Biology subjects

Hoskisson, P. A.

Publications and source records attributed to Hoskisson, P. A..

At least 19 recordsLinked to original sources

bioRxiv2026

A bacterial Rhesus transporter retunes a structurally conserved ammonium pore into a reversible nitrogen valve

How conserved proteins acquire new physiological functions is a central question in molecular evolution. Rather than inventing new architectures, evolution often repurposes existing scaffolds, preserving core structural features while retuning the molecular logic that connects mechanism to physiology. Membrane transporters offer a powerful test of this principle because substrate selectivity, directionality, flux and energetic cost must be coordinated at the interface between the cell and its environment. The Amt/Mep/Rh superfamily controls one of the most fundamental requirements of cellular life: the movement of reduced nitrogen across biological membranes. Despite sharing a highly conserved ammonium-conducting pore, these proteins support distinct physiological roles, including nitrogen acquisition, sensing and homeostatic control. Here, we combined targeted mutagenesis, electrophysiology, yeast complementation and molecular dynamics simulations to define the transport logic of NeRh50, a bacterial Rhesus protein from the ammonia-oxidising bacterium Nitrosomonas europaea. We show that NeRh50 is not simply an AmtB-like ammonium importer. Instead, it uses the conserved Amt/Mep/Rh pore as a branched transport system in which inward uptake and export-linked transport can be separated genetically and mechanistically. Two conserved pore residues define this division of labour. A residue at the external entrance couples ammonium recruitment to productive inward uptake, whereas a second residue deeper in the pore enables a distinct transport mode required for substrate release when intracellular nitrogen accumulates. Thus, conserved Amt/Mep/Rh pore landmarks do not impose a single mechanism. Their local chemistry can be reassigned to generate different transport outputs, allowing NeRh50 to function as a reversible nitrogen valve. These findings reveal how minimal retuning within an ancient membrane-protein scaffold can rewire transport directionality and adapt nitrogen handling to ecological and physiological demand.

biochemistry↗

bioRxiv2026

Genome evolution during the domestication of an antibiotic-producing Streptomyces strain

The domestication of Streptomyces species for antibiotic production involves long-term, iterative mutagenesis and selection, yet the genomic changes driving enhanced production remain unclear. Analysis of five strains from an industrial lineage of Streptomyces clavuligerus using comparative genomics, transcriptomics and phenotypic profiling for dynamic genome architectures with plasmid integration events and chromosomal rearrangements, alongside the accumulation of mutations affecting metabolic pathways and global gene regulation. These changes increased precursor supply and reprogrammed transcription leading to enhanced clavulanic acid production but reduced catabolic flexibility. Complementation experiments confirmed the functional impacts of specific mutations. These findings reveal that artificial selection shapes genome evolution in industrial strains, balancing production gains with metabolic trade-offs. This work will likely inform rational design of Streptomyces strains for improved natural product production in industry while highlighting the constraints imposed by domestication on metabolic versatility. More broadly it shows that many of the evolutionary processes in industrial strain improvement programmes mirror those at play during natural selection.

microbiology↗

bioRxiv2026

Genome mining of amylases and amylase inhibitors from Streptomyces

Most antibiotics are derived from Streptomyces bacteria and produced industrially via fermentations relying on food-grade feedstocks, which has a substantial environmental impact. Efficient utilisation of starch-rich organic wastes such as bread and potato waste as alternative feedstocks remains limited by incomplete knowledge of starch metabolism in Streptomyces. Here, a genus-wide analysis of 295 Streptomyces strains was performed, identifying 645 amylases grouped into pullulanases, -1,4-amylases, and cyclomaltodextrin-like amylases, alongside biosynthetic gene clusters encoding amylase inhibitors such as acarbose and tendamistat. Structural and sequence analyses revealed that inhibitor resistance arises from subtle modifications in the -amylase catalytic pocket. This comprehensive analysis of amylases and inhibitors provides a foundation for engineering inhibitor-resistant enzymes tailored to diverse starch substrates, facilitating the development of sustainable, starch-based Streptomyces fermentations. Impact statementPharmaceutical manufacturing contributes substantially to global carbon emissions, with feedstocks used in natural product fermentations being a major factor. Replacing conventional feedstocks with organic waste streams, such as starch-rich bread or potato waste, could reduce the environmental impact, but two key challenges remain: production strains may lack the enzymes required to efficiently degrade alternative substrates, and nutrient changes may trigger carbon catabolite repression, reducing product yields. While previous studies have linked carbohydrate-active enzymes with biosynthetic gene clusters, no comprehensive analysis has focused specifically on starch degradation in Streptomyces. To fill this gap, a genus-wide analysis of amylases and amylase inhibitors across 295 Streptomyces genomes was carried out. This created an atlas of 645 amylases which may serve as the foundation for the targeted selection of enzymes optimised for catabolising organic waste from various sources, thereby improving the sustainably of industrial natural product production such as antibiotics. Data summaryScripts used along with supplementary files and figures can be accessed through GitHub at https://github.com/ALawaetz/Amylases_and_amylase_inhibitors_in_Streptomycetes.

microbiology↗

bioRxiv2025

Adaptive radiation during long-term experimental evolution of the multicellular bacterium, Streptomyces

Adaptive radiation of a single lineage into novel ecological niches underpins the evolution of biodiversity. To study adaptation in the industrially and ecologically important bacterium Streptomyces, a long-term evolution experiment (LTEE) was undertaken in a strain lacking several antibiotic biosynthetic gene clusters, facilitating the study of epistasis in antibiotic biosynthesis. Streptomyces coelicolor is a filamentous soil organism that does not undergo sporulation under the LTEE conditions, affording the opportunity to study adaptation under relaxed selection on sporulation. Over 3000 generations, replicate populations showed parallel loss of sporulation, morphological transitions to fragmenting hyphae and diversification in carbon source utilisation. Genomic analyses revealed mutations in developmental and metabolic genes, while reintroduction of the actinorhodin cluster demonstrated pervasive negative epistasis affecting antibiotic production. These findings reveal adaptive diversification and antagonistic interactions in regulatory pathways under relaxed selection, providing insights into the evolution of complex multicellular bacteria and informing industrial antibiotic production strategies.

microbiology↗

bioRxiv2025

A simple silicone elastomer colonisation model highlights complexities of Candida albicans and Staphylococcus aureus interactions in biofilm formation.

Healthcare-associated infections (HAIs) significantly contribute to the burden of antimicrobial resistance (AMR). A major factor in HAIs is the colonisation of indwelling medical devices by biofilm-forming opportunistic pathogens such as Candida albicans and Staphylococcus aureus. These organisms frequently co-infect, resulting in synergistic interactions with enhanced virulence and resistance to treatment. C. albicans and S. aureus readily form dual-species biofilms on silicone elastomers, a commonly used medical device material, yet the colonisation phenotypes of these organisms on such surfaces remains poorly understood. We developed a simple, optically tractable model to mimic the colonisation of indwelling medical devices to investigate C. albicans and S. aureus biofilm formation. The system utilises discs of a silicone elastomer embedded in agar, reflecting device-associated conditions and enabling high-resolution imaging of biofilms formed by C. albicans and S. aureus co-culture. Initial results using the silicone elastomer colonisation model reveal robust biofilm formation. These biofilms exhibited morphological differences between dual species biofilms formed by S. aureus co-cultures with either yeast- or hyphal-form C. albicans, indicating the impact of differing C. albicans cell morphotypes in biofilm-associated medical device colonization on silicone elastomers. Quantification of biofilm formation by crystal violet staining provided further validation of the system. These findings underscore the importance of developing tools for biofilm study which more closely resemble the infectious microenvironment, with our work detailing such a system which can be employed in further study to improve strategies against device-related HAIs.

microbiology↗

bioRxiv2024

Oxygen Microenvironments in E. coli Biofilm Nutrient Transport Channels: Insights from Complementary Sensing Approaches

Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterised throughout the biofilm mass but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilisation throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualisations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches.

microbiology↗

bioRxiv2023

Fractal complexity of Escherichia coli nutrient transport channels is influenced by cell shape and growth environment

Recent mesoscopic characterisation of nutrient-transporting channels in E. coli has allowed the identification and measurement of individual channels in whole mature biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of biofilms formed by cell shape mutants of E. coli shows that channels have a high fractal complexity, regardless of cell phenotype or growth medium. In particular, biofilms formed by the mutant strain {Delta}ompR, which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentration in the medium. Osmotic stress leads to a dramatic reduction in {Delta}ompR cell size, but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli biofilms.

microbiology↗

bioRxiv2023

Time-lapse mesoscopy of Candida albicans and Staphylococcus aureus dual-species biofilms reveals a structural role for the hyphae of C. albicans in biofilm formation

Polymicrobial infection with Candida albicans and Staphylococcus aureus may result in a concomitant increase in virulence and resistance to antimicrobial drugs. This enhanced pathogenicity phenotype is mediated by numerous factors including metabolic processes and direct interaction of S. aureus with C. albicans hyphae. The overall structure of biofilms is known to contribute to their recalcitrance to treatment, however the dynamics of direct interaction between species and how it contributes to pathogenicity is poorly understood. To address this, a novel time-lapse mesoscopic optical imaging method was developed to enable the formation of C. albicans/S. aureus whole dual-species biofilms to be followed. It was found that yeast-form or hyphal-form C. albicans in the biofilm founder-population profoundly affects the structure of the biofilm as it matures. Different sub-populations of C. albicans and S. aureus arise within each biofilm as a result of the different C. albicans morphotypes, resulting in distinct sub-regions. These data reveal that C. albicans cell morphology is pivotal in the development of global biofilm architecture and the emergence of colony macrostructures and may temporally influence synergy in infection.

microbiology↗

bioRxiv2023

16S rRNA phylogeny and clustering is not a reliable proxy for genome-based taxonomy in Streptomyces

Although Streptomyces is one of the most extensively studied genera of bacteria, their taxonomy remains contested and is suspected to contain significant species-level misclassification. Resolving the classification of Streptomyces would benefit many areas of study and applied microbiology that rely heavily on having an accurate ground truth classification of similar and dissimilar organisms, including comparative genomics-based searches for novel antimicrobials in the fight against the ongoing antimicrobial resistance (AMR) crisis. To attempt a resolution, we investigate taxonomic conflicts between 16S rRNA and whole genome classifications using all available 48,981 full-length 16S rRNA Streptomyces sequences from the combined SILVA, Greengenes, Ribosomal Database Project (RDP) and NCBI (National Center for Biotechnology Information) databases, and 2,276 publicly available Streptomyces genome assemblies. We construct a 16S gene tree for 14,239 distinct Streptomyces 16S rRNA sequences, identifying three major lineages of Streptomyces, and find that existing taxonomic classifications are inconsistent with the tree topology. We also use these data to delineate 16S and whole genome landscapes for Streptomyces, finding that 16S and whole-genome classifications of Streptomyces strains are frequently in disagreement, and in particular that 16S zero-radius Operational Taxonomic Units (zOTUs) are often inconsistent with Average Nucleotide Identity (ANI)-based taxonomy. Our results strongly imply that 16S rRNA sequence data does not map to taxonomy sufficiently well to delineate Streptomyces species reliably, and we propose that alternative markers should instead be adopted by the community for classification and metabarcoding. As much of current Streptomyces taxonomy has been determined or supported by historical 16S sequence data and may in parts be in error, we also propose that reclassification of the genus by alternative approaches is required. Impact StatementAccurate classification of microbes, usually in the form of taxonomic assignments, provides a fundamental ground truth or reference point for many aspects of applied microbiology including comparative genomics, identification of strains for natural product discovery, and dereplication of strains. Bacteria belonging to the genus Streptomyces are an important source of bioactive metabolites and enzymes in biotechnology, and proper understanding of their phylogeny aids understanding of the evolution of industrially important gene products and metabolites, and prioritization of strains for industrial exploitation. Taxonomic classification in the genus Streptomyces is complex and contested, and there are clear conflicts between taxonomies inferred from 16S rRNA and from whole genome sequences. Despite this, 16S sequence-based classifications are still widely used to infer taxonomic identity, to determine community composition, and to prioritise strains for study. We investigate a diverse and comprehensive set of Streptomyces genomes using whole-genome Average Nucleotide Identity (ANI) and 16S sequence analysis to delineate and compare classifications made using these approaches. We outline the genomic and 16S sequence landscape of Streptomyces, demonstrating that (i) distinct taxonomic species may share identical full-length 16S sequences, and (ii) in some instances, isolates representing the same taxonomic species do not share any common 16S rRNA sequence. Our results strongly imply that 16S rRNA sequence variation does not map to taxonomy sufficiently well to delineate Streptomyces species reliably, and that alternative markers should instead be adopted by the community. Much of current Streptomyces taxonomy has been determined or supported by historical 16S sequence data, and we therefore propose that reclassification within this group by alternative approaches is required. Data summaryAll code, raw and supporting data are publicly available from GitHub (https://github.com/kiepczi/Kiepas_et_al_2023_16S) and Zenodo (https://doi.org/10.5281/zenodo.8223787). The flowchart provided in Supplementary File 28 provides an overview of analysis steps and serves as a guide through Supplementary Files generated during reconstruction of the 16S phylogeny. The flowchart in Supplementary File 29 outlines the workflow processes and supplementary materials used for analysis of 16S rRNA sequences from Streptomyces genomes. Supplementary DataSupplementary File 1: Generate figures using Python and R. ZIP file containing all data, Python and R scripts to generate figures for this manuscript. (ZIP 40.9MB) Supplementary File 2: Raw 16S rRNA public databases. Zip file containing four separate txt files with sequence IDs for public 16S rRNA databases used in this manuscript, and an additional txt file with Greengenes sequence taxonomy information, and a Python script used to map taxonomy information to sequences found in Greengenes v13.5. (ZIP 34.8MB) Supplementary File 3: Filtration of 16S rRNA public databases. Zip file containing Python script used for filtration of the raw databases, and generated outputs. (ZIP 7.2MB) Supplementary File 4: Cleaning of the filtrated 16S rRNA local. Zip file containing all bash and Python scripts used to clean the local full-length 16S rRNA local databases by removing redundant and poor quality 16S rRNA sequences. (ZIP 9MB) Supplementary File 5: Sequence Clustering. Zip file containing a bash script used to cluster the full-length cleaned local 16S rRNA Streptomyces local databases at various thresholds, and provides txt files with accessions for representative sequences, and cluster members for each clustering threshold. (ZIP 40.8MB) Supplementary File 6: Analysis of taxonomic composition for each clustering threshold. Zip file containing Python scripts, NCBI taxonomy input and all outputs generated used to determine the taxonomic composition for each clustering threshold. (ZIP 49.6) Supplementary File 7. Cluster sizes. Empirical cumulative frequency plot showing cluster sizes generated for all clustering thresholds. (PDF 44KB) Supplementary File 8. Cluster taxID abundance. Empirical cumulative frequency plot for unique numbers of taxID present at all clustering thresholds. (PDF 9KB) Supplementary File 9. MSA. Zip file containing all Python and bash scripts, and additional data needed to generate and clean MSA for phylogenetic analysis. (ZIP 4.2MB) Supplementary File 10. Phylogenetic reconstruction. ZIP file containing bash scripts used for phylogenetic reconstruction, and all generated outputs and log files. (ZIP 16.8MB). Supplementary File 11. Collapse branches. ZIP file containing jupyter notebook used for collapsing branches with the same species names, and the collapsed tree in newick format. (ZIP 385KB) Supplementary File 12. Phylogenetic tree. PDF file showing collapsed phylogenetic tree with marked branches with transfer bootstrap expectation support of >= 50%. (PDF 224KB) Supplementary File 13. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces albus and Streptomyces griseus. (PDF 229KB) Supplementary File 14. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces albulus, Streptomyces lydicus and Streptomyces venezuelae. (PDF 228KB) Supplementary File 15. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces clavuligerus and Streptomyces coelicolor. (PDF 227KB) Supplementary File 16. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces lavendulae, Streptomyces rimosus and Streptomyces scabiei. (PDF 228KB) Supplementary File 17. Streptomyces genomes. Zip file containing bash scripts used to download Streptomyces genomes, and Python scripts used to check assembly status. The ZIP file also contains two separate txt files with Streptomyces genomes used in this manuscript: one file with all initial candidates, and a second file with replaced genomes. (ZIP 2.6MB) Supplementary File 18. Extraction of full-length and ambiguity free 16S rRNA sequences from Streptomyces genomes. Zip file containing all Python and bash scripts used to extract full-length sequences from the filtered Streptomyces genomes. A single FASTA file with all extracted 16S rRNA sequences, and a single FASTA file with filtered sequences. A txt file with accession of genomes retained in the analysis. (ZIP 742KB) Supplementary File 19. ANI analysis among Streptomyces genomes with identical 16S rRNA sequences. ZIP file containing all Bash and Python scripts used to determine taxonomic boundaries among Streptomyces genomes sharing identical full-length 16S rRNA sequences. All output and pyANI log files. (ZIP 37.1MB) Supplementary File 20. Network analysis of genomes based on shared 16S sequences. ZIP file containing jupyter notebook with NetworkX analysis and all associated output files including. bash script for pyANI analysis runs on each connected component and all associated matrices, heatmaps and log files. (ZIP 29.3MB) Supplementary File 21. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S sequences with each node colour corresponding to the number of connections/degrees. (HTML 4.7MB) Supplementary File 22. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S sequences showing clique (blue) and non-clique (green) components. (HTML 4.7MB) Supplementary File 23. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S sequences showing number of unique genera within each connected component. Each candidate genus is represented as a single node colour within a connected component. (HTML 4.7MB) Supplementary File 24. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S rRNA sequences showing number of unique species within each connected component. Each candidate species is represented as a single node colour within a connected component. (HTML 4.7MB) Supplementary File 25 Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S rRNA sequences showing number of unique NCBI names within each connected component. Each NCBI assigned name is represented as a single node colour within a connected component. Gray nodes represent genomes currently lacking assigned species names. (HTML 4.7MB) Supplementary file 26. Intragenomic 16S rRNA heterogeneity within 1,369 Streptomyces genomes which exclusively contain only full-length and ambiguity symbol-free 16S rRNA sequences. A total of 811 genomes containing single 16S rRNA sequences are not shown. (PDF 8KB) Supplementary File 27. Distribution of 16S copies per genome with a distinction between unique and total copies for genomes at assembly level complete and chromosome. (PDF 7KB) Supplementary File 28. Schematic workflow for construction of the full-length 16S rRNA Streptomyces phylogeny. Each arrow represents a process and is annotated with script used and corresponding supplementary file. Output/data files, and the number of remaining sequences after each step, are indicated by rectangles. The green shading represents a single processing step of collecting and collating 16S database sequences. (PDF 91KB) Supplementary File 29. Schematic representation of the pipeline used to filter publicly available Streptomyces genomes. (PDF 59KB) Supplementary File 30. Sankey plot showing counts of taxonomic names in source databases, assigned at ranks from phylum to genus, to sequences identified with a key word Streptomyces in the taxonomy field. Note that Actinobacteria and Actinobacteriota are synonyms in LPSN for the correct Phylum name Actinomycetota, but that Actinomycetales and Streptomycetales are not taxonomic synonyms for each other. Streptomycetales is synonymous in LPSN with the correct name Kitasatosporales; Actinomycetales is a distinct taxonomic Order. The parent order of the Family Streptomycetaceae in LPSN is Kitasatosporales. (PDF 64KB) Supplementary File 31. Rectangular phylogram of the comprehensive maximum-likelihood tree of the genus Streptomyces based on the 16S sequence diversity of all 5,064 full-length 16S rRNA sequences with 100 TBE values. (PDF 194KB) Supplementary file 32. Genomes sharing identical 16S rRNA sequences are assigned different names in NCBI. A total of 1,030 singleton clusters are not shown. (PDF 8KB) Supplementary File 33. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Acintacidiphila genus. (PDF 228KB) Supplementary File 34. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Phaeacidiphilus genus. (PDF 228KB) Supplementary File 35. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Mangrovactinospora genus. (PDF 228KB) Supplementary File 36. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Wenjunlia genus. (PDF 228KB) Supplementary File 37. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Streptantibioticus genus. (PDF 228KB)

genomics↗

bioRxiv2023

Construction and Characterisation of a Structured, Tuneable, and Transparent 3D Culture Platform for Soil Bacteria

2.We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the metabolic profile of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms, Bacillus subtilis and Streptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of other difficult-to-culture bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as interaction, competition, and warfare. 3. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/546105v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1123425org.highwire.dtl.DTLVardef@1d6cc3org.highwire.dtl.DTLVardef@1cf950aorg.highwire.dtl.DTLVardef@141dc0b_HPS_FORMAT_FIGEXP M_FIG C_FIG A step-by-step method for creating a tailored 3D culture medium for study of soil microbes. The complete workflow can be split into three parts: Growth and observation, metabolic profiling to provide a stable refractive index matching solution, and production of the 3D soil environment. The 3D culture scaffold was created by cryomilling Nafion resin pellets and size filtration. Chemical processing altered the surface chemistry of Nafion particles and facilitated nutrient binding by titration of a defined liquid culture medium. Metabolic profiling determined non-metabolisable sugars and provided an inert refractive index matching substrate, which was added to the final nutrient titration. Inoculation and growth of the test strain allowed for downstream assessment of colonisation behaviours and community dynamics in situ by, for example, optical microscopy.

microbiology↗

bioRxiv2022

Biosynthesis of aurodox, a Type III secretion system inhibitor from Streptomyces goldiniensis

The global increase in antimicrobial-resistant infections means that there is a need to develop new antimicrobial molecules and strategies to combat the issue. Aurodox is a linear polyketide natural product that is produced by Streptomyces goldiniensis, yet little is known about aurodox biosynthesis or the nature of the biosynthetic gene cluster (BGC) that encodes its production. To gain a deeper understanding of aurodox biosynthesis by S. goldiniensis, the whole genome of the organism was sequenced, revealing the presence of an 87 kb hybrid Polyketide Synthase/Non-Ribosomal Peptide Synthetase (PKS/NRPS) BGC. The aurodox BGC shares significant homology with the kirromycin BGC from S. collinus T{upsilon} 365; however, the genetic organisation of the BGC differs significantly. The candidate aurodox gene cluster was cloned and expressed in a heterologous host to demonstrate that it was responsible for aurodox biosynthesis and disruption of the primary PKS gene (aurAI) abolished aurodox production. These data support a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis follow that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus T{upsilon} 365 enabled methylation of the pyridone group, suggesting this is the last step in biosynthesis. This methylation step is also sufficient to confer the unique Type III Secretion System inhibitory properties to aurodox. ImportanceEnterohaemorrhagic Escherichia coli (EHEC) is a significant global pathogen for which traditional antibiotic treatment is not recommended. Aurodox inhibits the ability of EHEC to establish infection in the host gut through the specific targeting of the Type III Secretion System, whilst circumventing the induction of toxin production associated with traditional antibiotics. These properties suggest aurodox could be a promising anti-virulence compound for EHEC, which merits further investigation. Here, we have characterised the aurodox biosynthetic gene cluster from Streptomyces goldiniensis and have established the key enzymatic steps of aurodox biosynthesis that give rise to the unique anti-virulence activity. These data provide the basis for future chemical and genetic approaches to produce aurodox derivatives with increased efficacy and the potential to engineer novel elfamycins.

microbiology↗

bioRxiv2021

Intra-colony channel morphology in Escherichia coli biofilms is governed by nutrient availability and substrate stiffness

Nutrient-transporting channels have been recently discovered in mature Escherichia coli biofilms, however the relationship between intra-colony channel structure and the surrounding environmental conditions is poorly understood. Using a combination of fluorescence mesoscopy and a purpose-designed open-source quantitative image analysis pipeline, we show that growth substrate composition and nutrient availability have a profound effect on the morphology of intra-colony channels in mature E. coli biofilms. Under all nutrient conditions, intra-colony channel width was observed to increase non-linearly with radial distance from the centre of the biofilm. Notably, the channels were around 25% wider at the centre of glucose-limited biofilms compared to ammonium-limited biofilms. Channel density also differed in colonies grown on rich and minimal media, with the former creating a network of tightly packed channels and the latter leading to well-separated, wider channels with defined edges. Our approach paves the way for measurement of internal patterns in a wide range of biofilms, offering the potential for new insights into infection and pathogenicity.

microbiology↗

bioRxiv2021

A mechanistic switch from selective transporter to an ion channel impairs the filamentation signalling capability of ammonium transceptors in yeast

Ammonium translocation through biological membranes by the ubiquitous Amt-Mep-Rh family of transporters plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of these transporters, forming the familys characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology, in vivo yeast functional complementation and in silico molecular dynamics simulations, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs the specificity in Amt-Mep transporters but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We further show that both mechanisms coexist in fungal Mep2 Twin-His variants, disrupting the transceptor function and so inhibiting the filamentation process. These data strongly support a transport mechanism-mediated signalling process in the long-standing debate on the sensory function of Mep2-like transporters.

microbiology↗

bioRxiv2021

Genome sequence of the aurodox-producing bacterium Streptomyces goldiniensis ATCC 21386

We report the genome sequence of Streptomyces goldiniensis ATCC 21386, a strain which produces the anti-bacterial and anti-virulence polyketide, aurodox. The genome of S. goldiniensis ATCC 21386 was sequenced using a multiplatform hybrid approach, revealing a linear genome of ~10 Mbp with a G+C content of 71 %. The genome sequence revealed 36 putative biosynthetic gene clusters (BGCs), including a large region of 271 Kbp that was rich in biosynthetic capability. The genome sequence is deposited in DDBJ/EMBL/GenBank with the accession number PRJNA602141.

genomics↗

bioRxiv2021

Frog nest foam as a drug delivery system

Foams have frequently been used as systems for the delivery of cosmetic and therapeutic molecules; however, there is high variability in the foamability and long-term stability of synthetic foams. The development of pharmaceutical foams that exhibit desirable foaming properties, delivering appropriate amounts of the active pharmaceutical ingredient (API) and that have excellent biocompatibility is of great interest. The production of stable foams is rare in the natural world; however, certain species of frogs have adopted foam production as a means of providing a protective environment for their eggs and larvae from a predators and parasites, to prevent desiccation, to control gaseous exchange, temperature extremes, and to reduce UV damage. These foams show great stability (up to 10 days in tropical environments) and are highly biocompatible due to the sensitive nature of amphibian skin. This work demonstrates for the first time, that nests of the Tungara frog (Engystomops pustulosus) is stable ex situ with useful physiochemical and biocompatible properties and is capable of encapsulating a range of compounds, including antibiotics. These protein foam mixtures share some properties with pharmaceutical foams and may find utility in a range of pharmaceutical applications such as a topical drug delivery systems (DDS).

bioengineering↗

bioRxiv2020

ActDES- a Curated Actinobacterial Database for Evolutionary Studies

Actinobacteria are a large and diverse phylum of bacteria that contains medically and ecologically relevant organisms. Many members are valuable sources of bioactive natural products and chemical precursors that are exploited in the clinic. These are made using the enzyme pathways encoded in their complex genomes. Whilst the number of sequenced genomes has increased rapidly in the last twenty years, the large size and complexity of many Actinobacterial genomes means that the sequences remain incomplete and consist of large numbers of contigs with poor annotation, which hinders large scale comparative genomics and evolutionary studies. To enable greater understanding and exploitation of Actinobacterial genomes, specialist genomic databases must be linked to high-quality genome sequences. Here we provide a curated database of 612 high-quality actinobacterial genomes from 80 genera, chosen to represent a broad phylogenetic group with equivalent genome reannotation. Utilising this database will provide researchers with a framework for evolutionary and metabolic studies, to enable a foundation for genome and metabolic engineering, to facilitate discovery of novel bioactive therapeutics and studies on gene family evolution. Significance as a bioresource to the communityThe Actinobacteria are a large diverse phylum of bacteria, often with large, complex genomes with a high G+C content. Sequence databases have great variation in the quality of sequences, equivalence of annotation and phylogenetic representation, which makes it challenging to undertake evolutionary and phylogenetic studies. To address this, we have assembled a curated, taxa-specific, non-redundant database to aid detailed comparative analysis of Actinobacteria. ActDES constitutes a novel resource for the community of Actinobacterial researchers that will be useful primarily for two types of analyses: (i) comparative genomic studies - facilitated by reliable identification of orthologs across a set of defined, phylogenetically-representative genomes, and (ii) phylogenomic studies which will be improved by identification of gene subsets at specified taxonomic level. These analyses can then act as a springboard for the studies of the evolution of virulence genes, the evolution of metabolism and identification of targets for metabolic engineering. Data summaryAll genome sequences used in this study can be found in the NCBI taxonomy browser https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/www.tax.cgi and are summarised along with Accession numbers in Table S1 All other data are available on Figshare https://doi.org/10.6084/m9.figshare.12167529 and https://doi.org/10.5281/zenodo.3830391 O_LIPerl script files available on GitHub https://github.com/nselem/ActDES including details of how to batch annotate genomes in RAST from the terminal https://github.com/nselem/myrast C_LIO_LISupp. Table S1 List of genomes from NCBI (Actinobacteria database.xlsx) https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LICVS genome annotation files including the FASTA files of nucleotide and amino acids sequences (individual .cvs files) https://doi.org/10.6084/m9.figshare.12167880 C_LIO_LIBLAST nucleotide database (.fasta file) https://doi.org/10.6084/m9.figshare.12167724 C_LIO_LIBLAST protein database (.fasta file) https://doi.org/10.6084/m9.figshare.12167724 C_LIO_LISupp. Table S2 Expansion table genus level (Expansion table.xlsx Tab Genus level) https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LISupp. Table S2 Expansion table species level (Expansion table.xlsx Tab species level) https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LIAll GlcP and Glk data - blast hits from ActDES database, MUSCLE Alignment files and .nwk tree files can be found at https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LIInteractive trees in Microreact for Glk tree https://microreact.org/project/w_KDfn1xA/90e6759e and associated files can be found at https://doi.org/10.6084/m9.figshare.12326441.v1 C_LIO_LIInteractive trees in Microreact for GlcP tree https://microreact.org/project/VBUdiQ5_k/0fc4622b and associated files can be found at https://doi.org/10.6084/m9.figshare.12326441.v1 C_LI

microbiology↗

bioRxiv2019

Functional channels in mature E. coli colonies

Biofilms are important in medicine, industry and the natural environment, however their structure is largely unexplored across multiple spatial scales. We have studied the architecture of mature Escherichia coli macro-colony biofilms by means of the Mesolens, an optical system which uniquely allows simultaneous imaging of individual bacteria and over one hundred cubic millimetres of its biofilm milieu. Our chief finding is the presence of intra-colony channels on the order of 10 m in diameter in E. coli biofilms. These channels have a characteristic structure and reform after total mechanical disaggregation of the colony. We present evidence that the channels transport particles and function to assist the absorption of nutrients. These channels potentially offer a new route for the delivery of dispersal agents or antimicrobial drugs to biofilms, ultimately lowering their impact on public health and industry.

microbiology↗

bioRxiv2019

Differential transcription of expanded gene families in central carbon metabolism of Streptomyces coelicolor A3(2)

BackgroundStreptomycete bacteria are prolific producers of specialised metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalisation or sub-functionalisation. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. ResultsRNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialised metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. ConclusionsExpression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters.

microbiology↗