these compounds in Gram-positive bacteria. We show that, in Bacillus subtilis, there is a single pathway to polyamines, starting from arginine, with agmatine as an intermediate. We first identified the structural gene of arginine decarboxylase, speA (formerly cad), and then described the speE speB operon, directing synthesis of spermidine synthase and agmatinase. This operon is transcribed into two messenger RNAs, a major one for the speE gene and a minor one for both speEand speB. The promoter of the operon was identified upstream from the speE gene by primer extension analysis. Transcription of this operon indicated that the level of agmatinase synthesis is very low, thus allowing a stringent control on the synthesis of putrescine and, therefore, of all polyamines. This is consistent with the level of polyamines measured in the cell.

Aspartate Carbamoyltransferase↗
PubMed1998

Indigo: a World-Wide-Web review of genomes and gene functions.

The present article describes a genome database reviewing gene-related knowledge of two model bacteria, Bacillus subtilis and Escherichia coli. The database, Indigo, is open through the World-Wide Web (http://indigo.genetique.uvsq.fr). The concept used for organising the data, the concept of neighbourhood, allows one to explore the database content in an efficient although somewhat unusual way. Here, genes are related to each other by a variety of neighbourhoods, including proximity in the chromosome, phylogenetic kinship, participation in a common metabolic pathway, common presence in an article of the literature, or similar use of the genetic code. Several examples illustrate how this concept of neighbourhood permits one to review the available knowledge about a given gene or gene family, and elaborate unexpected, but revealing, analyses about gene functions.

Bacillus subtilis↗
PubMed1998

Aeromonas hydrophila adenylyl cyclase 2: a new class of adenylyl cyclases with thermophilic properties and sequence similarities to proteins from hyperthermophilic archaebacteria.

Complementation of an Escherichia coli cya mutant with a genomic library from Aeromonas hydrophila allowed isolation of clones containing two different cya genes. Whereas one of these genes (cyaA) coded for an adenylyl cyclase (AC1) belonging to the previously described class I adenylyl cyclases (ACs), the second one (cyaB) coded for a protein (AC2) that did not match any previously characterized protein when compared to protein sequence databases. In particular, it did not align with any of members of the three known classes of ACs. The purified AC2 enzyme exhibited remarkable biochemical characteristics, namely, an optimum activity at a high temperature (65 degrees C) and at an alkalinic pH (9.5). In order to investigate the functions of both cyclases in A. hydrophila, each gene was inactivated in the chromosome and the resulting mutant strains were examined for physiological alterations. It was shown that, in contrast to cyaA, the cyaB gene was not expressed under usual laboratory growth conditions. However, introduction of a plasmid harboring the cyaB gene in a cyaA mutant, as well as in a cyaA cyaB mutant, allowed cyclic AMP production. AC2 is the first member of a new class of previously unrecognized ACs, and to date, no functional counterpart has been demonstrated in other organisms. However, scanning databases revealed a significant similarity between AC2 and the gene product of three hyperthermophilic archaebacteria: Methanobacterium thermoautotrophicum, Archaeglobus fulgidus, and Methanococcus jannaschii. The possibility of a gene transfer between such phylogenetically divergent bacteria is discussed.

Adenylyl Cyclases↗
PubMed1998

Mutational analysis of UMP kinase from Escherichia coli.

UMP kinase from Escherichia coli is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of E. coli UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).

Amino Acid Sequence↗
PubMed1997

Using codon usage to predict genes origin: is the Escherichia coli outer membrane a patchwork of products from different genomes?

Analysis of the codon usage of genes coding for the structural components of the outer membrane in Escherichia coli, is consistent with the requirement for high expression of these genes. Because porins (which constitute the major protein component of the outer membrane), and LPS (which constitute the major outermost constituent of the outer membrane), are synthesized from genes displaying widely different codon usage, it is possible to investigate the origin of the outer membrane. The analysis predicts that the outer membrane might originate from a genome other than the genome coding for the major part of the cell. Such a special origin would explain in structural terms, the likely lethality of porins if they were inadvertently inserted within the inner membrane, giving rise to the Gram-negative bacterial type, having an envelope comprising two membranes, instead of a single cytoplasmic membrane and a murein sacculus.

Bacterial Outer Membrane Proteins↗
PubMed1997

Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi.

The molecular karyotype of a murine isolate of Encephalitozoon cuniculi, a microsporidian with a wide range of mammalian hosts, comprises eleven chromosomes ranging in size between 217 and 315 kb. To determine specific chromosomal markers, a partial genomic library was constructed and cloned DNA fragments were hybridized to chromosomal bands separated by pulsed-field gel electrophoresis. Most probes were assigned to single chromosomes, indicating prevalence of low-copy number nucleotide sequences within the very small genome of E. cuniculi (2.9 Mb). A few probes were shown to hybridize to all chromosomes. These repetitive DNA fragments corresponded to either rRNA genes or some non-coding regions whose sequences were characterized by short micro- and minisatellites. The chromosomal locations of beta-tubulin genes and six newly identified protein-encoding genes were determined. Genes encoding dihydrofolate reductase, thymidylate synthase, serine hydroxymethyl transferase, a cdc2 kinase-like protein and helicase ERCC6-like protein were each located on a single chromosome whereas genes for both beta-tubulin and aminopeptidase were on two different chromosomes. The mapping will serve as a reference for further analysis of intraspecific karyotype polymorphism in different isolates from different host species.

Animals↗
PubMed1997

Role of Escherichia coli histone-like nucleoid-structuring protein in bacterial metabolism and stress response--identification of targets by two-dimensional electrophoresis.

The histone-like nucleoid-structuring protein, H-NS, is a major bacterial chromatin component which influences DNA structure and gene expression. Mutations in hns, the structural gene of H-NS protein, have been shown to result in highly pleiotropic effects in Escherichia coli cells. In this study, we have initiated an index of the proteins whose synthesis is, directly or indirectly regulated by H-NS. Using two-dimensional gel electrophoresis, we have examined the global changes in gene expression which occured in an hns background compared with its wild-type parent. In addition, we analysed the effects of mutations in two other genes i.e. lrp and pta, which are also involved in global regulatory pathways. Although these comparative analyses revealed several common differences, thus suggesting possible interactions between these regulatory mechanisms, i.e. H-NS, Lrp (leucine-responsive regulatory protein) and acetylphosphate, the most extensive modifications occurred in an hns mutant. Among the polypeptides whose level of synthesis was specifically altered in an hns mutant, several corresponded to H-NS targets previously identified by classical selection methods. Moreover, the present study allows us to characterize several H-NS targets, which were identified either by comparison with the E. coli two-dimensional reference maps or by microsequencing procedure. Many of these newly identified polypeptides are involved in adaptation of E. coli cells to environmental challenges, and one of them could be involved in bacterial virulence. Finally, synthesis of several proteins belonging to the heat-shock regulon, more particularly molecular chaperones, was induced in an hns mutant.

Amino Acid Sequence↗