Complete genome, catabolic sub-proteomes and key-metabolites of Desulfobacula toluolica Tol2, a marine, aromatic compound-degrading, sulfate-reducing bacterium.

Among the dominant deltaproteobacterial sulfate-reducing bacteria (SRB), members of the genus Desulfobacula are not only present in (hydrocarbon-rich) marine sediments, but occur also frequently in the anoxic water bodies encountered in marine upwelling areas. Here, we present the 5.2 Mbp genome of Desulfobacula toluolica Tol2, which is the first of an aromatic compound-degrading, marine SRB. The genome has apparently been shaped by viral attacks (e.g. CRISPRs) and its high plasticity is reflected by 163 detected genes related to transposases and integrases, a total of 494 paralogous genes and 24 group II introns. Prediction of the catabolic network of strain Tol2 was refined by differential proteome and metabolite analysis of substrate-adapted cells. Toluene and p-cresol are degraded by separate suites of specific enzymes for initial arylsuccinate formation via addition to fumarate (p-cresol-specific enzyme HbsA represents a new phylogenetic branch) as well as for subsequent modified ß-oxidation of arylsuccinates to the central intermediate benzoyl-CoA. Proteogenomic evidence suggests specific electron transfer (EtfAB) and membrane proteins to channel electrons from dehydrogenation of both arylsuccinates directly to the membrane redox pool. In contrast to the known anaerobic degradation pathways in other bacteria, strain Tol2 deaminates phenylalanine non-oxidatively to cinnamate by phenylalanine ammonia-lyase and subsequently forms phenylacetate (both metabolites identified in (13) C-labelling experiments). Benzoate degradation involves CoA activation, reductive dearomatization by a class II benzoyl-CoA reductase and hydrolytic ring cleavage as found in the obligate anaerobe Geobacter metallireducens GS-15. The catabolic sub-proteomes displayed high substrate specificity, reflecting the genomically predicted complex and fine-tuned regulatory network of strain Tol2. Despite the genetic equipment for a TCA cycle, proteomic evidence supports complete oxidation of acetyl-CoA to CO2 via the Wood-Ljungdahl pathway. Strain Tol2 possesses transmembrane redox complexes similar to that of other Desulfobacteraceae members. The multiple heterodisulfide reductase-like proteins (more than described for Desulfobacterium autotrophicum HRM2) may constitute a multifaceted cytoplasmic electron transfer network.

Long-term survival of hydrated resting eggs from Brachionus plicatilis.

BACKGROUND: Several organisms display dormancy and developmental arrest at embryonic stages. Long-term survival in the dormant form is usually associated with desiccation, orthodox plant seeds and Artemia cysts being well documented examples. Several aquatic invertebrates display dormancy during embryonic development and survive for tens or even hundreds of years in a hydrated form, raising the question of whether survival in the non-desiccated form of embryonic development depends on pathways similar to those occurring in desiccation tolerant forms. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, Illumina short read sequencing was used to generate transcription profiles from the resting and amictic eggs of an aquatic invertebrate, the rotifer, Brachionus plicatilis. These two types of egg have very different life histories, with the dormant or diapausing resting eggs, the result of the sexual cycle and amictic eggs, the non-dormant products of the asexual cycle. Significant transcriptional differences were found between the two types of egg, with amictic eggs rich in genes involved in the morphological development into a juvenile rotifer. In contrast, representatives of classical "stress" proteins: a small heat shock protein, ferritin and Late Embryogenesis Abundant (LEA) proteins were identified in resting eggs. More importantly however, was the identification of transcripts for messenger ribonucleoprotein particles which stabilise RNA. These inhibit translation and provide a valuable source of useful RNAs which can be rapidly activated on the exit from dormancy. Apoptotic genes were also present. Although apoptosis is inconsistent with maintenance of prolonged dormancy, an altered apoptotic pathway has been proposed for Artemia, and this may be the case with the rotifer. CONCLUSIONS: These data represent the first transcriptional profiling of molecular processes associated with dormancy in a non-desiccated form and indicate important similarities in the molecular pathways activated in resting eggs compared with desiccated dormant forms, specifically plant seeds and Artemia.

Directed sequencing and annotation of three Dicentrarchus labrax L. chromosomes by applying Sanger- and pyrosequencing technologies on pooled DNA of comparatively mapped BAC clones.

Dicentrarchus labrax is one of the major marine aquaculture species in the European Union. In this study, we have developed a directed-sequencing strategy to sequence three sea bass chromosomes and compared results with other teleosts. Three BAC DNA pools were created from sea bass BAC clones that mapped to stickleback chromosomes/groups V, XVII and XXI. The pools were sequenced to 17-39x coverage by pyrosequencing. Data assembly was supported by Sanger reads and mate pair data and resulted in superscaffolds of 13.2 Mb, 17.5 Mb and 13.7 Mb respectively. Annotation features of the superscaffolds include 1477 genes. We analyzed size change of exon, intron and intergenic sequence between teleost species and deduced a simple model for the evolution of genome composition in teleost lineage. Combination of second generation sequencing technologies, Sanger sequencing and genome partitioning strategies allows "high-quality draft assemblies" of chromosome-sized superscaffolds, which are crucial for the prediction and annotation of complete genes.

Comparative analysis of intronless genes in teleost fish genomes: insights into their evolution and molecular function.

This study assessed the relationship between the occurrence and function of intronless or single exon genes (SEG) in the genome of five teleost species and their phylogenetic distance. The results revealed that Takifugu rubripes, Tetraodon nigroviridis, Oryzias latipes, Gasterosteus aculeatus and Danio rerio genomes are respectively comprised of 2.83%, 3.42%, 4.49%, 4.35% and 4.02% SEGs. These SEGs encode for a variety of family proteins including claudins, olfactory receptors and histones that are essential for various biological functions. Subsequently, we predicted and annotated SEGs in three European sea bass, Dicentrarchus labrax chromosomes that we have sequenced, and compared results with those of stickleback (G. aculeatus) homologous chromosomes. While the annotation features of three D. labrax chromosomes revealed 78 (5.30%) intronless genes, comparisons with G. aculeatus showed that SEG composition and their order varied significantly among corresponding chromosomes, even for those with nearly complete synteny. More than half of SEGs identified in most of the species have at least one ortholog multiple exon gene in the same genome, which provides insight to their possible origin by retrotransposition. In spite of the fact that they belong to the same lineage, the fraction of predicted SEGs varied significantly between the genomes analyzed, and only a low fraction of proteins (4.1%) is conserved between all five species. Furthermore, the inter-specific distribution of SEGs as well as the functional categories shared by species did not reflect their phylogenetic relationships. These results indicate that new SEGs are continuously and independently generated after species divergence over evolutionary time as evidenced by the phylogenetic results of single exon claudins genes. Although the origin of SEGs cannot be inferred directly from the phylogeny, our results provide strong support for the idea that retrotransposition followed by tandem duplications is the most probable event that can explain the expansion of SEGs in eukaryotic organisms.

The European sea bass Dicentrarchus labrax genome puzzle: comparative BAC-mapping and low coverage shotgun sequencing.

BACKGROUND: Food supply from the ocean is constrained by the shortage of domesticated and selected fish. Development of genomic models of economically important fishes should assist with the removal of this bottleneck. European sea bass Dicentrarchus labrax L. (Moronidae, Perciformes, Teleostei) is one of the most important fishes in European marine aquaculture; growing genomic resources put it on its way to serve as an economic model. RESULTS: End sequencing of a sea bass genomic BAC-library enabled the comparative mapping of the sea bass genome using the three-spined stickleback Gasterosteus aculeatus genome as a reference. BAC-end sequences (102,690) were aligned to the stickleback genome. The number of mappable BACs was improved using a two-fold coverage WGS dataset of sea bass resulting in a comparative BAC-map covering 87% of stickleback chromosomes with 588 BAC-contigs. The minimum size of 83 contigs covering 50% of the reference was 1.2 Mbp; the largest BAC-contig comprised 8.86 Mbp. More than 22,000 BAC-clones aligned with both ends to the reference genome. Intra-chromosomal rearrangements between sea bass and stickleback were identified. Size distributions of mapped BACs were used to calculate that the genome of sea bass may be only 1.3 fold larger than the 460 Mbp stickleback genome. CONCLUSIONS: The BAC map is used for sequencing single BACs or BAC-pools covering defined genomic entities by second generation sequencing technologies. Together with the WGS dataset it initiates a sea bass genome sequencing project. This will allow the quantification of polymorphisms through resequencing, which is important for selecting highly performing domesticated fish.

Gilthead sea bream (Sparus auratus) and European sea bass (Dicentrarchus labrax) expressed sequence tags: Characterization, tissue-specific expression and gene markers.

The gilthead sea bream, Sparus auratus, and the European sea bass, Dicentrarchus labrax, are two of the most important marine species cultivated in Southern Europe. This study aimed at increasing genomic resources for the two species and produced and annotated two sets of 30,000 expressed sequence tags (EST) each from 14 normalized tissue-specific cDNA libraries from sea bream and sea bass. Clustering and assembly of the ESTs formed 5268 contigs and 12,928 singletons for sea bream and 4573 contigs and 13,143 singletons for sea bass, representing 18,196 and 17,716 putative unigenes, respectively. Assuming a similar number of genes in sea bass, sea bream and in the model fish Gasterosteus aculeatus genomes, it was estimated that approximately two thirds of the sea bream and the sea bass transcriptomes were covered by the unigene collections. BLAST sequence similarity searches (using a cut off of e-value <10(-5)) against fully the curated SwissProt (and TrEMBL) databases produced matches of 28%(37%) and 43%(53%) of the sea bream and sea bass unigene datasets respectively, allowing some putative designation of function. A comparative approach is described using human Ensembl peptide ID homolog's for functional annotation, which increased the number of unigenes with GO terms assigned and resulted in more GO terms assigned per unigene. This allowed the identification of tissue-specific genes using enrichment analysis for GO pathways and protein domains. The comparative annotation approach represents a good strategy for transferring more relevant biological information from highly studied species to genomic resource poorer species. It was possible to confirm by interspecies mRNA-to-genomic alignments 25 and 21 alternative splice events in sea bream and sea bass genes, respectively. Even using normalized cDNA from relatively few pooled individuals it was possible to identify 1145 SNPs and 1748 microsatellites loci for genetic marker development. The EST data are being applied to a range of projects, including the development microarrays, genetic and radiation hybrid maps and QTL genome scans. This highlights the important role of ESTs for generating genetic and genomic resources of aquaculture species.

The genome of Erwinia tasmaniensis strain Et1/99, a non-pathogenic bacterium in the genus Erwinia.

The complete genome of the bacterium Erwinia tasmaniensis strain Et1/99 consisting of a 3.9 Mb circular chromosome and five plasmids was sequenced. Strain Et1/99 represents an epiphytic plant bacterium related to Erwinia amylovora and E. pyrifoliae, which are responsible for the important plant diseases fire blight and Asian pear shoot blight, respectively. Strain Et1/99 is a non-pathogenic bacterium and is thought to compete with these and other bacteria when occupying the same habitat during initial colonization. Genome analysis revealed tools for colonization, cellular communication and defence modulation, as well as genes coding for the synthesis of levan and a not detected capsular exopolysaccharide. Strain Et1/99 may secrete indole-3-acetic acid to increase availability of nutrients provided on plant surfaces. These nutrients are subsequently accessed and metabolized. Secretion systems include the hypersensitive response type III pathway present in many pathogens. Differences or missing parts within the virulence-related factors distinguish strain Et1/99 from pathogens such as Pectobacterium atrosepticum and the related Erwinia spp. Strain Et1/99 completely lacks the sorbitol operon, which may also affect its inability to invade fire blight host plants. Erwinia amylovora in contrast depends for virulence on utilization of sorbitol, the dominant carbohydrate in rosaceous plants. The presence of other virulence-associated factors in strain Et1/99 indicates the ancestral genomic background of many plant-associated bacteria.

SNP and haplotype mapping for genetic analysis in the rat.

The laboratory rat is one of the most extensively studied model organisms. Inbred laboratory rat strains originated from limited Rattus norvegicus founder populations, and the inherited genetic variation provides an excellent resource for the correlation of genotype to phenotype. Here, we report a survey of genetic variation based on almost 3 million newly identified SNPs. We obtained accurate and complete genotypes for a subset of 20,238 SNPs across 167 distinct inbred rat strains, two rat recombinant inbred panels and an F2 intercross. Using 81% of these SNPs, we constructed high-density genetic maps, creating a large dataset of fully characterized SNPs for disease gene mapping. Our data characterize the population structure and illustrate the degree of linkage disequilibrium. We provide a detailed SNP map and demonstrate its utility for mapping of quantitative trait loci. This community resource is openly available and augments the genetic tools for this workhorse of physiological studies.

Comparative analysis of expressed sequence tag (EST) libraries in the seagrass Zostera marina subjected to temperature stress.

Global warming is associated with increasing stress and mortality on temperate seagrass beds, in particular during periods of high sea surface temperatures during summer months, adding to existing anthropogenic impacts, such as eutrophication and habitat destruction. We compare several expressed sequence tag (EST) in the ecologically important seagrass Zostera marina (eelgrass) to elucidate the molecular genetic basis of adaptation to environmental extremes. We compared the tentative unigene (TUG) frequencies of libraries derived from leaf and meristematic tissue from a control situation with two experimentally imposed temperature stress conditions and found that TUG composition is markedly different among these conditions (all P < 0.0001). Under heat stress, we find that 63 TUGs are differentially expressed (d.e.) at 25 degrees C compared with lower, no-stress condition temperatures (4 degrees C and 17 degrees C). Approximately one-third of d.e. eelgrass genes were characteristic for the stress response of the terrestrial plant model Arabidopsis thaliana. The changes in gene expression suggest complex photosynthetic adjustments among light-harvesting complexes, reaction center subunits of photosystem I and II, and components of the dark reaction. Heat shock encoding proteins and reactive oxygen scavengers also were identified, but their overall frequency was too low to perform statistical tests. In all conditions, the most abundant transcript (3-15%) was a putative metallothionein gene with unknown function. We also find evidence that heat stress may translate to enhanced infection by protists. A total of 210 TUGs contain one or more microsatellites as potential candidates for gene-linked genetic markers. Data are publicly available in a user-friendly database at http://www.uni-muenster.de/Evolution/ebb/Services/zostera .

Genome sequence of the chlorinated compound-respiring bacterium Dehalococcoides species strain CBDB1.

Dehalococcoides species are strictly anaerobic bacteria, which catabolize many of the most toxic and persistent chlorinated aromatics and aliphatics by reductive dechlorination and are used for in situ bioremediation of contaminated sites. Our sequencing of the complete 1,395,502 base pair genome of Dehalococcoides strain CBDB1 has revealed the presence of 32 reductive-dehalogenase-homologous (rdh) genes, possibly conferring on the bacteria an immense dehalogenating potential. Most rdh genes were associated with genes encoding transcription regulators such as two-component regulatory systems or transcription regulators of the MarR-type. Four new paralog groups of rdh-associated genes without known function were detected. Comparison with the recently sequenced genome of Dehalococcoides ethenogenes strain 195 reveals a high degree of gene context conservation (synteny) but exceptionally high plasticity in all regions containing rdh genes, suggesting that these regions are under intense evolutionary pressure.

A catabolic gene cluster for anaerobic benzoate degradation in methanotrophic microbial Black Sea mats.

A microbial mat from the Black Sea shelf was analyzed by a metagenomic approach. While the habitat and its microbial community are characterized by anaerobic methane oxidation, a 79 kb contiguous DNA sequence obtained from the same mat provided first evidence for the concomitant presence of the capacity for anaerobic benzoate degradation. Benzoyl-CoA is one central intermediate of anaerobic aromatic degradation, among others. Within a stretch of 31 kb, all genes required for the complete pathway of anaerobic benzoate degradation (catabolic island) were identified, including the four subunits of the key enzyme benzoyl-CoA reductase (bcrCBAD), which catalyzes the ATP-driven 2-electron reduction of the aromatic ring. Genes for a ketoacid:acceptor oxidoreductase (korABC) and a ferredoxin (fdx), which are required for generation of a suitable electron donor, were also detected. The majority of the identified catabolic gene products are most similar to their respective orthologs from the denitrifying freshwater bacterium Azoarcus evansii, and the genes are also similarly organized. Due to the lack of established markers, the phylogenetic affiliation of the source organism remains unclear. The presented findings indicate that the metabolic diversity of the Black Sea mat is wider than currently known and that probably other bacteria than those of the methane-oxidizing consortia contribute to aromatic degradation in this anoxic habitat.

The genome of the kinetoplastid parasite, Leishmania major.

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.

The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1.

Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process. Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons. A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins. Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters. The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1. Metabolic versatility is also reflected by the presence of multiple respiratory complexes. A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment. The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster. Supplementary material on sequence and annotation are provided at the Web page http://www.micro-genomes.mpg.de/ebn1/.

Genes involved in the anaerobic degradation of toluene in a denitrifying bacterium, strain EbN1.

The organization of all genes required for the anaerobic conversion of toluene to benzoyl-CoA was investigated in denitrifying Azoarcus-like strain EbN1. All of these genes are clustered within 25.3 kb of contiguous DNA sequence, which includes only a few intervening sequences. The toluene-catabolic genes are organized in two apparent operons. One contains the genes ( bssCAB) for the three subunits of benzylsuccinate synthase, which initiates anaerobic toluene degradation by converting toluene to ( R)-benzylsuccinate. The BssCAB proteins of strain EbN1 are most similar to those of Thauera aromatica strain K172. The bssCAB genes are part of a larger putative operon ( bssDCABEFGH), which contains the gene bssD, encoding the activase for benzylsuccinate synthase, and four genes ( bssEFGH) encoding proteins of unknown function. RT-PCR experiments showing continuation of transcription over the three largest intergenic regions of the bss operon support the assumed structure. Moreover, BssG was identified as toluene-induced protein. Downstream of the bss genes, another large putative operon ( bbsA- H) was identified that contains all genes required for beta-oxidation of benzylsuccinate to benzoyl-CoA, e.g. bbsEF, encoding succinyl-CoA:( R)-benzylsuccinate CoA-transferase. Immediately upstream of the bss operon, genes for a two-component regulatory system were identified; their products may sense toluene and induce the expression of both catabolic operons. The order and sequences of the bss and bbs genes are highly similar among toluene-degrading denitrifiers. The bss and bbs genes of the Fe(III)-reducing Geobacter metallireducens display less sequence similarity and are organized differently. The genes between the bss and bbs operons and in the flanking regions differ between strain EbN1 and the other strains.

Generation, annotation, evolutionary analysis, and database integration of 20,000 unique sea urchin EST clusters.

Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies, the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analyzed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalization by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalog comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the base of deuterostome evolution. Comparison of the number of gene copies in sea urchins to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. All cDNA clones and filters of all analyzed libraries are available from the resource center of the German genome project at http://www.rzpd.de.

Genes involved in the anaerobic degradation of ethylbenzene in a denitrifying bacterium, strain EbN1.

Genes involved in anaerobic degradation of the petroleum hydrocarbon ethylbenzene in the denitrifying Azoarcus-like strain EbN1 were identified on a 56-kb DNA contig obtained from shotgun sequencing. Ethylbenzene is first oxidized via ethylbenzene dehydrogenase to (S)-1-phenylethanol; this is converted by (S)-1-phenylethanol dehydrogenase to acetophenone. Further degradation probably involves acetophenone carboxylase forming benzoylacetate, a ligase forming benzoylacetyl-CoA, and a thiolase forming acetyl-CoA and benzoyl-CoA. Genes of this pathway were identified via N-terminal sequences of proteins isolated from strain EbN1 and by sequence similarities to proteins from other bacteria. Ethylbenzene dehydrogenase is encoded by three genes (ebdABC), in accordance with the heterotrimeric enzyme structure. Binding domains for a molybdenum cofactor (in subunit EbdA) and iron/sulfur-clusters (in subunits EbdA and EbdB) were identified. The previously observed periplasmic location of the enzyme was corroborated by the presence of a twin-arginine leader peptide characteristic of the Tat system for protein export. A fourth gene (ebdD) was identified, the product of which may act as an enzyme-specific chaperone in the maturation of the molybdenum-containing subunit. A distinct gene (ped) coding for (S)-1-phenylethanol dehydrogenase apparently forms an operon with the ebdABCD genes. The ped gene product with its characteristic NAD(P)-binding motif in the N-terminal domain belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. A further operon apparently contains five genes (apc1-5) suggested to code for subunits of acetophenone carboxylase. Four of the five gene products are similar to subunits of acetone carboxylase from Xanthobacter autotrophicus. Upstream of the apc genes, a single gene (bal) was identified which possibly codes for a benzoylacetate CoA-ligase and which is co-transcribed with the apc genes. In addition, an apparent operon containing almost all genes required for beta-oxidation of fatty acids was detected; one of the gene products may be involved in thiolytic cleavage of benzoylacetyl-CoA. The DNA fragment also included genes for regulatory systems; these were two sets of two-component systems, two LysR homologs, and a TetR homolog. Some of these proteins may be involved in ethylbenzene-dependent gene expression.