Complete Set Of Genes Research Articles

Expression of flagellin and key regulatory flagellar genes in the non-motile bacterium Piscirickettsia salmonis.

The Piscirickettsia salmonis genome was screened to evaluate potential flagella-related open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.

Expression map of a complete set of gustatory receptor genes in chemosensory organs of Bombyx mori

Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27–BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO2 receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for finding genes involved in plant-insect interactions in Lepidoptera and establishing correlations between these genes and vital insect behaviors like host plant selection and courtship for mating.

Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.)

BackgroundStarch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism.ResultsWe identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis.ConclusionsThis study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR.

A comprehensive custom panel design for routine hereditary cancer testing: preserving control, improving diagnostics and revealing a complex variation landscape

We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.

Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes

Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus “Kp15virus” within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between l-alanine and d-glutamic acid.

Genome analysis of 'Candidatus Ancillula trichonymphae', first representative of a deep-branching clade of Bifidobacteriales, strengthens evidence for convergent evolution in flagellate endosymbionts.

The flagellate protists in the hindgut of lower termites play an essential role in the digestion of lignocellulose. Most flagellate species are associated with host-specific symbionts from various bacterial lineages, which typically lack cultured representatives. In this study, we analyzed the genome of 'Candidatus Ancillula trichonymphae', an endosymbiont of Trichonympha flagellates from dry-wood termites, which represents a novel, family-level lineage of uncultured Actinobacteria encountered so far only in termite guts. The draft genome of 'Ca. A. trichonymphae' (ca. 1.48 Mbp; 95% complete) revealed a purely fermentative metabolism that is probably fueled by xylose, N-acetyl-glucosamine and glycerol 3-phosphate acquired from the flagellate host. The absence of fructose bisphosphate aldolase and the presence of a complete gene set encoding the phosphoketolase pathway underscore the sister position of the new lineage to Bifidobacteriaceae. The preservation of the pathways for the assimilation of ammonia and the synthesis of 18 amino acids and several cofactors and vitamins suggests that 'Ca. A. trichonymphae' - like other endosymbionts of termite gut flagellates - provides essential amino acids and vitamins to its host. Our findings corroborate the emerging concept that numerous lineages of unrelated flagellate endosymbionts have convergently evolved to fill similar ecological niches.

Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis

Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex fatty acid profile comprised of straight-chain fatty acids and branched-chain fatty acids (BCFAs), and encodes a complete set of genes required for fatty acid synthesis, there is still little known about the mechanism of BCFA synthesis. We reported that expression of Xcc fabH restores the growth of Ralstonia solanacearum fabH mutant, and this allows the R. solanacearum fabH mutant to produce BCFAs. Using in vitro assays, we demonstrated that Xcc FabH is able to condense branched-chain acyl-CoAs with malonyl-ACP to initiate BCFA synthesis. Moreover, although the fabH gene is essential for growth of Xcc, it can be replaced with Escherichia coli fabH, and Xcc mutants failed to produce BCFAs. These results suggest that Xcc does not have an obligatory requirement for BCFAs. Furthermore, Xcc mutants lost the ability to produce cis-11-methyl-2-dodecenoic acid, a diffusible signal factor (DSF) required for quorum sensing of Xcc, which confirms that the fatty acid synthetic pathway supplies the intermediates for DSF signal biosynthesis. Our study also showed that replacing Xcc fabH with E. coli fabH affected Xcc pathogenesis in host plants.

OGS2: genome re-annotation of the jewel wasp Nasonia vitripennis.

BackgroundNasonia vitripennis is an emerging insect model system with haplodiploid genetics. It holds a key position within the insect phylogeny for comparative, evolutionary and behavioral genetic studies. The draft genomes for N. vitripennis and two sibling species were published in 2010, yet a considerable amount of transcriptiome data have since been produced thereby enabling improvements to the original (OGS1.2) annotated gene set. We describe and apply the EvidentialGene method used to produce an updated gene set (OGS2). We also carry out comparative analyses showcasing the usefulness of the revised annotated gene set.ResultsThe revised annotation (OGS2) now consists of 24,388 genes with supporting evidence, compared to 18,850 for OGS1.2. Improvements include the nearly complete annotation of untranslated regions (UTR) for 97 % of the genes compared to 28 % of genes for OGS1.2. The fraction of RNA-Seq validated introns also grow from 85 to 98 % in this latest gene set. The EST and RNA-Seq expression data provide support for several non-protein coding loci and 7712 alternative transcripts for 4146 genes. Notably, we report 180 alternative transcripts for the gene lola.Nasonia now has among the most complete insect gene set; only 27 conserved single copy orthologs in arthropods are missing from OGS2. Its genome also contains 2.1-fold more duplicated genes and 1.4-fold more single copy genes than the Drosophila melanogaster genome. The Nasonia gene count is larger than those of other sequenced hymenopteran species, owing both to improvements in the genome annotation and to unique genes in the wasp lineage.We identify 1008 genes and 171 gene families that deviate significantly from other hymenopterans in their rates of protein evolution and duplication history, respectively. We also provide an analysis of alternative splicing that reveals that genes with no annotated isoforms are characterized by shorter transcripts, fewer introns, faster protein evolution and higher probabilities of duplication than genes having alternative transcripts.ConclusionsGenome-wide expression data greatly improves the annotation of the N. vitripennis genome, by increasing the gene count, reducing the number of missing genes and providing more comprehensive data on splicing and gene structure. The improved gene set identifies lineage-specific genomic features tied to Nasonia’s biology, as well as numerous novel genes.OGS2 and its associated search tools are available at http://arthropods.eugenes.org/EvidentialGene/nasonia/, www.hymenopteragenome.org/nasonia/ and waspAtlas: www.tinyURL.com/waspAtlas.The EvidentialGene pipeline is available at https://sourceforge.net/projects/evidentialgene/.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2886-9) contains supplementary material, which is available to authorized users.

Extensive complementarity between gene function prediction methods.

The number of sequenced genomes rises steadily but we still lack the knowledge about the biological roles of many genes. Automated function prediction (AFP) is thus a necessity. We hypothesized that AFP approaches that draw on distinct genome features may be useful for predicting different types of gene functions, motivating a systematic analysis of the benefits gained by obtaining and integrating such predictions. Our pipeline amalgamates 5133543 genes from 2071 genomes in a single massive analysis that evaluates five established genomic AFP methodologies. While 1227 Gene Ontology (GO) terms yielded reliable predictions, the majority of these functions were accessible to only one or two of the methods. Moreover, different methods tend to assign a GO term to non-overlapping sets of genes. Thus, inferences made by diverse genomic AFP methods display a striking complementary, both gene-wise and function-wise. Because of this, a viable integration strategy is to rely on a single most-confident prediction per gene/function, rather than enforcing agreement across multiple AFP methods. Using an information-theoretic approach, we estimate that current databases contain 29.2 bits/gene of known Escherichia coli gene functions. This can be increased by up to 5.5 bits/gene using individual AFP methods or by 11 additional bits/gene upon integration, thereby providing a highly-ranking predictor on the Critical Assessment of Function Annotation 2 community benchmark. Availability of more sequenced genomes boosts the predictive accuracy of AFP approaches and also the benefit from integrating them. The individual and integrated GO predictions for the complete set of genes are available from http://gorbi.irb.hr/ CONTACT: fran.supek@irb.hrSupplementary information: Supplementary materials are available at Bioinformatics online.

A Phenotypically Silent vanB2 Operon Carried on a Tn1549-Like Element in Clostridium difficile.

In the last decade, Clostridium difficile infection (CDI) has reached an epidemic state with increasing incidence and severity in both health care and community settings. Vancomycin is an important first-line therapy for CDI, and the emergence of resistance would have significant clinical consequences. In this study, we describe for the first time a vanB2 vancomycin resistance operon in C. difficile, isolated from an Australian veal calf at slaughter. The operon was carried on an ~42-kb element showing significant homology and synteny to Tn1549, a conjugative transposon linked with the emergence and global dissemination of vancomycin-resistant enterococci (VRE). Notably, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly as a result of an aberrant vanRB gene. As observed for other anaerobic species of the animal gut microbiota, C. difficile may be a reservoir of clinically important vancomycin resistance genes. IMPORTANCE In an era when the development of new antimicrobial drugs is slow, vancomycin remains the preferred antimicrobial therapy for Clostridium difficile infection (CDI), the most important health care-related infection in the world today. The emergence of resistance to vancomycin would have significant consequences in relation to treating patients with CDI. In this paper, we describe for the first time a complete set of vancomycin resistance genes in C. difficile. The genes were very similar to genes found in vancomycin-resistant enterococci (VRE) that were associated with the emergence and global dissemination of this organism. Fortunately, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly because of a small difference in one gene. However, this observation signals that we may be very close to seeing a fully vancomycin-resistant strain of C. difficile.

Gene set analysis for interpreting genetic studies.

Interpretation of genome-wide association study (GWAS) results is lacking behind the discovery of new genetic associations. Consequently, there is an urgent need for data-driven methods for interpreting genetic association studies. Gene set analysis (GSA) can identify aetiologic pathways and functional annotations and may hence point towards novel biological insights. However, despite the growing availability of GSA tools, the sizeable amount of variants identified for a vast number of complex traits, and many irrefutably trait-associated gene sets, the gap between discovery and interpretation remains. More efficient interpretation requires more complete and consistent gene set representations of biological pathways, phenotypes and functional annotations. In this review, I examine different types of gene sets, discuss how inconsistencies in gene set definitions impact GSA, describe how GSA has helped to elucidate biology and outline potential future directions.

Quorum sensing systems differentially regulate the production of phenazine-1-carboxylic acid in the rhizobacterium Pseudomonas aeruginosa PA1201

Pseudomonas aeruginosa strain PA1201 is a newly identified rhizobacterium that produces high levels of the secondary metabolite phenazine-1-carboxylic acid (PCA), the newly registered biopesticide Shenqinmycin. PCA production in liquid batch cultures utilizing a specialized PCA-promoting medium (PPM) typically occurs after the period of most rapid growth, and production is regulated in a quorum sensing (QS)-dependent manner. PA1201 contains two PCA biosynthetic gene clusters phz1 and phz2; both clusters contribute to PCA production, with phz2 making a greater contribution. PA1201 also contains a complete set of genes for four QS systems (LasI/LasR, RhlI/RhlR, PQS/MvfR, and IQS). By using several methods including gene deletion, the construction of promoter-lacZ fusion reporter strains, and RNA-Seq analysis, this study investigated the effects of the four QS systems on bacterial growth, QS signal production, the expression of phz1 and phz2, and PCA production. The possible mechanisms for the strain- and condition-dependent expression of phz1 and phz2 were discussed, and a schematic model was proposed. These findings provide a basis for further genetic engineering of the QS systems to improve PCA production.

Genome Analysis of Thermosulfurimonas dismutans, the First Thermophilic Sulfur-Disproportionating Bacterium of the Phylum Thermodesulfobacteria.

Thermosulfurimonas dismutans S95T, isolated from a deep-sea hydrothermal vent is the first bacterium of the phylum Thermodesulfobacteria reported to grow by the disproportionation of elemental sulfur, sulfite, or thiosulfate with carbon dioxide as the sole carbon source. In contrast to its phylogenetically close relatives, which are dissimilatory sulfate-reducers, T. dismutans is unable to grow by sulfate respiration. The features of this organism and its 2,1 Mb draft genome sequence are described in this report. Genome analysis revealed that the T. dismutans genome contains the set of genes for dissimilatory sulfate reduction including ATP sulfurylase, the AprA and B subunits of adenosine-5′-phosphosulfate reductase, and dissimilatory sulfite reductase. The oxidation of elemental sulfur to sulfite could be enabled by APS reductase-associated electron transfer complex QmoABC and heterodisulfide reductase. The genome also contains several membrane-linked molybdopterin oxidoreductases that are thought to be involved in sulfur metabolism as subunits of thiosulfate, polysulfide, or tetrathionate reductases. Nitrate could be used as an electron acceptor and reduced to ammonium, as indicated by the presence of periplasmic nitrate and nitrite reductases. Autotrophic carbon fixation is enabled by the Wood–Ljungdahl pathway, and the complete set of genes that is required for nitrogen fixation is also present in T. dismutans. Overall, our results provide genomic insights into energy and carbon metabolism of chemolithoautotrophic sulfur-disproportionating bacterium that could be important primary producer in microbial communities of deep-sea hydrothermal vents.

Inferring active regulatory networks from gene expression data using a combination of prior knowledge and enrichment analysis.

BackgroundUnder both physiological and pathological conditions gene expression programs are shaped through the interplay of regulatory proteins and their gene targets, interactions between which form intricate gene regulatory networks (GRN). While the assessment of genome-wide expression for the complete set of genes at a given condition has become rather straight-forward and is performed routinely, we are still far from being able to infer the topology of gene regulation simply by analyzing its “descendant” expression profile. In this work we are trying to overcome the existing limitations for the inference and study of such regulatory networks. We are combining our approach with state-of-the-art gene set enrichment analyses in order to create a tool, called Regulatory Network Enrichment Analysis (RNEA) that will prioritize regulatory and functional characteristics of a genome-wide expression experiment.ResultsRNEA combines prior knowledge, originating from manual literature curation and small-scale experimental data, to construct a reference network of interactions and then uses enrichment analysis coupled with a two-level hierarchical parsing of the network, to infer the most relevant subnetwork for a given experimental setting. It is implemented as an R package, currently supporting human and mouse datasets and was herein tested on one test case for each of the two organisms. In both cases, RNEA’s gene set enrichment analysis was comparable to state-of-the-art methodologies. Moreover, through its distinguishing feature of regulatory subnetwork reconstruction, RNEA was able to define the key transcriptional regulators for the studied systems as supported from the literature.ConclusionsRNEA constitutes a novel computational approach to obtain regulatory interactions directly from a genome-wide expression profile. Its simple implementation, with minimal requirements from the user is coupled with easy-to-parse enrichment lists and a subnetwork file that may be readily visualized to reveal the most important components of the regulatory hierarchy. The combination of prior information and novel concept of a hierarchical reconstruction of regulatory interactions makes RNEA a very useful tool for a first-level interpretation of gene expression profiles.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1040-7) contains supplementary material, which is available to authorized users.

Persistent Alterations in Microglial Enhancers in a Model of Chronic Pain.

Chronic pain is a common and devastating condition that induces well-characterized changes in neurons and microglia. One major unanswered question is why these changes should persist long after the precipitating injury has healed. Here, we suggest that some of the longer-lasting consequences of nerve injury may be hidden in the epigenome. Cell sorting and sequencing techniques were used to characterize the spinal cord immune response in a mouse model of chronic neuropathic pain. Infiltration of peripheral myeloid cells was found to be absent, and RNA sequencing (RNA-seq) of central microglia revealed transient gene expression changes in response to nerve ligation. Conversely, examination of microglial enhancers revealed persistent, post-injury alterations in close proximity to transcriptionally regulated genes. Enhancers are regions of open chromatin that define a cell's transcription factor binding profile. We hypothesize that changes at enhancers may constitute a mechanism by which painful experiences are recorded at a molecular level.

'Candidatus Adiutrix intracellularis', an endosymbiont of termite gut flagellates, is the first representative of a deep-branching clade of Deltaproteobacteria and a putative homoacetogen.

Termite gut flagellates are typically colonized by specific bacterial symbionts. Here we describe the phylogeny, ultrastructure and subcellular location of 'Candidatus Adiutrix intracellularis', an intracellular symbiont of Trichonympha collaris in the termite Zootermopsis nevadensis. It represents a novel, deep-branching clade of uncultured Deltaproteobacteria widely distributed in intestinal tracts of termites and cockroaches. Fluorescence in situ hybridization and transmission electron microscopy localized the endosymbiont near hydrogenosomes in the posterior part and near the ectosymbiont 'Candidatus Desulfovibrio trichonymphae' in the anterior part of the host cell. The draft genome of 'Ca. Adiutrix intracellularis' obtained from a metagenomic library revealed the presence of a complete gene set encoding the Wood-Ljungdahl pathway, including two homologs of fdhF encoding hydrogenase-linked formate dehydrogenases (FDHH ) and all other components of the recently described hydrogen-dependent carbon dioxide reductase (HDCR) complex, which substantiates previous claims that the symbiont is capable of reductive acetogenesis from CO2 and H2 . The close phylogenetic relationship between the HDCR components and their homologs in homoacetogenic Firmicutes and Spirochaetes suggests that the deltaproteobacterium acquired the capacity for homoacetogenesis via lateral gene transfer. The presence of genes for nitrogen fixation and the biosynthesis of amino acids and cofactors indicate the nutritional nature of the symbiosis.

Biomineralization of atrazine and analysis of 16S rRNA and catabolic genes of atrazine degraders in a former pesticide mixing site and a machinery washing area

The purpose of this study was to determine the first-order rate constants and half-lives of aerobic and anaerobic biomineralization of atrazine in soil samples from an agricultural farm site that had been previously used for mixing pesticide formulations and washing application equipment. Atrazine catabolic genes and atrazine-degrading bacteria in the soil samples were analyzed by molecular methods. Biomineralization of atrazine was measured in soil samples with a [U-ring-14C]-atrazine biometer technique in soil samples. Enrichment cultures growing with atrazine were derived from soil samples and they were analyzed for bacterial diversity by constructing 16S rDNA clone libraries and sequencing. Bacterial isolates were also obtained and they were screened for atrazine catabolic genes. The soils contained active atrazine-metabolizing microbial communities and both aerobic and anaerobic biomineralization of [U-ring-14C]-atrazine to 14CO2 was demonstrated. In contrast to aerobic incubations, anaerobic biometers displayed considerable differences in the kinetics of atrazine mineralization between duplicates. Sequence analysis of 16S rDNA clone libraries constructed from the enrichment cultures revealed a preponderance of Variovorax spp. (51 %) and Schlesneria (16 %). Analysis of 16S rRNA gene sequences from pure cultures (n = 12) isolated from enrichment cultures yielded almost exclusively Arthrobacter spp. (83 %; 10/12 isolates). PCR screening of pure culture isolates for atrazine catabolic genes detected atzB, atzC, trzD, trzN, and possibly atzA. The presence of a complete metabolic pathway was not demonstrated by the amplification of catabolic genes among these isolates. The soils contained active atrazine-metabolizing microbial communities. The anaerobic biometer data showed variable response of atrazine biomineralization to external electron acceptor conditions. Partial pathways are inevitable in soil microbial communities, with metabolites linking into other catabolic and assimilative pathways of carbon and nitrogen. There was no evidence for the complete set of functional genes of the known pathways of atrazine biomineralization among the isolates.

Characterization and analysis of ribosomal proteins in two marine calanoid copepods

Copepods are among the most abundant and successful metazoans in the marine ecosystem. However, genomic resources related to fundamental cellular processes are still limited in this particular group of crustaceans. Ribosomal proteins are the building blocks of ribosomes, the primary site for protein synthesis. In this study, we characterized and analyzed the cDNAs of cytoplasmic ribosomal proteins (cRPs) of two calanoid copepods, Pseudodiaptomus poplesia and Acartia pacifica. We obtained 79 cRP cDNAs from P. poplesia and 67 from A. pacifica by cDNA library construction/sequencing and rapid amplification of cDNA ends. Analysis of the nucleic acid composition showed that the copepod cRP-encoding genes had higher GC content in the protein-coding regions (CDSs) than in the untranslated regions (UTRs), and single nucleotide repeats (>3 repeats) were common, with “A” repeats being the most frequent, especially in the CDSs. The 3′-UTRs of the cRP genes were significantly longer than the 5′-UTRs. Codon usage analysis showed that the third positions of the codons were dominated by C or G. The deduced amino acid sequences of the cRPs contained high proportions of positively charged residues and had high pI values. This is the first report of a complete set of cRP-encoding genes from copepods. Our results shed light on the characteristics of cRPs in copepods, and provide fundamental data for further studies of protein synthesis in copepods. The copepod cRP information revealed in this study indicates that additional comparisons and analysis should be performed on different taxonomic categories such as orders and families.

Sodium-Driven Energy Conversion for Flagellar Rotation of the Earliest Divergent Hyperthermophilic Bacterium

Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here, we cultured this hyperthermophilic bacterium and observed motile behavior with a variable–temperature chamber for optical microscopy [1, 2]. The swimming of the cells was about 90 µm s−1 at 85 °C, and decreased with decreasing temperature of the chamber [3]. Next, we expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na+. As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na+ for energy coupling of the flagellar motor. The Na+-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.[1] Nishiyama, M. and Sowa Y. 2012. Microscopic Analysis of Bacterial Motility at High Pressure. Biophys. J. 102, 1872-1880.[2] Nishiyama, M. et al. 2013. High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor. J. Bactetiol. 195, 1809-1814.[3] Takekawa, N. et al. 2015. Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium. Sci. Rep. 5, 12711.

Structural and functional annotation of the MADS-box transcription factor family in grapevine.

BackgroundMADS-box genes encode transcription factors that are involved in developmental control and signal transduction in eukaryotes. In plants, they are associated to numerous development processes most notably those related to reproductive development: flowering induction, specification of inflorescence and flower meristems, establishment of flower organ identity, as well as regulation of fruit, seed and embryo development. Genomic analyses of MADS-box genes in different plant species are providing new relevant information on the function and evolution of this transcriptional factor family. We have performed a true genome-wide analysis of the complete set of MADS-box genes in grapevine (Vitis vinifera), analyzed their expression pattern and establish their phylogenetic relationships (including MIKC* and type I MADS-box) with genes from 16 other plant species. This study was integrated to previous works on the family in grapevine.ResultsA total of 90 MADS-box genes were detected in the grapevine reference genome by completing current gene annotations with a genome-wide analysis based on sequence similarity. We performed a thorough in-depth curation of all gene models and combined the results with gene expression information including RNAseq data to clarifying the expression of newly identified genes and improve their functional characterization. Curated data were uploaded to the ORCAE database for grapevine in the frame of the grapevine genome curation effort. This approach resulted in the identification of 30 additional MADS box genes. Among them, ten new MIKCC genes were identified, including a potential new group of short proteins similar to the SVP protein subfamily. The MIKC* subgroup contains six genes in grapevine that can be grouped in the S (4 genes) and P (2 genes) clades, showing less redundancy than that observed in Arabidopsis thaliana. Expression pattern of these genes in grapevine is compatible with a role in male gametophyte development. Most of the identified new genes belong to the type I MADS-box genes and were classified as members of the Mα and Mγ subclasses. Ours analyses indicate that only few members of type I genes in grapevine have homology in other species and that species-specific clades appeared both in the Mα and Mγ subclasses. On the other hand, as deduced from the phylogenetic analysis with other plant species, genes that can be crucial for development of central cell, endosperm and embryos seems to be conserved in plants.ConclusionsThe genome analysis of MADS-box genes in grapevine, the characterization of their pattern of expression and the phylogenetic analysis with other plant species allowed the identification of new MADS-box genes not yet described in other plant species as well as basic characterization of their possible role, particularly in the case of type I and MIKC* genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2398-7) contains supplementary material, which is available to authorized users.