Prisca Viehöver

The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analysed by the 454-pyrosequencing technology

Composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was analysed by means of a metagenomic approach applying the ultrafast 454-pyrosequencing technology. Sequencing of isolated total community DNA on a Genome Sequencer FLX System resulted in 616,072 reads with an average read length of 230 bases accounting for 141,664,289 bases sequence information. Assignment of obtained single reads to COG (Clusters of Orthologous Groups of proteins) categories revealed a genetic profile characteristic for an anaerobic microbial consortium conducting fermentative metabolic pathways. Assembly of single reads resulted in the formation of 8752 contigs larger than 500 bases in size. Contigs longer than 10kb mainly encode house-keeping proteins, e.g. DNA polymerase, recombinase, DNA ligase, sigma factor RpoD and genes involved in sugar and amino acid metabolism. A significant portion of contigs was allocated to the genome sequence of the archaeal methanogen Methanoculleus marisnigri JR1. Mapping of single reads to the M. marisnigri JR1 genome revealed that approximately 64% of the reference genome including methanogenesis gene regions are deeply covered. These results suggest that species related to those of the genus Methanoculleus play a dominant role in methanogenesis in the analysed fermentation sample. Moreover, assignment of numerous contig sequences to clostridial genomes including gene regions for cellulolytic functions indicates that clostridia are important for hydrolysis of cellulosic plant biomass in the biogas fermenter under study. Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production.

Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing

The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

A unique transcriptome: 1782 positions of RNA editing alter 1406 codon identities in mitochondrial mRNAs of the lycophyte Isoetes engelmannii

The analysis of the mitochondrial DNA of Isoetes engelmannii as a first representative of the lycophytes recently revealed very small introns and indications for extremely frequent RNA editing. To analyze functionality of intron splicing and the extent of RNA editing in I. engelmannii, we performed a comprehensive analysis of its mitochondrial transcriptome. All 30 groups I and II introns were found to be correctly removed, showing that intron size reduction does not impede splicing. We find that mRNA editing affects 1782 sites, which lead to a total of 1406 changes in codon meanings. This includes the removal of stop codons from 23 of the 25 mitochondrial protein encoding genes. Comprehensive sequence analysis of multiple cDNAs per locus allowed classification of partially edited sites as either inefficiently edited but relevant or as non-specifically edited at mostly low frequencies. Abundant RNA editing was also found to affect tRNAs in hitherto unseen frequency, taking place at 41 positions in tRNA-precursors, including the first identification of U-to-C exchanges in two tRNA species. We finally investigated the four group II introns of the nad7 gene and could identify 27 sites of editing, most of which improve base pairing for proper secondary structure formation.

Elongation‐related functions of LEAFY COTYLEDON1 during the development of Arabidopsis thaliana

The transcription factor LEAFY COTYLEDON1 (LEC1) controls aspects of early embryogenesis and seed maturation in Arabidopsis thaliana. To identify components of the LEC1 regulon, transgenic plants were derived in which LEC1 expression was inducible by dexamethasone treatment. The cotyledon-like leaves and swollen root tips developed by these plants contained seed-storage compounds and resemble the phenotypes produced by increased auxin levels. In agreement with this, LEC1 was found to mediate up-regulation of the auxin synthesis gene YUCCA10. Auxin accumulated primarily in the elongation zone at the root-hypocotyl junction (collet). This accumulation correlates with hypocotyl growth, which is either inhibited in LEC1-induced embryonic seedlings or stimulated in the LEC1-induced long-hypocotyl phenotype, therefore resembling etiolated seedlings. Chromatin immunoprecipitation analysis revealed a number of phytohormone- and elongation-related genes among the putative LEC1 target genes. LEC1 appears to be an integrator of various regulatory events, involving the transcription factor itself as well as light and hormone signalling, especially during somatic and early zygotic embryogenesis. Furthermore, the data suggest non-embryonic functions for LEC1 during post-germinative etiolation.

Comparative sequence analysis of Solanum and Arabidopsis in a hot spot for pathogen resistance on potato chromosome V reveals a patchwork of conserved and rapidly evolving genome segments

BackgroundQuantitative phenotypic variation of agronomic characters in crop plants is controlled by environmental and genetic factors (quantitative trait loci = QTL). To understand the molecular basis of such QTL, the identification of the underlying genes is of primary interest and DNA sequence analysis of the genomic regions harboring QTL is a prerequisite for that. QTL mapping in potato (Solanum tuberosum) has identified a region on chromosome V tagged by DNA markers GP21 and GP179, which contains a number of important QTL, among others QTL for resistance to late blight caused by the oomycete Phytophthora infestans and to root cyst nematodes.ResultsTo obtain genomic sequence for the targeted region on chromosome V, two local BAC (bacterial artificial chromosome) contigs were constructed and sequenced, which corresponded to parts of the homologous chromosomes of the diploid, heterozygous genotype P6/210. Two contiguous sequences of 417,445 and 202,781 base pairs were assembled and annotated. Gene-by-gene co-linearity was disrupted by non-allelic insertions of retrotransposon elements, stretches of diverged intergenic sequences, differences in gene content and gene order. The latter was caused by inversion of a 70 kbp genomic fragment. These features were also found in comparison to orthologous sequence contigs from three homeologous chromosomes of Solanum demissum, a wild tuber bearing species. Functional annotation of the sequence identified 48 putative open reading frames (ORF) in one contig and 22 in the other, with an average of one ORF every 9 kbp. Ten ORFs were classified as resistance-gene-like, 11 as F-box-containing genes, 13 as transposable elements and three as transcription factors. Comparing potato to Arabidopsis thaliana annotated proteins revealed five micro-syntenic blocks of three to seven ORFs with A. thaliana chromosomes 1, 3 and 5.ConclusionComparative sequence analysis revealed highly conserved collinear regions that flank regions showing high variability and tandem duplicated genes. Sequence annotation revealed that the majority of the ORFs were members of multiple gene families. Comparing potato to Arabidopsis thaliana annotated proteins suggested fragmented structural conservation between these distantly related plant species.

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction.

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

Natural variation in flavonol accumulation in Arabidopsis is determined by the flavonol glucosyltransferase BGLU6

Highlight BGLU6 is a single trait locus that causes natural qualitative variation in the accumulation of flavonol 3-O-gentiobioside 7-O-rhamnoside in Arabidopsis thaliana. BGLU6 encodes a glycoside hydrolase-type flavonol 3-O-glucoside: 6″-O-glucosyltransferase.

IncP-1β plasmids of Comamonas sp. and Delftia sp. strains isolated from a wastewater treatment plant mediate resistance to and decolorization of the triphenylmethane dye crystal violet.

The application of toxic triphenylmethane dyes such as crystal violet (CV) in various industrial processes leads to large amounts of dye-contaminated sludges that need to be detoxified. Specific bacteria residing in wastewater treatment plants (WWTPs) are able to degrade triphenylmethane dyes. The objective of this work was to gain insights into the genetic background of bacterial strains capable of CV degradation. Three bacterial strains isolated from a municipal WWTP harboured IncP-1β plasmids mediating resistance to and decolorization of CV. These isolates were assigned to the genera Comamonas and Delftia. The CV-resistance plasmid pKV29 from Delftia sp. KV29 was completely sequenced. In addition, nucleotide sequences of the accessory regions involved in conferring CV resistance were determined for plasmids pKV11 and pKV36 from the other two isolates. Plasmid pKV29 contains typical IncP-1β backbone modules that are highly similar to those of previously sequenced IncP-1β plasmids that confer antibiotic resistance, degradative capabilities or mercury resistance. The accessory regions located between the conjugative transfer (tra) and mating pair formation modules (trb) of all three plasmids analysed share common modules and include a triphenylmethane reductase gene, tmr, that is responsible for decolorization of CV. Moreover, these accessory regions encode other enzymes that are dispensable for CV degradation and hence are involved in so-far-unknown metabolic pathways. Analysis of plasmid-mediated degradation of CV in Escherichia coli by ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight MS revealed that leuco crystal violet was the first degradation product. Michlers ketone and 4-dimethylaminobenzaldehyde appeared as secondary degradation metabolites. Enzymes encoded in the E. coli chromosome seem to be responsible for cleavage of leuco crystal violet. Plasmid-mediated degradation of triphenylmethane dyes such as CV is an option for the biotechnological treatment of sludges contaminated with these dyes.

Reliable in silico identification of sequence polymorphisms and their application for extending the genetic map of sugar beet (Beta vulgaris).

Molecular markers are a highly valuable tool for creating genetic maps. Like in many other crops, sugar beet (Beta vulgaris L.) breeding is increasingly supported by the application of such genetic markers. Single nucleotide polymorphism (SNP) based markers have a high potential for automated analysis and high-throughput genotyping. We developed a bioinformatics workflow that uses Sanger and 2nd-generation sequence data for detection, evaluation and verification of new transcript-associated SNPs from sugar beet. RNAseq data from one parent of an established mapping population were produced by 454-FLX sequencing and compared to Sanger ESTs derived from the other parent. The workflow established for SNP detection considers the quality values of both types of reads, provides polymorphic alignments as well as selection criteria for reliable SNP detection and allows painless generation of new genetic markers within genes. We obtained a total of 14,323 genic SNPs and InDels. According to empirically optimised settings for the quality parameters, we classified these SNPs into four usability categories. Validation of a subset of the in silico detected SNPs by genotyping the mapping population indicated a high success rate of the SNP detection. Finally, a total of 307 new markers were integrated with existing data into a new genetic map of sugar beet which offers improved resolution and the integration of terminal markers.

A De Novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny

Arabidopsis thaliana is the most important model organism for fundamental plant biology. The genome diversity of different accessions of this species has been intensively studied, for example in the 1001 genome project which led to the identification of many small nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). In addition, presence/absence variation (PAV), copy number variation (CNV) and mobile genetic elements contribute to genomic differences between A. thaliana accessions. To address larger genome rearrangements between the A. thaliana reference accession Columbia-0 (Col-0) and another accession of about average distance to Col-0, we created a de novo next generation sequencing (NGS)-based assembly from the accession Niederzenz-1 (Nd-1). The result was evaluated with respect to assembly strategy and synteny to Col-0. We provide a high quality genome sequence of the A. thaliana accession (Nd-1, LXSY01000000). The assembly displays an N50 of 0.590 Mbp and covers 99% of the Col-0 reference sequence. Scaffolds from the de novo assembly were positioned on the basis of sequence similarity to the reference. Errors in this automatic scaffold anchoring were manually corrected based on analyzing reciprocal best BLAST hits (RBHs) of genes. Comparison of the final Nd-1 assembly to the reference revealed duplications and deletions (PAV). We identified 826 insertions and 746 deletions in Nd-1. Randomly selected candidates of PAV were experimentally validated. Our Nd-1 de novo assembly allowed reliable identification of larger genic and intergenic variants, which was difficult or error-prone by short read mapping approaches alone. While overall sequence similarity as well as synteny is very high, we detected short and larger (affecting more than 100 bp) differences between Col-0 and Nd-1 based on bi-directional comparisons. The de novo assembly provided here and additional assemblies that will certainly be published in the future will allow to describe the pan-genome of A. thaliana.