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Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a "mycobacterial" gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the "non mycobacterial" factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.

Background: The outermost layer of the bacterial surface is of crucial importance because it is in constant interaction with the host. Glycopeptidolipids (GPLs) are major surface glycolipids present on various mycobacterial species. In the fast-grower model organism Mycobacterium smegmatis, GPL biosynthesis involves approximately 30 genes all mapping to a single region of 65 kb., Results: We have recently sequenced the complete genomes of two fast-growers causing human infections, Mycobacterium abscessus (CIP 104536T) and M. chelonae (CIP 104535T). We show here that these two species contain genes corresponding to all those of the M. smegmatis "GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in M. chelonae and M. abscessus. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (atf1 and atf2) in M. abscessus and M. chelonae has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (M. chelonae, 900 kb; M. abscessus, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins., Conclusion: Although these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in M. smegmatis represents the ancestral form and that evolution has scattered various pieces throughout the genome in M. abscessus and M. chelonae.

Most proteins comprise one or several domains. New domain architectures can be created by combining previously existing domains. The elementary events that create new domain architectures may be categorized into three classes, namely domain(s) insertion or deletion (indel), exchange and repetition. Using 'DomainTeam', a tool dedicated to the search for microsyntenies of domains, we quantified the relative contribution of these events. This tool allowed us to collect homologous bacterial genes encoding proteins that have obviously evolved by modular assembly of domains. We show that indels are the most frequent elementary events and that they occur in most cases at either the N- or C-terminus of the proteins. As revealed by the genomic neighbourhood/context of the corresponding genes, we show that a substantial number of these terminal indels are the consequence of gene fusions/fissions. We provide evidence showing that the contribution of gene fusion/fission to the evolution of multi-domain bacterial proteins is lower-bounded by 27% and upper-bounded by 64%. We conclude that gene fusion/fission is a major contributor to the evolution of multi-domain bacterial proteins.

The detection, across several genomes, of local conservation of gene content and proximity considerably helps the prediction of features of interest, such as gene fusions or physical and functional interactions. Here, we want to process realistic models of chromosomes, in which genes (or genomic segments of several genes) can be duplicated within a chromosome, or be absent from some other chromosome(s). Our approach adopts the technique of temporarily forgetting genes and working directly with protein "domains" such as those found in Pfam. This allows the detection of strings of domains that are conserved in their content, but not necessarily in their order, which we refer to as domain teams. The prominent feature of the method is that it relaxes the rigidity of the orthology criterion and avoids many of the pitfalls of gene-families identification methods, often hampered by multidomain proteins or low levels of sequence similarity. This approach, that allows both inter- and intrachromosomal comparisons, proves to be more sensitive than the classical methods based on pairwise sequence comparisons, particularly in the simultaneous treatment of many species. The automated and fast detection of domain teams, together with its increased sensitivity at identifying segments of identical (protein-coding) gene contents as well as gene fusions, should prove a useful complement to other existing methods.

Genomic projects heavily depend on genome annotations and are limited by the current deficiencies in the published predictions of gene structure and function. It follows that, improved annotation will allow better data mining of genomes, and more secure planning and design of experiments. The purpose of the GeneFarm project is to obtain homogeneous, reliable, documented and traceable annotations for Arabidopsis nuclear genes and gene products, and to enter them into an added-value database. This re-annotation project is being performed exhaustively on every member of each gene family. Performing a family-wide annotation makes the task easier and more efficient than a gene-by-gene approach since many features obtained for one gene can be extrapolated to some or all the other genes of a family. A complete annotation procedure based on the most efficient prediction tools available is being used by 16 partner laboratories, each contributing annotated families from its field of expertise. A database, named GeneFarm, and an associated user-friendly interface to query the annotations have been developed. More than 3000 genes distributed over 300 families have been annotated and are available at http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data into the protein knowledgebase Swiss-Prot.

All the protein sequences from plants (including Arabidopsis thaliana) available from SwissProt/TrEMBL have been the subject of an all-by-all systematic comparison and grouped into clusters of related proteins. Within each cluster, the sequences have been submitted to pyramidal classification; in the case where two or several subfamilies have been grouped together, the pyramidal tree helps in finding which sequences make the links between subfamilies. In addition, the 'domains' that are common to two or more sequences within a cluster were determined and displayed à la ProDom. The resulting graphical representations proved to be quite efficient in pinpointing those protein sequences suffering from a probable error in the annotation of their genes. The clusters can be searched through various criteria and their pyramidal classifications and their domain representations can be displayed by querying http://genoplante-info. infobiogen.fr/phytoprot. The user can also launch a BLAST search of a query sequence against all the clusters.

An all-by-all comparison of all the publicly available protein sequences from plants has been performed, followed by a clusterization process. Within each of the 1064 resulting clusters-containing sequences that are orthologous as well as paralogous-the sequences have been submitted to a pyramidal classification and their domains delineated by an automated procedure à la. This process provides a means for easily checking for any apparent inconsistency in a cluster, for example, whether one sequence is shorter or longer than the others, one domain is missing, etc. In such cases, the alignment of the DNA sequence of the gene with that of a close homologous protein often reveals (in 10% of the clusters) probable sequencing errors (leading to frameshifts) or probable wrong intron/exon predictions. The composition of the clusters, their pyramidal classifications, and domain decomposition, as well as our comments when appropriate, are available from http://chlora.infobiogen.fr:1234/PHYTOPROT.

We propose and study a new approach for the analysis of families of protein sequences. This method is related to the LogDet distances used in phylogenetic reconstructions; it can be viewed as an attempt to embed these distances into a multidimensional framework. The proposed method starts by associating a Markov matrix to each pairwise alignment deduced from a given multiple alignment. The central objects under consideration here are matrix-valued logarithms L of these Markov matrices, which exist under conditions that are compatible with fairly large divergence between the sequences. These logarithms allow us to compare data from a family of aligned proteins with simple models (in particular, continuous reversible Markov models) and to test the adequacy of such models. If one neglects fluctuations arising from the finite length of sequences, any continuous reversible Markov model with a single rate matrix Q over an arbitrary tree predicts that all the observed matrices L are multiples of Q. Our method exploits this fact, without relying on any tree estimation. We test this prediction on a family of proteins encoded by the mitochondrial genome of 26 multicellular animals, which include vertebrates, arthropods, echinoderms, molluscs, and nematodes. A principal component analysis of the observed matrices L shows that a single rate model can be used as a rough approximation to the data, but that systematic deviations from any such model are unmistakable and related to the evolutionary history of the species under consideration.

Genome annotation requires explicit identification of gene function. This task frequently uses protein sequence alignments with examples having a known function. Genetic drift, co-evolution of subunits in protein complexes and a variety of other constraints interfere with the relevance of alignments. Using a specific class of proteins, it is shown that a simple data analysis approach can help solve some of the problems posed. The origin of ureohydrolases has been explored by comparing sequence similarity trees, maximizing amino acid alignment conservation. The trees separate agmatinases from arginases but suggest the presence of unknown biases responsible for unexpected positions of some enzymes. Using factorial correspondence analysis, a distance tree between sequences was established, comparing regions with gaps in the alignments. The gap tree gives a consistent picture of functional kinship, perhaps reflecting some aspects of phylogeny, with a clear domain of enzymes encoding two types of ureohydrolases (agmatinases and arginases) and activities related to, but different from ureohydrolases. Several annotated genes appeared to correspond to a wrong assignment if the trees were significant. They were cloned and their products expressed and identified biochemically. This substantiated the validity of the gap tree. Its organization suggests a very ancient origin of ureohydrolases. Some enzymes of eukaryotic origin are spread throughout the arginase part of the trees: they might have been derived from the genes found in the early symbiotic bacteria that became the organelles. They were transferred to the nucleus when symbiotic genes had to escape Muller's ratchet. This work also shows that arginases and agmatinases share the same two manganese-ion-binding sites and exhibit only subtle differences that can be accounted for knowing the three-dimensional structure of arginases. In the absence of explicit biochemical data, extreme caution is needed when annotating genes having similarities to ureohydrolases.

In spite of many efforts, the prediction of the location of proteins in eukaryotic cells (cytoplasm, mitochondrion or chloroplast) is still far from straightforward. In some cases (e.g. ribosomal proteins and aminoacyl-tRNA synthetases) both the cytoplasmic proteins and their organellar counterparts are encoded by the nuclear genome. A factorial correspondence analysis of the codon usage in yeast and Caenorhabditis elegans shows that the codon usage of those nuclear genes encoding ribosomal proteins or aminoacyl-tRNA synthetases is markedly different, depending on the final location of the proteins (cytoplasmic or mitochondrial). As a consequence, the location of such proteins-whose sequences are now frequently determined by systematic genomic sequencing-can be easily and quickly predicted. A WWW interface has been developed, aimed at providing a user-friendly tool for codon usage pattern analysis. It is available from http://www.genetique.uvsq.fr/afc.html

A cloning of hepatic cDNAs associated with the early phase of an acute, systemic inflammation was carried out by differential screening of arrayed cDNA clones from rat livers obtained at 4-8 h postchallenge with Freund's complete adjuvant. End sequencing of 174 selected clones provided three cDNA groups that coded for: (i) 23 known acute-phase proteins, (ii) 31 known proteins whose change in hepatic synthesis during an acute phase was so far unsuspected, and (iii) 36 novel proteins whose cDNAs were completely sequenced. For 16 proteins in the third group the hepatic mRNA could be detected and quantitated by Northern blot hybridization in Freund's adjuvant-challenged animals, and an extrahepatic expression in healthy animals was further investigated. Matching the open reading frames of the 36 novel proteins with general and specialized data libraries indicated the potential relationships of 16 of these proteins with known protein families/superfamilies and/or the presence of functional domains previously described in other proteins. Overall, our search for novel inflammation-associated proteins selected mostly known or as yet undescribed proteins with an intracellular or membrane location, which extends our knowledge of the proteins involved in the intracellular metabolism of hepatic cells during a systemic, acute-phase response. Finally, some of the cDNAs above allowed us to successfully identify hepatic mRNAs that are differentially expressed in acute vs chronic (polyarthritis) inflammatory conditions in rat., (Copyright 1999 Academic Press.)

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

All of the aminoacyl-tRNA synthetase (aaRS) sequences currently available in the data banks have been subjected to a systematic analysis aimed at finding gene duplications, genetic recombinations, and horizontal transfers. Evidence is provided for the occurrence (or probable occurrence) of such phenomena within this class of enzymes. In particular, it is suggested that the monomeric PheRS from the yeast mitochondrion is a chimera of the alpha and beta chains of the standard tetrameric protein. In addition, it is proposed that the dimeric and tetrameric forms of GlyRS are the result of a double and independent acquisition of the same specificity within two different subclasses of aaRS. The phylogenetic reconstructions of the evolutionary histories of the genes encoding aaRS are shown to be extremely diverse. While large segments of the population are consistent with the broad grouping into the three Woesian domains, some phylogenetic reconstructions do not place the Archae and the Eucarya as sister groups but, rather, show a gram-negative bacteria/eukaryote clustering. In addition, many individual genes pose difficulties that preclude any simple evolutionary scheme. Thus, aaRS's are clearly a paradigm of F. Jacob's "odd jobs of evolution" but, on the whole, do not call into question the evolutionary scenario originally proposed by Woese and subsequently refined by others.

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.

The inter-alpha-inhibitor (I alpha I) family is comprised of the plasma protease inhibitors I alpha I, inter-alpha-like inhibitor (I alpha LI), pre-alpha-inhibitor (P alpha I) and bikunin. I alpha I, I alpha LI and P alpha I are distinct assemblies of bikunin with one of three heavy (H) chains designated H1, H2 and H3. These H chains and bikunin are respectively encoded by a set of three H genes and an alpha 1-microglobulin/bikunin precursor (AMBP) gene. All four gene products undergo maturation steps from precursor polypeptides. The full-length cDNAs for the H1-, H2- and H3-chain precursors were cloned from a mouse liver cDNA library and sequenced. Extensive searches of amino acid sequence similarities to other proteins in databanks revealed (i) a highly significant similarity of the C-terminal sequence in the three H-chain precursors to the multicopper-binding domain in the group of multicopper oxidase proteins and (ii) the presence of von Willebrand type-A domains in the mature H chains. Amino acid sequence comparisons between the three mouse H1-, H2- and H3-chain precursors and their human counterparts allowed us to appraise the timing and order of occurrence of the three H-chain genes from a shared ancestor during mammalian evolution. Owing to a multiple alignment of the six mouse and human nucleotide sequences for these H-chain precursors, a reverse transcriptase PCR assay with degenerate oligonucleotides was designed, allowing us to (i) present evidence that no mRNAs for further H genes exist in mouse liver and (ii) demonstrate a previously undescribed transcription of the H2- and H3-chain mRNAs in mouse brain, which contrasts with the expression of all four, H1, H2, H3 and AMBP, mRNAs in liver.

Fast sequence databanks search algorithms generally make use of hash tables and look for exactly matching words. An increased sensitivity--at the expense of a decreased selectivity--can be attained in the case of proteins by using a reduced amino acid alphabet. We propose here an alphabet reduced to 10 symbols, that we used in modified versions of the FASTP and SCAN programs. An application to the aminoacyl-tRNA synthetases shows that this technique may be useful in detecting distant relationships between proteins.

A method aimed at classifying protein sequences without resorting to pairwise alignment is presented. Called DOCMA (DOt-plot Comparisons by Multivariate Analysis), it is based on a multivariate analysis of the pairwise dot-plots between all the sequences in the set. The dot-plots are first simplified by considering only the projections of the 'diagonal' segments of similarity onto the axes. From these projections a data matrix is built, in which each column is representative of the comparisons of one given sequence with all the other ones. This data matrix is then transformed into a distance matrix by a chi-squared analysis, from which the coordinates of the sequences in an orthonormal Euclidean space are obtained. The sequences are finally classified by a dynamic clustering procedure followed by a search for strong clusters. Application of this method to protein families such as the globins, the cytochromes c and the aminoacyl-tRNA synthetases shows that it is quite effective in delineating subgroups that contain even distantly related sequences.

The present work describes an attempt to identify reliable criteria which could be used as distance indices between protein sequences. Seven different criteria have been tested: i and ii) the scores of the alignments as given by the BESTFIT and the FASTA programs; iii) the ratio parameter, i.e. the BESTFIT score divided by the length of the aligned peptides; iv and v) the statistical significance (Z-scores) of the scores calculated by BESTFIT and FASTA, as obtained by comparison with shuffled sequences; vi) the Z-scores provided by the program RELATE which performs a segment-by-segment comparison of 2 sequences, and vii) an original distance index calculated by the program DOCMA from all the pairwise dotplots between the sequences. These 7 criteria have been tested against the aminoacid sequences of 39 globins and those of the 20 aminoacyl-tRNA synthetases from E. coli. The distances between the sequences were analyzed by the multivariate analysis techniques. The results show that the distances calculated from the scores of the pairwise alignments are not adequately sensitive. The Z-score from RELATE is not selective enough and too demanding in computer time. Three criteria gave a classification consistent with the known similarities between the sequences in the sets, namely the Z-scores from BESTFIT and FASTA and the multiple dotplot comparison distance index from DOCMA.

Detailed comparisons between the structures of the tRNA-bound Escherichia coli glutaminyl-tRNA (Gln-tRNA) synthetase [L-glutamine:tRNA(Gln) ligase (AMP-forming), EC 6.1.1.18] and recently refined E. coli methionyl-tRNA (Met-tRNA) synthetase [L-methionine:tRNA(Met) ligase (AMP-forming), EC 6.1.1.10] reveal significant similarities beyond the anticipated correspondence of their respective dinucleotide-fold domains. One similarity comprises a 23-amino acid alpha-helix-turn-beta-strand motif found in each enzyme within a domain that is inserted between the two halves of the dinucleotide binding fold. A second correspondence, which consists of two alpha-helices connected by a large loop and beta-strand, is located in the Gln-tRNA synthetase within a region that binds the inside corner of the "L"-shaped tRNA molecule. This structural motif contains a long alpha-helix, which extends along the entire length of the D and anticodon stems of the complexed tRNA. We suggest that the positioning of this helix relative to the dinucleotide fold plays a critical role in ensuring the proper global orientation of tRNA(Gln) on the surface of the enzyme. The structural correspondences suggest a similar overall orientation of binding of tRNA(Met) and tRNA(Gln) to their respective synthetases.

The crystal structure of a small calcium-binding protein, the parvalbumin IIIf from Opsanus tau in which Tb was substituted for Ca, has been analysed by multiwavelength anomalous diffraction. Data at a resolution of 2.3 A were collected at three wavelengths near the L3 absorption edge of Tb (1.645-1.650 A), using the synchrotron radiation emitted by a storage ring and a multiwire proportional counter. The phases of the reflections were determined from this single derivative, without native data. Prior to any refinement, the resulting electron density map shows a good agreement with the model of the homologous carp parvalbumin in regions of identical amino-acid sequence.