Untersuchung der Klassifizierung der bakteriellen Begleitflora in veterinärmedizinischen Proben

Basierend auf 16S rRNA Gensequenzanalysen konnte für die Bakterienstämme 256 II, 408, 1000 und 2224 gezeigt werden, dass sie zur B. cereus-Gruppe gehören. Der PCR-Nachweis für Cereulid (B. cereus spezifisches hitzestabiles Toxin) war negativ. Daher können diese Stämme zumindest vorerst als harmlos angesehen werden. Eine zuverlässige Identifizierung auf Artebene konnte mit den verwendeten Techniken allerdings nicht erreicht werden. Die Isolate 1600 (S. xylosus), 2289 (M. odoratimimus), 2388 (M. odoratimimus) konnten auf Grund der Ergebnisse der Fingerabdrücke eindeutig einer bestimmten Art zugeordnet werden. Für das Isolat 410 hatte schon die Analyse des 16S rRNA Gens angedeutet, dass es sich um eine neue Art des Genus Corynebacterium handelt. Die weiteren Ergebnisse der durchgeführten Analysen bestätigten dies und die gesammelten Daten wurden in einem Manuskript zusammengefast, das im Anhang zu finden ist. Ein Teil dieser Untersuchungen wurde von den in der Autorenliste aufgeführten Kollegen durchgeführt. Die Isolate 256-I, 793, 886, 2236 und 1656 konnten mit Hilfe der verwendeten Analysen nicht zu einer bestimmten Art zugeordnet werden. Die Zuordnung zu den Gattungen Pseudomonas (256-I), Comamonas (793), Acinetobacter (888), Kurthia (2236) und Solibacillus (1656) kann aber als abgesichert gelten. Es ist gut zu erkennen, dass die verwendeten Methoden ausreichen, um in vielen Fällen eine Artzugehörigkeit aufzuzeigen. Allerdings wären in anderen Fällen weitere Untersuchungen nötig gewesen, die Isolate zuverlässig einer Art zuzuordnen. In diesen Fällen kann aber auch nicht ausgeschlossen werden, dass das jeweilige Isolat zu einer noch nicht beschriebenen Art gehört was dann natürlich eine Identifizierung auf Artniveau unmöglich macht. Allgemein ist über die Normalflora beim Menschen viel bekannt. Für Tiere ist zu diesem Thema allerdings kaum Literatur zu finden. Die im Rahmen dieser Arbeit durchgeführten Untersuchungen deuten aber nicht an, dass sich die Normalflora beim Tier substantiell von der beim Menschen unterscheidet

Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction

Abstract Background The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei, the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. Results Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1, and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. Conclusion We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose expression is absent in QM9978. We propose that in T. reesei, as in Neurospora crassa, vib1 is involved in cellulase induction, although the exact mechanism remains to be elucidated. The data presented here show an example of a combined genome sequencing and transcriptomic approach to explain a specific trait, in this case the QM9978 cellulase-negative phenotype, and how it helps to better understand the mechanisms during cellulase gene regulation. When focusing on mutations on the single base-pair level, changes on the chromosome level can be easily overlooked and through this work we provide an example that stresses the importance of the big picture of the genomic landscape during analysis of sequencing data

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species.

The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for "hot topic" research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway