Background: The use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. The aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organismCorynebacterium glutamicumby RNAseq in order to improve its genome annotation and to describe important features for transcription and translation.Results: RNAseq data sets were obtained by two methods, one that focuses on 5′-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3′-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5′-UTRs (untranslated regions) for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. Interestingly, a high number of mRNAs (33%) is transcribed as leaderless transcripts. From the data, consensus promoter and ribosome binding site (RBS) motifs were identified and it was shown that the majority of genes inC. glutamicumare transcribed monocistronically, but operons containing up to 16 genes are also present.Conclusions: The comprehensive transcriptome map ofC. glutamicumestablished in this study represents a major step forward towards a complete definition of genetic elements (e.g. promoter regions, gene starts and stops, 5′-UTRs, RBSs, transcript starts and ends) and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. The methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future.

Genome mining tools have enabled us to predict biosynthetic gene clusters that might encode compounds with valuable functions for industrial and medical applications. With the continuously increasing number of genomes sequenced, we are confronted with an overwhelming number of predicted clusters. In order to guide the effective prioritization of biosynthetic gene clusters towards finding the most promising compounds, knowledge about diversity, phylogenetic relationships and distribution patterns of biosynthetic gene clusters is necessary. Here, we provide a comprehensive analysis of the model actinobacterial genus Amycolatopsis and its potential for the production of secondary metabolites. A phylogenetic characterization, together with a pan-genome analysis showed that within this highly diverse genus, four major lineages could be distinguished which differed in their potential to produce secondary metabolites. Furthermore, we were able to distinguish gene cluster families whose distribution correlated with phylogeny, indicating that vertical gene transfer plays a major role in the evolution of secondary metabolite gene clusters. Still, the vast majority of the diverse biosynthetic gene clusters were derived from clusters unique to the genus, and also unique in comparison to a database of known compounds. Our study on the locations of biosynthetic gene clusters in the genomes of Amycolatopsis’ strains showed that clusters acquired by horizontal gene transfer tend to be incorporated into non-conserved regions of the genome thereby allowing us to distinguish core and hypervariable regions in Amycolatopsis genomes. Using a comparative genomics approach, it was possible to determine the potential of the genus Amycolatopsis to produce a huge diversity of secondary metabolites. Furthermore, the analysis demonstrates that horizontal and vertical gene transfer play an important role in the acquisition and maintenance of valuable secondary metabolites. Our results cast light on the interconnections between secondary metabolite gene clusters and provide a way to prioritize biosynthetic pathways in the search and discovery of novel compounds.

Summary: l ‐Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l ‐histidine biosynthesis has been studied mainly in E scherichia coli and S almonella typhimurium , revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid‐producing bacterium C orynebacterium glutamicum , revealing similarities to E . coli and S . typhimurium , as well as differences. This review summarizes the current knowledge of l ‐histidine biosynthesis in C . glutamicum . The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol‐phosphate synthase ( HisFH ) and the histidinol‐phosphate phosphatase ( HisN ). The transcriptional organization of his genes in C . glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C . glutamicum strains for the industrial l ‐histidine production.

The Hypoxylaceae (Xylariales, Ascomycota) is a diverse family of mainly saprotrophic fungi, which commonly occur in angiosperm-dominated forests around the world. Despite their importance in forest and plant ecology as well as a prolific source of secondary metabolites and enzymes, genome sequences of related taxa are scarce and usually derived from environmental isolates. To address this lack of knowledge thirteen taxonomically well-defined representatives of the family and one member of the closely related Xylariaceae were genome sequenced using combinations of Illumina and Oxford nanopore technologies or PacBio sequencing. The workflow leads to high quality draft genome sequences with an average N50 of 3.0 Mbp. A backbone phylogenomic tree was calculated based on the amino acid sequences of 4912 core genes reflecting the current accepted taxonomic concept of the Hypoxylaceae. A Percentage of Conserved Proteins (POCP) analysis revealed that 70% of the proteins are conserved within the family, a value with potential application for the definition of family boundaries within the order Xylariales. Also, Hypomontagnella spongiphila is proposed as a new marine derived lineage of Hypom. monticulosa based on in-depth genomic comparison and morphological differences of the cultures. The results showed that both species share 95% of their genes corresponding to more than 700 strain-specific proteins. This difference is not reflected by standard taxonomic assessments (morphology of sexual and asexual morph, chemotaxonomy, phylogeny), preventing species delimitation based on traditional concepts. Genetic changes are likely to be the result of environmental adaptations and selective pressure, the driving force of speciation. These data provide an important starting point for the establishment of a stable phylogeny of the Xylariales; they enable studies on evolution, ecological behavior and biosynthesis of natural products; and they significantly advance the taxonomy of fungi.

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of ‘superfolder’ green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 μg/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens.

Background: The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results: Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 ± 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions: Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense.