Genome, transcriptome and phenotype analyses of Corynebacteria with biotechnological relevance

Al-Dilaimi A. Genome, transcriptome and phenotype analyses of Corynebacteria with biotechnological relevance. Bielefeld: Universität Bielefeld; 2016.In the course of the pan-genome sequencing project for the genus Corynebacterium, two bacteria, Corynebacterium glyciniphilum and Corynebacterium vitaeruminis, attracted attention due to their strong historical background towards L-serine and vitamin B production, respectively. In this context, the aim of this thesis was to characterize these bacteria based on their genome, transcriptome and phenotype by applying state-of-the-art high-throughput technologies, metabolic reconstructions and identification of specific genetic features. The first accomplishment of this study was the valid description of C. glyciniphilum as a new species of the genus Corynebacterium based on chemotaxonomic and phylogenetic as well as enzymatic and morphological studies. Genome sequencing revealed that this species possesses the largest genome of its genus. In addition, an approach combining phenotypic, genomic and transcriptomic data allowed to the optimization of genome annotation and provided insights into the extended metabolic repertoire of this bacterium. Due to the common background in amino acid production, the transcriptome of C. glyciniphilum was compared to the previously published transcriptome of C. glutamicum. Thereby, parallels were identified in terms of the transcriptional architecture, such as promoter and ribosome-binding site motifs. However, the two bacteria reflected divergence in terms of the regulation on the transcriptome and translatome level. While the transcriptome of C. glutamicum reflects a wider range of cis-regulatory elements influencing the translation of transcripts, C. glyciniphilum possesses an enlarged protein-coding regulatory repertoire, such as sigma factors and response regulators. Nonetheless, the existence of two glycine riboswitches arranged in tandem and located in the leader sequence of the glycine cleavage system transcript provides an explanation for the previously described high glycine tolerance of C. glyciniphilum that supports an enhanced L-serine production. The second bacterium investigated in this thesis was the cow rumen isolate C. vitaeruminis. The characterization of this bacterium by correlating phenotype and genome features revealed a high adaptation to the ruminal habitat. The analysis of the genome sequence architecture revealed the presence of two prophage regions in addition to two CRISPR loci, of which one is the largest known within the phylum Actinobacteria. In addition, a gene cluster encoding a complete CRISPR-Cas9 system has been identified within the two CRISPR loci. Recently, this CRISPR-Cas system type became famous in the field of targeted gene editing due to its specificity, simplicity and versatility. Further, comparative gene content analysis of C. vitaeruminis and closely related bacteria as well as C. glutamicum reflected an adaptation to low oxygen levels and the competence for persistence and colonization of the rumen. Complementary analyses on the phenotype and genome level pointed out a mutualistic relationship between C. vitaeruminis, the microbial community in the rumen and its host. Screening of metabolized carbon sources revealed preferences for mono- and di-carboxylic acids, which are the by-products of a normal fiber digesting rumen microbiota. Moreover, the demonstrated production of significant amounts of riboflavin and niacin under anaerobic conditions indicates a potential supplementation of the rumen with this vitamin by C. vitaeruminis. The combination of complementary information derived from the genome, transcriptome and phenotype applied in this thesis resulted in an extensive in-depth characterization of two bacteria. Both possess biotechnologically interesting features that can be used to enhance the yield of amino acids, like L-serine and L-methionine, or B vitamins, like riboflavin and niacin, in other bacteria. Furthermore, the identified CRISRP-Cas9 system of C. vitaeruminis provides opportunity for a customized application in corynebacteria. Taking together, this thesis demonstrates that a complementary application of state-of-the-art technologies on different omics levels in combination with sophisticated bioinformatic application tools provides a valuable tool for a precise characterization and gain of new biological perspectives on the lifestyle of organisms