Biology subjects
Publications and source records attributed to Booth, T. J..
An unusually divergent Plasmodium vivax parasite was reported in a Swedish patient with recent travel. Using whole-genome sequencing, we show that the infection was caused by a strain of ape P. vivax-like, providing evidence of the zoonotic potential of these parasites that infect wild gorillas and chimpanzees in central Africa.
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
Embleya is a genus within the family Streptomycetaceae, a group of actinobacteria with outstanding capacity for production of specialised metabolites and a strikingly complex life cycle. In this work, we sequenced the complete genome of the new species Embleya australiensis MST-11070 and validated the assembly using optical mapping. The genome of E. australiensis MST-11070 consists of a 7.1 Mb linear chromosome and three additional replicons, including a 4.2 Mb linear replicon, EEC1, significantly larger than all previously described secondary replicons from bacteria. EEC1 is typified by its similar composition to the chromosome in terms of GC-content, codon usage and gene functions. It also carries terminal inverted repeats identical to the chromosome. EEC1 is enriched in biosynthetic gene clusters (BGCs), including the only copy of the BGCs for the spore pigment and the surfactant peptide SapB, metabolites essential for the organisms lifecycle. EEC1 contains an origin of replication with at least some chromosomal properties, and its replication is likely to depend on functions provided by chromosomally located genes. Further comparison of Embleya spp. genomes suggests that EEC1-like replicons are conserved across the genus, in contrast to other known large linear extrachromosomal replicons (megaplasmids) in the order. EEC1 is thus a hallmark of the Embleya genus and is central to its evolution within the Streptomycetaceae family. We propose EEC1 as a secondary chromosome, distinct from previously described secondary chromosomes that utilise plasmid-like replication mechanisms (chromids) and the largest secondary replicon reported in bacteria, to date.
The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds. Advances in molecular engineering tools, such as CRISPR, has provided some access to the metabolic potential of Streptomyces, but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols. In this paper, we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp. using the affordable and open-sourced Opentrons (OT-2) robotics platform. To increase the accessibility of the workflow we provide an open-source protocol-creator, ActinoMation. ActinoMation is a literate programming environment using Python in Jupyter Notebook. We validated the method by transforming Streptomyces coelicolor (M1152 and M1146), S. albidoflavus (J1047), and S. venezuelae (DSM40230) with the plasmids pSETGUS and pIJ12551. We demonstrate conjugation efficiencies of 3.33*10-3 for M1152 with pSETGUS and pIJ12551; 2.96*10-3 for M1146 with pSETGUS and pIJ12551; 1.21*10-5 for J1047 with pSETGUS and 4.70*10-4 with pIJ12551, and 4.97*10-2 for DSM40230 with pSETGUS and 6.13*10-2 with pIJ12551 with a false positive rate between 8.33% and 54.54%. Automation of the conjugation workflow improves consistency when handling large sample sizes that facilitates easy reproducibility on a larger scale.
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.
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.
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.
MotivationPhylogenetic trees are the primary tool for visualising evolutionary relationships. Traditionally, phylogenies are inferred from manually curated sets of marker genes. As available genomic data increases, there is increasing demand for tools to automatically build phylogenies from assembled genomes. Existing tools rely on reference databases of preselected marker genes, limiting their taxonomic scope. We sought to develop a tool that could quickly build phylogeny from input genomes alone. ResultsWe developed getphylo, a tool to automatically generate multi-locus phylogenetic trees from GenBank files. It has a low barrier to entry with minimal dependencies. getphylo uses a parallelised, heuristic workflow to keep runtime and system requirements as low as possible. getphylo consistently produces trees with topologies comparable to other tools in less time. Furthermore, as getphylo does not rely on reference databases, it has a virtually unlimited scope in terms of taxonomy (e.g., not limited to bacteria) and genetic scale (e.g., can analyse plasmids, prophage, and gene clusters). This combination of speed and flexibility makes getphylo a valuable addition to the phylogenetics toolkit. Availabilitygetphylo is freely available and is downloadable through the Python Package Index (pip install getphylo; https://pypi.org/project/getphylo/) and GitHub (https://github.com/drboothtj/getphylo).
BackgroundCo-localized sets of genes that encode specialized functions are common across microbial genomes and occur in genomes of larger eukaryotes as well. Important examples include Biosynthetic Gene Clusters (BGCs) that produce specialized metabolites with medicinal, agricultural, and industrial value (e.g. antimicrobials). Comparative analysis of BGCs can aid in the discovery of novel metabolites by highlighting distribution and identifying variants in public genomes. Unfortunately, gene-cluster-level homology detection remains inaccessible, time-consuming and difficult to interpret. ResultsThe comparative gene cluster analysis toolbox (CAGECAT) is a rapid and user-friendly platform to mitigate difficulties in comparative analysis of whole gene clusters. The software provides homology searches and downstream analyses without the need for command-line or programming expertise. By leveraging remote BLAST databases, which always provide up-to-date results, CAGECAT can yield relevant matches that aid in the comparison, taxonomic distribution, or evolution of an unknown query. The service is extensible and interoperable and implements the cblaster and clinker pipelines to perform homology search, filtering, gene neighbourhood estimation, and dynamic visualisation of resulting variant BGCs. With the visualisation module, publication-quality figures can be customized directly from a web-browser, which greatly accelerates their interpretation via informative overlays to identify conserved genes in a BGC query. ConclusionOverall, CAGECAT is an extensible software that can be interfaced via a standard web-browser for whole region homology searches and comparison on continually updated genomes from NCBI. The public web server and installable docker image are open source and freely available without registration at: https://cagecat.bioinformatics.nl
Reprogramming biosynthetic assembly-lines is a topic of intense interest. This is unsurprising as the scaffolds of most antibiotics in current clinical use are produced by such pathways. The modular nature of assembly-lines provides a direct relationship between the sequence of enzymatic domains and the chemical structure of the product, but rational reprogramming efforts have been met with limited success. To gain greater insight into the design process, we wanted to examine how Nature creates assembly-lines and searched for biosynthetic pathways that might represent evolutionary transitions. By examining the biosynthesis of the anti-tubercular wollamides, we show how whole gene duplication and neofunctionalization can result in pathway bifurcation. Importantly, we show that neofunctionalization occurs primarily through intragenomic recombination. This pathway bifurcation leads to redundancy, providing the genetic robustness required to enable large structural changes during the evolution of antibiotic structures. Should the new product be none-functional, gene loss can restore the original genotype. However, if the new product confers an advantage, depreciation and eventual loss of the original gene creates a new linear pathway. This provides the blind watchmaker equivalent to the design, build, test cycle of synthetic biology.
Genes involved in coordinated biological pathways, including metabolism, drug resistance and virulence, are often collocalised as gene clusters. Identifying homologous gene clusters aids in the study of their function and evolution, however existing tools are limited to searching local sequence databases. Tools for remotely searching public databases are necessary to keep pace with the rapid growth of online genomic data. Here, we present cblaster, a Python based tool to rapidly detect collocated genes in local and remote databases. cblaster is easy to use, offering both a command line and a user-friendly graphical user interface (GUI). It generates outputs that enable intuitive visualisations of large datasets, and can be readily incorporated into larger bioinformatic pipelines. cblaster is a significant update to the comparative genomics toolbox. cblaster source code and documentation is freely available from GitHub under the MIT license (github.com/gamcil/cblaster).
The roles of migration, admixture and acculturation in the European transition to farming have been debated for over 100 years. Genome-wide ancient DNA studies indicate predominantly Anatolian ancestry for continental Neolithic farmers, but also variable admixture with local Mesolithic hunter-gatherers1-9. Neolithic cultures first appear in Britain c. 6000 years ago (kBP), a millennium after they appear in adjacent areas of northwestern continental Europe. However, the pattern and process of the British Neolithic transition remains unclear10-15. We assembled genome-wide data from six Mesolithic and 67 Neolithic individuals found in Britain, dating from 10.5-4.5 kBP, a dataset that includes 22 newly reported individuals and the first genomic data from British Mesolithic hunter-gatherers. Our analyses reveals persistent genetic affinities between Mesolithic British and Western European hunter-gatherers over a period spanning Britains separation from continental Europe. We find overwhelming support for agriculture being introduced by incoming continental farmers, with small and geographically structured levels of additional hunter-gatherer introgression. We find genetic affinity between British and Iberian Neolithic populations indicating that British Neolithic people derived much of their ancestry from Anatolian farmers who originally followed the Mediterranean route of dispersal and likely entered Britain from northwestern mainland Europe.