Biology subjects

Jorgensen, T. S.

Publications and source records attributed to Jorgensen, T. S..

15 recordsLinked to original sources

bioRxiv2026

A long-hidden prevalent PCR artifact and its application in DNA library screening

PCR artifacts can lead to aberrant DNA products and confound result interpretation, posing significant challenges in research and diagnostics. Traditionally, it has been blamed to misannealing of primers on unintended sites. However, by sequencing, we find that most PCR byproducts and artifacts actually have both primers bound correctly but lack a middle part of the target, a phenomenon we named as PCR leaping. The leaping occurs at random positions, is independent of sequence repeats and requires a single piece template, indicating an unprecedented mechanism. It is observed with various polymerases, template qualities, GC contents, and PCR programs, thus necessitating a reset of our PCR understanding and careful consideration in future studies, for example in CRISPR editing validations. On the other hand, it provides a simple way of DNA end pairing, based on which we develop a high throughput DNA library screening method for natural product discovery at substantially reduced cost.

molecular biology↗

bioRxiv2025

Tying up loose ends: Recovering thousands of missing telomeres from Streptomyces and other Streptomycetaceae genomes

Members of the Gram-positive Streptomycetaceae family of bacteria have linear chromosomes and carry linear plasmids, which end in telomeres bound by proteins. In a large-scale analysis of 762 linear complete genomes, we discovered that the telomeres were truncated in most assemblies, as they are not captured by Oxford Nanopore sequencing. To address this issue, we present Telomore, a tool to reconstitute this missing telomeric sequence using ONT and Illumina data. In the studied dataset, Telomore increased detection of archetypal telomeres from 0% to 37%, which could be near the occurrence rate in nature. Combining these reconstituted telomeres with previously published telomeres and all complete Streptomycetaceae RefSeq genomes, we created a compendium of more than 2000 telomeres. Similarity-based clustering identified 137 telomere clusters. We find that 78% of Telomore-extended chromosomes encode both telomeres, while this is only the case for 15% of comparable RefSeq chromosomes. Therefore, most assignments of "complete" to Streptomycetaceae are erroneous. Finally, we mined the 762 genomes for known telomeric maintenance proteins and used those to identify a plasmid-specific archetypal telomere and to identify a previously unidentified protein family likely involved with the maintenance of Sg2247-class telomeres. Together, these results highlight a common issue assembling complete linear Streptomycetaceae genomes and provide a programmatic solution and identify a candidate for a new telomeric protein. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/682034v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1adbb96org.highwire.dtl.DTLVardef@f68519org.highwire.dtl.DTLVardef@9c50b4org.highwire.dtl.DTLVardef@768c00_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Spirolactone, an unprecedented antifungal β-lactone spiroketal macrolide from Streptomyces iranensis

Fungal infections pose a great threat to public health and there are limited antifungal medicaments. Streptomyces is an important source of antibiotics, represented by the clinical drug amphotericin B. The rapamycin-producer Streptomyces iranensis harbors an unparalleled Type I polyketide synthase, which codes for a novel antifungal macrolide alligamycin A (1), the structure of which was confirmed by NMR, MS, and X-ray crystallography. Alligamycin A harbors an undescribed carbon skeleton with 13 chiral centers, featuring a ({beta}-lactone moiety, a [6,6]-spiroketal ring, and an unprecedented 7-oxo-octylmalonyl-CoA extender unit incorporated by a potential novel crotonyl-CoA carboxylase/reductase. The ali biosynthetic gene cluster was confirmed through CRISPR-based gene editing. Alligamycin A displayed profound antifungal effects against numerous clinically relevant filamentous fungi, including Talaromyces and Aspergillus species. ({beta}-Lactone ring is essential for the antifungal activity and alligamycin B (2) with disruption in the ring abolished the antifungal effect. Proteomics analysis revealed alligamycin A potentially disrupted the integrity of fungal cell walls and induced the expression of stress-response proteins in Aspergillus niger. Alligamycins represent a new class of potential drug candidate to combat fungal infections.

biochemistry↗

bioRxiv2024

Pangenome mining of the Streptomyces genus redefines their biosynthetic potential

BackgroundStreptomyces is a highly diverse genus known for the production of secondary or specialized metabolites with a wide range of applications in the medical and agricultural industries. Several thousand complete or nearly-complete Streptomyces genome sequences are now available, affording the opportunity to deeply investigate the biosynthetic potential within these organisms and to advance natural product discovery initiatives. ResultWe performed pangenome analysis on 2,371 Streptomyces genomes, including approximately 1,200 complete assemblies. Employing a data-driven approach based on genome similarities, the Streptomyces genus was classified into 7 primary and 42 secondary MASH-clusters, forming the basis for a comprehensive pangenome mining. A refined workflow for grouping biosynthetic gene clusters (BGCs) redefined their diversity across different MASH-clusters. This workflow also reassigned 2,729 known BGC families to only 440 families, a reduction caused by inaccuracies in BGC boundary detections. When the genomic location of BGCs is included in the analysis, a conserved genomic structure (synteny) among BGCs becomes apparent within species and MASH-clusters. This synteny suggests that vertical inheritance is a major factor in the acquisition of new BGCs. ConclusionOur analysis of a genomic dataset at a scale of thousands of genomes refined predictions of BGC diversity using MASH-clusters as a basis for pangenome analysis. The observed conservation in the order of BGCs genomic locations showed that the BGCs are vertically inherited. The presented workflow and the in-depth analysis pave the way for large-scale pangenome investigations and enhance our understanding of the biosynthetic potential of the Streptomyces genus.

systems biology↗

bioRxiv2024

A treasure trove of 1,034 actinomycete genomes

Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters benefit from or require chromosome-level assemblies. Here, we provide DNA sequences from more than 1,000 isolates: 881 complete genomes and 153 near-complete genomes, representing 28 genera and 389 species, including 244 likely novel species. All genomes are from filamentous isolates of the class Actinomycetia from the NBC culture collection. The largest genus is Streptomyces with 886 genomes including 742 complete assemblies. We use this data to show that analysis of complete genomes can bring biological understanding not previously derived from more fragmented sequences or less systematic datasets. We document the central and structured location of core genes and distal location of specialized metabolite biosynthetic gene clusters and duplicate core genes on the linear Streptomyces chromosome, and analyze the content and length of the terminal inverted repeats which are characteristic for Streptomyces. We then analyze the diversity of trans-AT polyketide synthase biosynthetic gene clusters, which encodes the machinery of a biotechnologically highly interesting compound class. These insights have both ecological and biotechnological implications in understanding the importance of high quality genomic resources and the complex role synteny plays in Actinomycetia biology.

microbiology↗

bioRxiv2023

Biosynthesis of the azoxy compound azodyrecin from Streptomyces mirabilis P8-A2

Azoxy compounds are a distinctive group of bioactive secondary metabolites, characterized by a unique RN=N+(O-)R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S. albidoflavus J1074 and S. coelicolor M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knock-out using CRISPR-Cas9 base editing in the native producer, S. mirabilis P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to azo compound; followed by further oxygenation. Additionally, N-oxygenase and dehydrogenase activities were confirmed among 8 core biosynthetic genes and five helper genes. Moreover, the distribution of the azoxy biosynthetic gene clusters across Streptomyces spp. genomes is explored, highlighting the presence of these clusters in over 20% of the Streptomyces spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A like clusters are widely distributed across the phylogenetic tree.

microbiology↗

bioRxiv2021

Identification of the 216kbp gene cluster and structure elucidation of gargantulides B and C, new complex 52-membered macrolides from Amycolatopsis sp.

Gargantulides B and C, two new and highly complex 52-membered glycosylated macrolactones, were isolated from Amycolatopsis sp. strain CA-230715 during an antibacterial screening campaign. The structures of these giant macrolides were elucidated by 2D NMR spectroscopy and shown to be related to gargantulide A, although containing additional {beta}-glucopyranose and/or -arabinofuranose monosaccharides separately attached to their backbones. Genome sequencing allowed the identification of a strikingly large 216 kbp biosynthetic gene cluster, among the largest type I PKS clusters described so far, and the proposal of a biosynthetic pathway for gargantulides A-C. Additionally, genes putatively responsible for the biosynthesis of the amino sugar {beta}-3,6-deoxy-3-methylamino glucose, reported exclusively in gargantulide macrolides, were also found in the cluster and described in this work. The absolute configurations of gargantulides B and C were assigned based on a combination of NMR and bioinformatics analysis of ketoreductase and enoylreductase domains within the multimodular type I PKS. Furthermore, the absolute stereochemistry of the related macrolide gargantulide A has now been revised and completed. Gargantulides B and C display potent antibacterial activity against a set of drug-resistant Gram-positive bacteria and moderate activity against the clinically relevant Gram-negative pathogen Acinetobacter baumannii.

microbiology↗

bioRxiv2021

Critical Assessment of Metagenome Interpretation - the second round of challenges

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the community-driven initiative for the Critical Assessment of Metagenome Interpretation (CAMI). In its second challenge, CAMI engaged the community to assess their methods on realistic and complex metagenomic datasets with long and short reads, created from [~]1,700 novel and known microbial genomes, as well as [~]600 novel plasmids and viruses. Altogether 5,002 results by 76 program versions were analyzed, representing a 22x increase in results. Substantial improvements were seen in metagenome assembly, some due to using long-read data. The presence of related strains still was challenging for assembly and genome binning, as was assembly quality for the latter. Taxon profilers demonstrated a marked maturation, with taxon profilers and binners excelling at higher bacterial taxonomic ranks, but underperforming for viruses and archaea. Assessment of clinical pathogen detection techniques revealed a need to improve reproducibility. Analysis of program runtimes and memory usage identified highly efficient programs, including some top performers with other metrics. The CAMI II results identify current challenges, but also guide researchers in selecting methods for specific analyses.

bioinformatics↗

bioRxiv2020

Characterization and Engineering of Streptomyces griseofuscus DSM 40191 as a Potential Host for Heterologous Expression of Biosynthetic Gene Clusters

Streptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Using CRISPR-BEST technology, core genes of 4 biosynthetic gene clusters (BGCs) that are situated on the chromosome arms were inactivated and the outcomes of the inactivations were tested. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5,19% genome reduction. Sequencing confirmed that DEL2 does not bear any crucial off-target effects or rearrangements in its genome. It can be characterized by faster growth and inability to produce three main native metabolites of S. griseofuscus: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to confirm the production of actinorhodin by both S. griseofuscus wild type and DEL2. We believe that this strain will serve as a good chassis for heterologous expression of BGCs. ImportanceThe rise of antibacterial resistance calls on the development of the next generation of antibiotics, majority of which are derived from natural compounds, produced by actinomycetes. The manipulation, refactoring and expression of BGCs coding for such natural products is a promising approach in secondary metabolite discovery. Thus, the development of a versatile panel of heterologous hosts for the expression of BGCs is essential. We believe that first-to-date systematic, detailed characterisation of S. griseofuscus, a highly promising chassis strain, will not only facilitate the further development of this particular strain, but also will set a blueprint for characterisation of other potential hosts.

microbiology↗

bioRxiv2020

CRISPR-nRAGE, a Cas9 nickase-reverse transcriptase assisted versatile genetic engineering toolkit for E. coli

In most prokaryotes, missing and poorly active non-homologous end joining (NHEJ) DNA repair pathways heavily restrict the direct application of CRISPR-Cas for DNA double-strand break (DSB)-based genome engineering without providing editing templates. CRISPR base editors, on the other hand, can be directly used for genome engineering in a number of bacteria, including E. coli, showing advantages over CRISPR-Cas9, since they do not require DSBs. However, as the current CRISPR base editors can only engineer DNA by A to G or C to T/G/A substitutions, they are incapable of mediating deletions, insertions, and combinations of deletions, insertions and substitutions. To address these challenges, we developed a Cas9 nickase (Cas9n)-reverse transcriptase (Moloney Murine Leukemia Virus, M-MLV) mediated, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes, termed CRISPR-nRAGE (CRISPR-Cas9n Reverse transcriptase Assisted Genome Engineering) system. CRISPR-nRAGE can be used to introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli. Notably, small sized-deletion shows better editing efficiency compared to other kinds of DNA engineering. CRISPR-nRAGE has been used to delete and insert DNA fragments up to 97 bp and 33 bp, respectively. Efficiencies, however, drop sharply with the increase of the fragment size. It is not only a useful addition to the genome engineering arsenal for E. coli, but also may be the basis for the development of similar toolkits for other organisms.

bioengineering↗

bioRxiv2020

Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing. In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

microbiology↗

bioRxiv2020

Distribution of Epsilon-Polylysine Synthetases in Coryneform Bacteria Isolated from Cheese and Human Skin

Epsilon-polylysine ({varepsilon}-PL) is an antimicrobial commercially produced by Streptomyces fermentation and widely used in Asian countries for food preservation. Here we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the {varepsilon}-PL synthetase from Streptomyces in terms of enzymatic domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus {varepsilon}-PL synthetase promoter, we confirmed that its product is indeed {varepsilon}-PL. A comprehensive sequence analysis suggests that {varepsilon}-PL synthetases are widely spread among coryneform bacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain Pls gene. This discovery raises the possibility that {varepsilon}-PL as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCEEvery year, microbial contamination causes billions of tons of food wasted and millions of cases of foodborne illness. {varepsilon}-PL is an excellent food preservative as it is potent, wide spectrum and is heat stable and biodegradable. It has not been accepted by all countries (e.g those in the EU) partially because it was not a natural composition of food but rather originated from the soil bacteria Streptomyces, a famous producer of various antibiotic drugs and toxins. The unexpected finding of {varepsilon}-PL synthetases in cheese and skin bacteria suggests that {varepsilon}-PL may naturally exist in cheese and on our skin.

microbiology↗

bioRxiv2019

The genome and mRNA transcriptome of the cosmopolitan calanoid copepod Acartia tonsa Dana improve the understanding of copepod genome size evolution

Members of the crustacean subclass Copepoda are likely the most abundant metazoans worldwide. Pelagic marine species are critical in converting planktonic microalgae to animal biomass, supporting oceanic food webs. Despite their abundance and ecological importance, only five copepod genomes are publicly available, owing to a number of factors including large genome size, repetitiveness, GC-content, and small animal size. Here, we report the sixth representative copepod genome and the first genome and transcriptome from the calanoid copepod species Acartia tonsa Dana, which is among the most numerous mesozooplankton in boreal coastal and estuarine waters. The ecology, physiology and behavior of A. tonsa has been studied extensively. The genetic resources contributed in this work will allow researchers to link experimental results to molecular mechanisms. From PCRfree WGS and mRNA Illumina data, we assemble the largest copepod genome to date. We estimate A. tonsa has a total genome size of 2.5 Gb including repetitive elements we could not resolve. The non-repetitive fraction of the genome assembly is estimated to be 566Mb. Our DNA sequencing-based analyses suggest there is a 14-fold difference in genome size between the six members of Copepoda with available genomic information through NCBI. This finding complements nucleus staining genome size estimations, where 100-fold difference has been reported within 70 species. We briefly analyze the repeat structure in the existing copepod WGS datasets. The information presented here confirms the evolution of genome size in Copepoda and expands the scope for evolutionary inferences in Copepoda by providing several levels of genetic information from a key planktonic crustacean species.

genomics↗

bioRxiv2018

The whole genome sequence and mRNA transcriptome of the tropical cyclopoid copepod Apocyclops royi

Copepoda is one of the most ecologically important animal groups on Earth, yet very few genetic resources are available for this Subclass. Here, we present the first whole genome sequence (WGS, acc. UYDY01) and the first mRNA transcriptome assembly (TSA, Acc. GHAJ01) for the tropical cyclopoid copepod species Apocyclops royi. Until now, only the 18S small subunit of ribosomal RNA gene and the COI gene has been available from A. royi, and only one other cyclopoid copepod had WGS resources available. Overall, the provided resources are the 7th copepod species to have WGS available and the 19th copepod species with TSA information available. We analyze the length and GC content of the provided WGS scaffolds as well as the coverage and gene content of both the WGS and the TSA assembly. Finally, we place the resources within the copepod order Cyclopoida as a member of the Apocyclops genus. We estimate the total genome size of A. royi to 450 Mb, with 181 Mb assembled nonrepetitive, 76 Mb assembled repeats and 193Mb unassembled sequence. The TSA assembly consists of 29,737 genes and an additional 45,756 isoforms. In the WGS and TSA assemblies, >80% and >95% of core genes can be found, though many in fragmented versions. The provided resources will allow researchers to conduct physiological experiments on A. royi, and also increase the possibilities for copepod gene set analysis, as it adds substantially to the copepod datasets available.

genomics↗

bioRxiv2017

Plasmids, Viruses, And Other Circular Elements In Rat Gut

Circular DNA such as plasmids and some viruses is the major source of genetic variation in bacteria and thus has the same important evolutionary function as sexual reproduction in eukaryotic species: It allows dissemination of advantageous traits through bacterial populations. Here, we present the largest collection of novel complete extrachromosomal genetic elements to date, and compare the diversity, distribution, and content of circular sequences from 12 rat cecum samples from the pristine Falkland Islands and Danish hospital sewers, two environments with contrasting anthropogenic impact. Using a validated pipeline, we find 1,869 complete, circular, non-redundant sequences, of which only 114 are previously described. While sequences of similar size from the two environments share general features, the size distribution of the elements between environments differs significantly, with hospital sewer samples hosting larger circular elements than Falkland Island samples, a possible consequence of the massive anthropogenic influence in the hospital sewer environment. Several antibiotic resistance genes have been identified with a notably larger diversity in hospital sewer samples than in Falkland Islands samples in concordance with expectations. Our findings suggest that even though sequences of similar length carry similar traits, the mobilome of rat gut bacteria are affected by human activities in that sewer rats have larger elements and more diverse large elements than pristine island rats. More than 1000 novel and not classified small sequences was identified and hint the existence of a biological unit not previously described on a community level.\n\nList of figures O_LISampling sites and Rat gut anatomy\nC_LIO_LIRarefaction curves of circular elements\nC_LIO_LIElements shared between environments\nC_LIO_LISize distribution plots of rep, mob, stab, capsid carrying elements.\nC_LIO_LIGraphical representation of representative elements\nC_LIO_LIReplication genes content, diversity\nC_LIO_LIRep_2 plasmid replication gene phylogeny\nC_LIO_LITable Resfams findings\nC_LI\n\nList of appendices O_LIbasic statistics table\nC_LIO_LIPhylogeny of plasmid replication genes\nC_LIO_LIList of known plasmids and circular sequences found in 1869 elements\nC_LIO_LIAutomatic identification of identical circular elements with different breaking points\nC_LIO_LITest of separation of plasmids and viruses based on predicted, annotated genes\nC_LIO_LINo false positive circular sequences from MG1655 genomic sequencing.\nC_LI

microbiology↗