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Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

Abstract Background Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome. Results P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood. Conclusions The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.

Abstract Background Chloroflexus aurantiacus is a thermophilic filamentous anoxygenic phototrophic (FAP) bacterium, and can grow phototrophically under anaerobic conditions or chemotrophically under aerobic and dark conditions. According to 16S rRNA analysis, Chloroflexi species are the earliest branching bacteria capable of photosynthesis, and Cfl. aurantiacus has been long regarded as a key organism to resolve the obscurity of the origin and early evolution of photosynthesis. Cfl. aurantiacus contains a chimeric photosystem that comprises some characters of green sulfur bacteria and purple photosynthetic bacteria, and also has some unique electron transport proteins compared to other photosynthetic bacteria. Methods The complete genomic sequence of Cfl. aurantiacus has been determined, analyzed and compared to the genomes of other photosynthetic bacteria. Results Abundant genomic evidence suggests that there have been numerous gene adaptations/replacements in Cfl. aurantiacus to facilitate life under both anaerobic and aerobic conditions, including duplicate genes and gene clusters for the alternative complex III (ACIII), auracyanin and NADH:quinone oxidoreductase; and several aerobic/anaerobic enzyme pairs in central carbon metabolism and tetrapyrroles and nucleic acids biosynthesis. Overall, genomic information is consistent with a high tolerance for oxygen that has been reported in the growth of Cfl. aurantiacus. Genes for the chimeric photosystem, photosynthetic electron transport chain, the 3-hydroxypropionate autotrophic carbon fixation cycle, CO2-anaplerotic pathways, glyoxylate cycle, and sulfur reduction pathway are present. The central carbon metabolism and sulfur assimilation pathways in Cfl. aurantiacus are discussed. Some features of the Cfl. aurantiacus genome are compared with those of the Roseiflexus castenholzii genome. Roseiflexus castenholzii is a recently characterized FAP bacterium and phylogenetically closely related to Cfl. aurantiacus. According to previous reports and the genomic information, perspectives of Cfl. aurantiacus in the evolution of photosynthesis are also discussed. Conclusions The genomic analyses presented in this report, along with previous physiological, ecological and biochemical studies, indicate that the anoxygenic phototroph Cfl. aurantiacus has many interesting and certain unique features in its metabolic pathways. The complete genome may also shed light on possible evolutionary connections of photosynthesis.

Abstract Background Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains. Results With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains. Conclusions In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.

Abstract Background Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process. Results Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae. Conclusions Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.

Abstract Background Initial interest in Dechloromonas aromatica strain RCB arose from its ability to anaerobically degrade benzene. It is also able to reduce perchlorate and oxidize chlorobenzoate, toluene, and xylene, creating interest in using this organism for bioremediation. Little physiological data has been published for this microbe. It is considered to be a free-living organism. Results The a priori prediction that the D. aromatica genome would contain previously characterized "central" enzymes to support anaerobic aromatic degradation of benzene proved to be false, suggesting the presence of novel anaerobic aromatic degradation pathways in this species. These missing pathways include the benzylsuccinate synthase (bssABC) genes (responsible for fumarate addition to toluene) and the central benzoyl-CoA pathway for monoaromatics. In depth analyses using existing TIGRfam, COG, and InterPro models, and the creation of de novo HMM models, indicate a highly complex lifestyle with a large number of environmental sensors and signaling pathways, including a relatively large number of GGDEF domain signal receptors and multiple quorum sensors. A number of proteins indicate interactions with an as yet unknown host, as indicated by the presence of predicted cell host remodeling enzymes, effector enzymes, hemolysin-like proteins, adhesins, NO reductase, and both type III and type VI secretory complexes. Evidence of biofilm formation including a proposed exopolysaccharide complex and exosortase (epsH) are also present. Annotation described in this paper also reveals evidence for several metabolic pathways that have yet to be observed experimentally, including a sulphur oxidation (soxFCDYZAXB) gene cluster, Calvin cycle enzymes, and proteins involved in nitrogen fixation in other species (including RubisCo, ribulose-phosphate 3-epimerase, and nif gene families, respectively). Conclusion Analysis of the D. aromatica genome indicates there is much to be learned regarding the metabolic capabilities, and life-style, for this microbial species. Examples of recent gene duplication events in signaling as well as dioxygenase clusters are present, indicating selective gene family expansion as a relatively recent event in D. aromatica's evolutionary history. Gene families that constitute metabolic cycles presumed to create D. aromatica's environmental 'foot-print' indicate a high level of diversification between its predicted capabilities and those of its close relatives, A. aromaticum str EbN1 and Azoarcus BH72.

Abstract Background The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. Results The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second putative succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. Conclusion The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae.

Abstract Background Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. Results The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced – Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. Conclusion The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

Introduction: Soil microbiome is a major source of physiologically active microorganisms, which can be potentially mobilized by adding various nutrients. To study this process, a long-term experiment was conducted on the decomposition of oat straw and leaf litter using soil as a microbial inoculum., Methods: Combined analyses of enzymatic activity and NGS data for 16S rRNA gene amplicon and full metagenome sequencing were applied to study taxonomic, carbohydrate-active enzyme (CAZy), and polysaccharide utilization loci (PULs) composition of microbial communities at different stages of decomposition between substrates., Results: In straw degradation, the microbial community demonstrated higher amylase, protease, catalase, and cellulase activities, while peroxidase, invertase, and polyphenol oxidase were more active in leaf litter. Consistent with this, the metagenome analysis showed that the microbiome of straw compost was enriched in genes for metabolic pathways of simpler compounds. At the same time, there were more genes for aromatic compound degradation pathways in leaf litter compost. We identified nine metagenome-assembled genomes (MAGs) as the most promising prokaryotic decomposers due to their abnormally high quantity of PULs for their genome sizes, which were confirmed by 16S rRNA gene amplicon sequencing to constitute the bulk of the community at all stages of substrate degradation. MAGs from Bacteroidota ( Chitinophaga and Ohtaekwangia ) and Actinomycetota ( Streptomyces ) were found in both composts, while those from Bacillota ( Pristimantibacillus ) were specific for leaf litter. The most frequently identified PULs were specialized on xylans and pectins, but not cellulose, suggesting that PUL databases may be underrepresented in clusters for complex substrates., Discussion: Our study explores microbial communities from natural ecosystems, such as soil and lignocellulosic waste, which are capable of decomposing lignocellulosic substrates. Using a comprehensive approach with chemical analyses of the substrates, amplicon, and full metagenome sequencing data, we have shown that such communities may be a source of identifying the highly effective decomposing species with novel PULs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Kimeklis, Gladkov, Orlova, Lisina, Afonin, Aksenova, Kichko, Lapidus, Abakumov and Andronov.)

For the targeted selection of microbial communities that provide cellulose degradation, soil samples containing cellulolytic microorganisms and specific plant residues as a substrate can be used. The details of this process have not been studied: in particular, whether the use of different soils determines the varying efficiency of communities; whether these established cellulolytic communities will have substrate specificity, and other factors. To answer these questions, four soil microbial communities with different cellulolytic activity (Podzol and the soil of Chernevaya taiga) and substrates (oat straw and hemp shives) with different levels of cellulose availability were used, followed by trained communities that were tested on botrooth substrates (in all possible combinations). Based on the analysis of the taxonomic structure of all communities and their efficiency across all substrates (decomposition level, carbon, and nitrogen content), it was shown that the most important taxa of all trained microbial cellulolytic communities are recruited from secondary soil taxa. The original soil does not affect the efficiency of cellulose decomposition: both soils produce equally active communities. Unexpectedly, the resulting communities trained on oats were more effective on hemp than the communities trained on hemp. In general, the usage of pre-trained microbial communities increases the efficiency of decomposition.

Annotating newly sequenced genomes and determining alternative isoforms from long-read RNA data are complex and incompletely solved problems. Here we present IsoQuant-a computational tool using intron graphs that accurately reconstructs transcripts both with and without reference genome annotation. For novel transcript discovery, IsoQuant reduces the false-positive rate fivefold and 2.5-fold for Oxford Nanopore reference-based or reference-free mode, respectively. IsoQuant also improves performance for Pacific Biosciences data., (© 2023. The Author(s).)

While metagenome sequencing may provide insights on the genome sequences and composition of microbial communities, metatranscriptome analysis can be useful for studying the functional activity of a microbiome. RNA-Seq data provides the possibility to determine active genes in the community and how their expression levels depend on external conditions. Although the field of metatranscriptomics is relatively young, the number of projects related to metatranscriptome analysis increases every year and the scope of its applications expands. However, there are several problems that complicate metatranscriptome analysis: complexity of microbial communities, wide dynamic range of transcriptome expression and importantly, the lack of high-quality computational methods for assembling meta-RNA sequencing data. These factors deteriorate the contiguity and completeness of metatranscriptome assemblies, therefore affecting further downstream analysis. Here we present MetaGT, a pipeline for de novo assembly of metatranscriptomes, which is based on the idea of combining both metatranscriptomic and metagenomic data sequenced from the same sample. MetaGT assembles metatranscriptomic contigs and fills in missing regions based on their alignments to metagenome assembly. This approach allows to overcome described complexities and obtain complete RNA sequences, and additionally estimate their abundances. Using various publicly available real and simulated datasets, we demonstrate that MetaGT yields significant improvement in coverage and completeness of metatranscriptome assemblies compared to existing methods that do not exploit metagenomic data. The pipeline is implemented in NextFlow and is freely available from https://github.com/ablab/metaGT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shafranskaya, Kale, Finn, Lapidus, Korobeynikov and Prjibelski.)

Metagenomics is a segment of conventional microbial genomics dedicated to the sequencing and analysis of combined genomic DNA of entire environmental samples. The most critical step of the metagenomic data analysis is the reconstruction of individual genes and genomes of the microorganisms in the communities using metagenomic assemblers - computational programs that put together small fragments of sequenced DNA generated by sequencing instruments. Here, we describe the challenges of metagenomic assembly, a wide spectrum of applications in which metagenomic assemblies were used to better understand the ecology and evolution of microbial ecosystems, and present one of the most efficient microbial assemblers, SPAdes that was upgraded to become applicable for metagenomics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lapidus and Korobeynikov.)

Dethiobacter alkaliphilus strain AHT1 T is an anaerobic, sulfidogenic, moderately salt-tolerant alkaliphilic chemolithotroph isolated from hypersaline soda lake sediments in northeastern Mongolia. It is a Gram-positive bacterium with low GC content, within the phylum Firmicutes . Here we report its draft genome sequence, which consists of 34 contigs with a total sequence length of 3.12 Mbp. D. alkaliphilus strain AHT1 T was sequenced by the Joint Genome Institute (JGI) as part of the Community Science Program due to its relevance to bioremediation and biotechnological applications.

Desulfurivibrio alkaliphilus strain AHT2(T) is a strictly anaerobic sulfidogenic haloalkaliphile isolated from a composite sediment sample of eight hypersaline alkaline lakes in the Wadi al Natrun valley in the Egyptian Libyan Desert. D. alkaliphilus AHT2(T) is Gram-negative and belongs to the family Desulfobulbaceae within the Deltaproteobacteria. Here we report its genome sequence, which contains a 3.10 Mbp chromosome. D. alkaliphilus AHT2(T) is adapted to survive under highly alkaline and moderately saline conditions and therefore, is relevant to the biotechnology industry and life under extreme conditions. For these reasons, D. alkaliphilus AHT2(T) was sequenced by the DOE Joint Genome Institute as part of the Community Science Program.

Novel nanohybrid materials were prepared by immobilizing Co nanoparticles on a microporous framework MIL-53(Al) as a porous host matrix. The synthesized cobalt-containing materials were characterized by XRD, STEM, and oxygen titration. The catalytic performance of Co@MIL-53(Al) nanohybrids was examined in Fischer-Tropsch synthesis (FTS) for the first time. A higher selectivity to C5+ hydrocarbons and lower selectivity to methane for Co@MIL-53(Al) as compared to conventional Co/Al2O3 were observed.

Methanospirillum hungatei strain JF1 (DSM 864) is a methane-producing archaeon and is the type species of the genus Methanospirillum, which belongs to the family Methanospirillaceae within the order Methanomicrobiales. Its genome was selected for sequencing due to its ability to utilize hydrogen and carbon dioxide and/or formate as a sole source of energy. Ecologically, M. hungatei functions as the hydrogen- and/or formate-using partner with many species of syntrophic bacteria. Its morphology is distinct from other methanogens with the ability to form long chains of cells (up to 100 μm in length), which are enclosed within a sheath-like structure, and terminal cells with polar flagella. The genome of M. hungatei strain JF1 is the first completely sequenced genome of the family Methanospirillaceae, and it has a circular genome of 3,544,738 bp containing 3,239 protein coding and 68 RNA genes. The large genome of M. hungatei JF1 suggests the presence of unrecognized biochemical/physiological properties that likely extend to the other Methanospirillaceae and include the ability to form the unusual sheath-like structure and to successfully interact with syntrophic bacteria.

The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 °C versus 60-65 °C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported--on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A., Database: Structural data are available in the Protein Data Bank under the accession numbers 5AMP, 4ZZV, 4ZZW, 4ZZT, and 4ZZU. The Geotrichum candidum GH family 7 cellobiohydrolase nucleotide sequence is available in GenBank under accession number KJ958925., Enzymes: Glycoside hydrolase family 7 reducing end acting cellobiohydrolase., (© 2015 FEBS.)

Bacteroides barnesiae Lan et al. 2006 is a species of the genus Bacteroides, which belongs to the family Bacteroidaceae. Strain BL2(T) is of interest because it was isolated from the gut of a chicken and the growing awareness that the anaerobic microbiota of the caecum is of benefit for the host and may impact poultry farming. The 3,621,509 bp long genome with its 3,059 protein-coding and 97 RNA genes is a part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.

Desulfovibrio carbinoliphilus subsp. oakridgensis FW-101-2B is an anaerobic, organic acid/alcohol-oxidizing, sulfate-reducing δ-proteobacterium. FW-101-2B was isolated from contaminated groundwater at The Field Research Center at Oak Ridge National Lab after in situ stimulation for heavy metal-reducing conditions. The genome will help elucidate the metabolic potential of sulfate-reducing bacteria during uranium reduction., (Copyright © 2015 Ramsay et al.)

Background: Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome., Description: Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes., Conclusions: Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains.

Syntrophobacter fumaroxidans strain MPOB(T) is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project.

Desulfovibrio alaskensis G20 (formerly Desulfovibrio desulfuricans G20) is a Gram-negative mesophilic sulfate-reducing bacterium (SRB), known to corrode ferrous metals and to reduce toxic radionuclides and metals such as uranium and chromium to sparingly soluble and less toxic forms. We present the 3.7-Mb genome sequence to provide insights into its physiology.

Desulfovibrio desulfuricans strain ND132 is an anaerobic sulfate-reducing bacterium (SRB) capable of producing methylmercury (MeHg), a potent human neurotoxin. The mechanism of methylation by this and other organisms is unknown. We present the 3.8-Mb genome sequence to provide further insight into microbial mercury methylation.

Syntrophomonas wolfei is a specialist, evolutionarily adapted for syntrophic growth with methanogens and other hydrogen- and/or formate-using microorganisms. This slow-growing anaerobe has three putative ribosome RNA operons, each of which has 16S rRNA and 23S rRNA genes of different length and multiple 5S rRNA genes. The genome also contains 10 RNA-directed, DNA polymerase genes. Genomic analysis shows that S. wolfei relies solely on the reduction of protons, bicarbonate or unsaturated fatty acids to re-oxidize reduced cofactors. Syntrophomonas wolfei lacks the genes needed for aerobic or anaerobic respiration and has an exceptionally limited ability to create ion gradients. An ATP synthase and a pyrophosphatase were the only systems detected capable of creating an ion gradient. Multiple homologues for β-oxidation genes were present even though S. wolfei uses a limited range of fatty acids from four to eight carbons in length.Syntrophomonas wolfei, other syntrophic metabolizers with completed genomic sequences, and thermophilic anaerobes known to produce high molar ratios of hydrogen from glucose have genes to produce H(2) from NADH by an electron bifurcation mechanism. Comparative genomic analysis also suggests that formate production from NADH may involve electron bifurcation. A membrane-bound, iron-sulfur oxidoreductase found in S. wolfei and Syntrophus aciditrophicus may be uniquely involved in reverse electron transport during syntrophic fatty acid metabolism. The genome sequence of S. wolfei reveals several core reactions that may be characteristic of syntrophic fatty acid metabolism and illustrates how biological systems produce hydrogen from thermodynamically difficult reactions., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

We have developed a new method for frameshift detection, a combination of ab initio and alignment-based algorithms, that can serve as a useful tool for sequencing quality control in the next generation sequencing. We evaluated the method's accuracy on test sets of annotated genomic sequences with artificial frameshifts in protein coding regions. These tests have shown that the new method performs comparably to the earlier developed FrameD. On the sets of sequences produced by 454 pyrosequencing with sequence errors recovered by Sanger re-sequencing the accuracy of the method was shown to hold at the same level.

A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121-126]. In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and a low Cop at 28 degrees C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42 degrees C. Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-1 beta (hIL-1 beta) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1 beta (re-hIL-1 beta) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the water-soluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1 beta. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3-5 mg of re-hIL-1 beta/g of wet cells. The re-hIL-1 beta specific activity was about 2 x 10(8) units/mg, coinciding with that of the authentic hIL-1 beta.

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human angiogenin has been synthesized in Escherichia coli with the aid of a human angiogenin gene (hAng) cloned by Neznanov et al (1990) from a human complementary DNA (cDNA) library. The gene has been expressed by use of a new type of expression vector called a 'TGATG vector' (plasmid pPR-TGATG-1; Mashko et al 1990a). The highest level of accumulation of the recombinant angiogenin (6%-8% of the total cell protein) was observed in E. coli strain BL21 carrying a temperature-amplifiable version of the plasmid. The synthesized polypeptide carries an additional serine residue at its N terminus in comparison with natural angiogenin. Furthermore, the initiator methionine residue of the recombinant protein is removed with high efficiency by E. coli terminal aminopeptidase. Simple procedures for purification of the recombinant angiogenin from the insoluble fraction of cell protein, and for refolding the protein allowed the isolation of almost 5 mg recombinant angiogenin g-1 wet bacterial biomass. The recombinant Ser-(-1) angiogenin displayed the same biological properties (specific RNAase activity and the ability to induce blood vessel growth on the sclera of experimental animals) as its natural counterpart isolated from human blood.

A new method of optimization of foreign gene expression in E. coli, based on the construction of hybrid operons with partially overlapping genes is described. The partial overlapping of the translation termination and initiation sites in the formed operon must provide translational coupling of appropriate gene product synthesis. Such an approach has provided the synthesis of human interferon alpha F in E. coli cells under the control of the lacUV5-promotor up to about (3-4).10(7) units per liter of bacterial culture. The reinitiation of the distal gene translation is shown to take place in the intercistronic region. Substitution of the lacUV5 promotor by the more efficient tac one allowed to increase the synthesis level of interferon alpha F to (1-2).10(8) units per liter. The conclusion is made about the equimolarity of distal and proximal to the promotor genes products syntheses when the intercistronic region of E. coli trpE-trpD genes are used for translational coupling.

The trpOP, lacUV5, tacOP, PR and PL-promotors were cloned in the previously obtained pML4 vector plasmid. The expression of structural gene cat was studied by the chloramphenicolacetyltransferase determination in cell extracts. The level of protein synthesis by appropriate recombinant plasmids was analysed in vivo and in vitro. It was shown that the efficiency of the gene expression is determined by both the "strength" of the promotors and mRNA translation specificity. The obtained collection of the plasmids might be used for the determination of the promotor strength by the hybridization of pulse-labeled mRNA with DNA and an effective expression of the genes by means of "hybrid protein gene" and "hybrid operon" constructions.

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.

The possibility of creating artificial overlappons was studied on the model of two genes, that coding for the N-terminal part of lambda cro protein and the cat of E. coli. To test the dependence of translational coupling efficiency on the intercistronic region a series of recombinant DNA molecules carrying different hybrid operons with partially overlapping genes was constructed. The translational efficiency of the distal to the promoter gene was shown to depend on the intercistronic region structure: overlapping of the AUG codon with the terminating one of the proximal gene in the UGAUG manner is optimal for the translational coupling, and the displacement of AUG at several nucleotides in both directions decreases the translational reinitiation efficiency for the ribosomes, that have synthesised the first gene product.

The basic results of the studies on expression of the genes of human alpha F- and beta 1-interferons in E. coli cells are presented. To synthesize the fibroblast interferon, the respective fragment of the human chromosome was cloned, the complete nucleotide sequence of the structural moiety of mature beta-interferon was determined and the genes of "hybrid (interferon-like) proteins" and "hybrid sites of ribosome binding" were constructed with control of the beta-interferon gene by the prokaryotic regulatory areas. Synthesis of beta-interferon was achieved (1.10(7)-5.10(7) IU per 1 l of the bacterial culture) with the use of the tryptophan operon promoter. A new procedure for optimization of allogenic genetic information in E. coli cells: constructing of "hybrid operons with partially overlapping genes" or artificial "overlappons" was developed following the example of the alpha F-interferon gene cloned in the Laboratory headed by E. D. Sverdlov at the Institute of Bioorganic Chemistry of the USSR Academy of Sciences. The use of this procedure enabled production of up to 5.10(7) IU/l of alpha F-interferon under the control of the lacUV5-promoter. On the basis of the newly constructed vector molecules expression of the genes of alpha F- and beta 1-interferons was amplified with the "overlappon" procedure.

The study on the rate of initiation of model gene cat transcription under the control of E. coli (Plac UV5, Ptrp, Pcat, Ptac), phage lambda (PL, PR), phi X174 (PD) promotors was carried out by means of hybridization of pulse labelled in vivo mRNA with the DNA coding parts. The presence of gene bla(Apr) with its own constitutive promoter in all the recombinants permitted to use the level of appropriate mRNA in the cell as an internal standard. This method allowed to evaluate the true efficiency of the promoters in question. The strength of the promoters studied is shown to be equal within the limit of one order value.

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.