Oliver Rupp

The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins

The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g. of L-glutamate and L-lysine was determined. The C. glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs. Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g. a segment of DNA from C. diphtheriae and a prophage-containing region. After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins. These analyses confirm the taxonomic position of C. glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil. As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.

Insights into Genome Plasticity and Pathogenicity of the Plant Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria Revealed by the Complete Genome Sequence

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation–related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world’s annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714–758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant’s potential in energy biotechnology.

Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid

Agrobacterium sp. H13-3, formerly known as Rhizobium lupini H13-3, is a soil bacterium that was isolated from the rhizosphere of Lupinus luteus. The isolate has been established as a model system for studying novel features of flagellum structure, motility and chemotaxis within the family Rhizobiaceae. The complete genome sequence of Agrobacterium sp. H13-3 has been established and the genome structure and phylogenetic assignment of the organism was analysed. For de novo sequencing of the Agrobacterium sp. H13-3 genome, a combined strategy comprising 454-pyrosequencing on the Genome Sequencer FLX platform and PCR-based amplicon sequencing for gap closure was applied. The finished genome consists of three replicons and comprises 5,573,770 bases. Based on phylogenetic analyses, the isolate could be assigned to the genus Agrobacterium biovar I and represents a genomic species G1 strain within this biovariety. The highly conserved circular chromosome (2.82 Mb) of Agrobacterium sp. H13-3 mainly encodes housekeeping functions characteristic for an aerobic, heterotrophic bacterium. Agrobacterium sp. H13-3 is a motile bacterium driven by the rotation of several complex flagella. Its behaviour towards external stimuli is regulated by a large chemotaxis regulon and a total of 17 chemoreceptors. Comparable to the genome of Agrobacterium tumefaciens C58, Agrobacterium sp. H13-3 possesses a linear chromosome (2.15 Mb) that is related to its reference replicon and features chromosomal and plasmid-like properties. The accessory plasmid pAspH13-3a (0.6 Mb) is only distantly related to the plasmid pAtC58 of A. tumefaciens C58 and shows a mosaic structure. A tumor-inducing Ti-plasmid is missing in the sequenced strain H13-3 indicating that it is a non-virulent isolate.

Unraveling the Chinese hamster ovary cell line transcriptome by next-generation sequencing.

The pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences with the Newbler Assembler were used to achieve a major step forward to unravel the transcriptome of Chinese hamster ovary (CHO) cells. Normalized cDNA libraries originating from several cell lines and diverse culture conditions were sequenced and the resulting 1.84 million reads were assembled into 32,801 contiguous sequences, 29,184 isotigs, and 24,576 isogroups. A taxonomic classification of the isotigs showed that more than 70% of the assembled data is most similar to the transcriptome of Mus musculus, with most of the remaining isotigs being homologous to DNA sequences from Rattus norvegicus. Mapping of the CHO cell line contigs to the mouse transcriptome demonstrated that 9124 mouse transcripts, representing 6701 genes, are covered by more than 95% of their sequence length. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway of CHO cells were detected. The present manuscript represents a data set of assembled and annotated genes for CHO cells that can now be used for a detailed analysis of the molecular functioning of CHO cell lines.

Construction of a Public CHO Cell Line Transcript Database Using Versatile Bioinformatics Analysis Pipelines

Chinese hamster ovary (CHO) cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins. In this context, detailed knowledge of the CHO cell transcriptome might help to improve biotechnological processes conducted by specific cell lines. Nevertheless, very few assembled cDNA sequences of CHO cells were publicly released until recently, which puts a severe limitation on biotechnological research. Two extended annotation systems and web-based tools, one for browsing eukaryotic genomes (GenDBE) and one for viewing eukaryotic transcriptomes (SAMS), were established as the first step towards a publicly usable CHO cell genome/transcriptome analysis platform. This is complemented by the development of a new strategy to assemble the ca. 100 million reads, sequenced from a broad range of diverse transcripts, to a high quality CHO cell transcript set. The cDNA libraries were constructed from different CHO cell lines grown under various culture conditions and sequenced using Roche/454 and Illumina sequencing technologies in addition to sequencing reads from a previous study. Two pipelines to extend and improve the CHO cell line transcripts were established. First, de novo assemblies were carried out with the Trinity and Oases assemblers, using varying k-mer sizes. The resulting contigs were screened for potential CDS using ESTScan. Redundant contigs were filtered out using cd-hit-est. The remaining CDS contigs were re-assembled with CAP3. Second, a reference-based assembly with the TopHat/Cufflinks pipeline was performed, using the recently published draft genome sequence of CHO-K1 as reference. Additionally, the de novo contigs were mapped to the reference genome using GMAP and merged with the Cufflinks assembly using the cuffmerge software. With this approach 28,874 transcripts located on 16,492 gene loci could be assembled. Combining the results of both approaches, 65,561 transcripts were identified for CHO cell lines, which could be clustered by sequence identity into 17,598 gene clusters.

Utilization and evaluation of CHO-specific sequence databases for mass spectrometry based proteomics†

Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40–50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter‐species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome. Biotechnol. Bioeng. 2012; 109:1386–1394.

Computational identification of microRNA gene loci and precursor microRNA sequences in CHO cell lines

Highlights ► We mapped all known mature CHO miRNAs to two CHO-K1 reference genomes. ► 212 unique genomic miRNA loci and the respective precursor miRNA sequences were identified. ► The genomic loci of 4 polycistronic miRNA cluster were confirmed by PCR. ► The identified sequences were analyzed for SNPs and conservation compared to mouse. ► Sequence data have been prepared for submission to miRBase miRNA sequence repository.

The more the better – polyandry and genetic similarity are positively linked to reproductive success in a natural population of terrestrial salamanders (Salamandra salamandra)

Although classically thought to be rare, female polyandry is widespread and may entail significant fitness benefits. If females store sperm over extended periods of time, the consequences of polyandry will depend on the pattern of sperm storage, and some of the potential benefits of polyandry can only be realized if sperm from different males is mixed. Our study aimed to determine patterns and consequences of polyandry in an amphibian species, the fire salamander, under fully natural conditions. Fire salamanders are ideal study objects, because mating, fertilization and larval deposition are temporally decoupled, females store sperm for several months, and larvae are deposited in the order of fertilization. Based on 18 microsatellite loci, we conducted paternity analysis of 24 female‐offspring arrays with, in total, over 600 larvae fertilized under complete natural conditions. More than one‐third of females were polyandrous and up to four males were found as sires. Our data clearly show that sperm from multiple males is mixed in the females spermatheca. Nevertheless, paternity is biased, and the most successful male sires on average 70% of the larvae, suggesting a ‘topping off’ mechanism with first‐male precedence. Female reproductive success increased with the number of sires, most probably because multiple mating ensured high fertilization success. In contrast, offspring number was unaffected by female condition and genetic characteristics, but surprisingly, it increased with the degree of genetic relatedness between females and their sires. Sires of polyandrous females tended to be genetically similar to each other, indicating a role for active female choice.