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.
Xenovulene A is a complex fungal meroterpenoid, produced by the organism hitherto known as Acremonium strictum IMI 501407, for which limited biosynthetic evidence exists. Here, we generate a draft ...genome and show that the producing organism is previously unknown and should be renamed as Sarocladium schorii. A biosynthetic gene cluster is discovered which bears resemblance to those involved in the biosynthesis of fungal tropolones, with additional genes of unknown function. Heterologous reconstruction of the entire pathway in Aspergillus oryzae allows the chemical steps of biosynthesis to be dissected. The pathway shows very limited similarity to the biosynthesis of other fungal meroterpenoids. The pathway features: the initial formation of tropolone intermediates; the likely involvement of a hetero Diels-Alder enzyme; a terpene cyclase with no significant sequence homology to any known terpene cyclase and two enzymes catalysing oxidative-ring contractions.
In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from
Cricetulus griseus
remain the most commonly used host cell for recombinant protein production, especially antibodies. ...Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species.
Graphical Abstract
Key points
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First-time comprehensive protein and miRNA characterization of CHO exosomes.
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Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles.
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CHO-derived exosomes also contain numerous piRNA species of yet unknown function.
It is currently assumed that around 100 million years ago, the common ancestor to the Fabales, Fagales, Rosales and Cucurbitales in Gondwana, developed a root nodule symbiosis with a nitrogen-fixing ...bacterium. The symbiotic trait evolved first in Frankia cluster-2; thus, strains belonging to this cluster are the best extant representatives of this original symbiont. Most cluster-2 strains could not be cultured to date, except for Frankia coriariae, and therefore many aspects of the symbiosis are still elusive. Based on phylogenetics of cluster-2 metagenome-assembled genomes (MAGs), it has been shown that the genomes of strains originating in Eurasia are highly conserved. These MAGs are more closely related to Frankia cluster-2 in North America than to the single genome available thus far from the southern hemisphere, i.e., from Papua New Guinea.
The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) has been established (and is generally accepted) as a primary “universal” genetic barcode for fungi for many years, but the ...actual value for taxonomy has been heavily disputed among mycologists. Recently, twelve draft genome sequences, mainly derived from type species of the family Hypoxylaceae (Xylariales, Ascomycota) and the ex-epitype strain of
Xylaria hypoxylon
have become available during the course of a large phylogenomic study that was primarily aimed at establishing a correlation between the existing multi-gene-based genealogy with a genome-based phylogeny and the discovery of novel biosynthetic gene clusters encoding for secondary metabolites. The genome sequences were obtained using combinations of Illumina and Oxford nanopore technologies or PacBio sequencing, respectively, and resulted in high-quality sequences with an average N50 of 3.2 Mbp. While the main results will be published concurrently in a separate paper, the current case study was dedicated to the detection of ITS nrDNA copies in the genomes, in an attempt to explain certain incongruities and apparent mismatches between phenotypes and genotypes that had been observed during previous polyphasic studies. The results revealed that all of the studied strains had at least three copies of rDNA in their genomes, with
Hypoxylon fragiforme
having at least 19 copies of the ITS region, followed by
Xylaria hypoxylon
with at least 13 copies. Several of the genomes contained 2–3 copies that were nearly identical, but in some cases drastic differences, below 97% identity were observed. In one case, ascribable to the presence of a pseudogene, the deviations of the ITS sequences from the same genome resulted in only ca. 90% of overall homology. These results are discussed in the scope of the current trends to use ITS data for species recognition and segregation of fungi. We propose that additional genomes should be checked for such ITS polymorphisms to reassess the validity of this non-coding part of the fungal DNA for molecular identification.
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.
Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of ...metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.
The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors ...of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response.
Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed.
The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have overcome the stress condition. Here we propose a model of the regulation of oxidative and heat stress response including redox homeostasis by SigH, RshA and the thioredoxin system.
The isolation and sequencing of new strains of Pseudomonas aeruginosa created an extensive dataset of closed genomes. Many of the publicly available genomes are only used in their original ...publication while additional in silico information, based on comparison to previously published genomes, is not being explored. In this study, we defined and investigated the genome of the environmental isolate P. aeruginosa KRP1 and compared it to more than 100 publicly available closed P. aeruginosa genomes. By using different genomic island prediction programs, we could identify a total of 17 genomic islands and 8 genomic islets, marking the majority of the accessory genome that covers ~ 12% of the total genome. Based on intra-strain comparisons, we are able to predict the pathogenic potential of this environmental isolate. It shares a substantial amount of genomic information with the highly virulent PSE9 and LESB58 strains. For both of these, the increased virulence has been directly linked to their accessory genome before. Hence, the integrated use of previously published data can help to minimize expensive and time consuming wetlab work to determine the pathogenetic potential.
Histone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ...ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of
in the fungus
is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in
, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.
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