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Recent mesoscopic characterization of nutrient-transporting channels in Escherichia coli has allowed the identification and measurement of individual channels in whole mature colony biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of colony biofilms formed by cell shape mutants of E. coli shows that channels have high fractal complexity, regardless of cell phenotype or growth medium. In particular, colony biofilms formed by the mutant strain Δ 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 concentrations in the medium. Osmotic stress leads to a dramatic reduction in the Δ 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 colony biofilms.

We provide a brief review of the development and application of the Mesolens and its impact on microbiology. Microbial specimens such as infected tissue samples, colonies surfaces, and biofilms are routinely collected at the mesoscale. This means that they are relatively large multimillimetre-sized samples which contain microscopic detail that must be observed to answer important questions across various sectors. The Mesolens presents the ideal imaging method to study these specimens as no other optical microscope can thanks to its unique combination of low magnification and high numerical aperture providing large field-of-view, high-resolution imaging. We demonstrate the current applications of the Mesolens to microbial imaging and go on to outline the huge potential of the Mesolens to impact other key areas of microbiology., (© 2023 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Streptomyces is among the most extensively studied genera of bacteria but its complex taxonomy remains contested and is suspected to contain significant species-level misclassification. Resolving the classification of Streptomyces would benefit many areas of applied microbiology that rely on an accurate ground truth for grouping of related organisms, including comparative genomics-based searches for novel antimicrobials. We survey taxonomic conflicts between 16S rRNA and whole genome-based Streptomyces classifications using 2276 publicly available Streptomyces genome assemblies and 48 981 publicly available full-length 16S rRNA Streptomyces sequences from silva, Greengenes, Ribosomal Database Project (RDP), and NCBI (National Centre for Biotechnology Information) databases. We construct a full-length 16S gene tree for 14 239 distinct Streptomyces sequences that resolves three major lineages of Streptomyces , but whose topology is not consistent with existing taxonomic assignments. We use these sequence data to delineate 16S and whole genome landscapes for Streptomyces , demonstrating that 16S and whole-genome classifications are frequently in disagreement, and 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 routinely. We propose that alternative marker sequences should be adopted by the community for classification and metabarcoding. Insofar as Streptomyces taxonomy has been determined or supported by 16S sequence data and may in parts be in error, we also propose that reclassification of the genus by alternative approaches may benefit the Streptomyces community.

Although ammonium is the preferred nitrogen source for microbes and plants, in animal cells it is a toxic product of nitrogen metabolism that needs to be excreted. Thus, ammonium movement across biological membranes, whether for uptake or excretion, is a fundamental and ubiquitous biological process catalysed by the superfamily of the Amt/Mep/Rh transporters. A remarkable feature of the Amt/Mep/Rh family is that they are ubiquitous and, despite sharing low amino acid sequence identity, are highly structurally conserved. Despite sharing a common structure, these proteins have become involved in a diverse range of physiological process spanning all domains of life, with reports describing their involvement in diverse biological processes being published regularly. In this context, we exhaustively present their range of biological roles across the domains of life and after explore current hypotheses concerning their evolution to help to understand how and why the conserved structure fulfils diverse physiological functions., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Nucleosides are pervasive building blocks that are found throughout nature and used extensively in medicinal chemistry and biotechnology. However, the preparation of base-modified analogues using conventional synthetic methodology poses challenges in scale-up and purification. In this work, an integrated approach involving structural analysis, screening and reaction optimization, is established to prepare 2'-deoxyribonucleoside analogues catalysed by the type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus leichmannii ( Ll NDT-2). Structural analysis in combination with substrate profiling, identified the constraints on pyrimidine and purine acceptor bases by Ll NDT2. A solvent screen identifies pure water as a suitable solvent for the preparation of high value purine and pyrimidine 2'-deoxyribonucleoside analogues on a gram scale under optimized reaction conditions. This approach provides the basis to establish a convergent, step-efficient chemoenzymatic platform for the preparation of high value 2'-deoxyribonucleosides., Competing Interests: The authors declare no competing financial conflict of interest., (This journal is © The Royal Society of Chemistry.)

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., Competing Interests: Competing Interests We declare no competing interests

Conjugation of plasmids from Escherichia coli is essential for the genetic manipulation of Streptomyces spp. To facilitate intergeneric conjugation from E. coli to Streptomyces the conjugative machinery required for genetic transfer is usually provided by the non-transferable helper plasmid, pUZ8002. Here we present the complete nucleotide sequence of pUZ8002, describe the previously undocumented creation process, and provide details of the sequence relative to the parental pUZ8 plasmid and another previously published pUZ8002 sequence., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors.)

Streptomyces are a large genus of multicellular bacteria best known for their prolific production of bioactive natural products. In addition, they play key roles in the mineralisation of insoluble resources, such as chitin and cellulose. Because of their multicellular mode of growth, colonies of interconnected hyphae extend over a large area that may experience different conditions in different parts of the colony. Here, we argue that within-colony phenotypic heterogeneity can allow colonies to simultaneously respond to divergent inputs from resources or competitors that are spatially and temporally dynamic. We discuss causal drivers of heterogeneity, including competitors, precursor availability, metabolic diversity and division of labour, that facilitate divergent phenotypes within Streptomyces colonies. We discuss the adaptive causes and consequences of within-colony heterogeneity, highlight current knowledge (gaps) and outline key questions for future studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

tGrowth of microorganisms and interpretation of growth data are core skills required by microbiologists. While science moves forward, it is of paramount importance that essential skills are not lost. The bacterial growth curve and the information that can gleaned from it is of great value to all of microbiology, whether this be a simple growth experiment, comparison of mutant strains or the establishment of conditions for a large-scale multi-omics experiment. Increasingly, the basics of plotting and interpreting growth curves and growth data are being overlooked. This primer article serves as a refresher for microbiologists on the fundamentals of microbial growth kinetics.

The parasitic worm-derived immunomodulator, ES-62 rescues defective levels of IL-10-producing regulatory B cells (Bregs) and suppresses chronic Th1/Th17-driven inflammation to protect against joint destruction in the mouse collagen-induced arthritis (CIA) model of rheumatoid arthritis. Such autoimmune arthritis is also associated with dysbiosis of the gut microbiota and disruption of intestinal barrier integrity. We recently further exploited the CIA model to show that ES-62's prevention of joint destruction is associated with protection of intestinal barrier integrity and normalization of the gut microbiota, thereby suppressing the gut pathology that precedes the onset of autoimmunity and joint damage in CIA-mice. As the status of the gut microbiota impacts on immune responses by influencing haematopoiesis, we have therefore investigated whether ES-62 harnesses the homeostatic mechanisms regulating this gut-bone marrow (BM) axis to resolve the chronic inflammation promoting autoimmunity and joint destruction in CIA. Reflecting this, ES-62 was found to counteract the BM myeloid/lymphoid bias typically associated with chronic inflammation and infection. This was achieved primarily by ES-62 acting to maintain the levels of lymphoid lineages (B220 + and CD3 + cells) observed in naïve, healthy mice but lost from the BM of CIA-mice. Moreover, ES-62's ability to prevent bone-destroying osteoclastogenesis was found to be associated with its suppression of CIA-induced upregulation of osteoclast progenitors (OCPs) in the BM. Critically, and supporting ES-62's targeting of the gut-BM axis, this rewiring of inflammatory haematopoiesis was lost in mice with a depleted microbiome. Underlining the importance of ES-62's actions in restoring steady-state haematopoiesis, the BM levels of B and T lymphoid cells were shown to be inversely correlated, whilst the levels of OCPs positively correlated, with the severity of joint damage in CIA-mice., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The exchange of ammonium across cellular membranes is a fundamental process in all domains of life and is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. Remarkably, despite a high structural conservation in all domains of life, these proteins have gained various biological functions during evolution. It is tempting to hypothesise that the physiological functions gained by these proteins may be explained at least in part by differences in the energetics of their translocation mechanisms. Therefore, in this review, we will explore our current knowledge of energetics of the Amt/Mep/Rh family, discuss variations in observations between different organisms, and highlight some technical drawbacks which have hampered effects at mechanistic characterisation. Through the review, we aim to provide a comprehensive overview of current understanding of the mechanism of transport of this unique and extraordinary Amt/Mep/Rh superfamily of ammonium transporters., (© 2024 The Author(s).)

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, although 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.

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 carbon catabolism 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 bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as competition and warfare.

Extra-intestinal pathogenic Escherichia coli (ExPEC) can cause a variety of infections outside of the intestine and are a major causative agent of urinary tract infections. Treatment of these infections is increasingly frustrated by antimicrobial resistance (AMR) diminishing the number of effective therapies available to clinicians. Incidence of multidrug resistance (MDR) is not uniform across the phylogenetic spectrum of E. coli. Instead, AMR is concentrated in select lineages, such as ST131, which are MDR pandemic clones that have spread AMR globally. Using a gnotobiotic mouse model, we demonstrate that an MDR E. coli ST131 is capable of out-competing and displacing non-MDR E. coli from the gut in vivo. This is achieved in the absence of antibiotic treatment mediating a selective advantage. In mice colonised with non-MDR E. coli strains, challenge with MDR E. coli either by oral gavage or co-housing with MDR E. coli colonised mice results in displacement and dominant intestinal colonisation by MDR E. coli ST131. To investigate the genetic basis of this superior gut colonisation ability by MDR E. coli, we assayed the metabolic capabilities of our strains using a Biolog phenotypic microarray revealing altered carbon metabolism. Functional pangenomic analysis of 19,571 E. coli genomes revealed that carriage of AMR genes is associated with increased diversity in carbohydrate metabolism genes. The data presented here demonstrate that independent of antibiotic selective pressures, MDR E. coli display a competitive advantage to colonise the mammalian gut and points to a vital role of metabolism in the evolution and success of MDR lineages of E. coli via carriage and spread., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Connor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The exchange of ammonium across cellular membranes is a fundamental process in all domains of life. In plants, bacteria and fungi, ammonium represents a vital source of nitrogen, which is scavenged from the external environment. In contrast, in animal cells ammonium is a cytotoxic metabolic waste product and must be excreted to prevent cell death. Transport of ammonium is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. In addition to their function as transporters, Amt/Mep/Rh proteins play roles in a diverse array of biological processes and human physiopathology. Despite this clear physiological importance and medical relevance, the molecular mechanism of Amt/Mep/Rh proteins has remained elusive. Crystal structures of bacterial Amt/Rh proteins suggest electroneutral transport, whilst functional evidence supports an electrogenic mechanism. Here, focusing on bacterial members of the family, we summarize the structure of Amt/Rh proteins and what three decades of research tells us concerning the general mechanisms of ammonium translocation, in particular the possibility that the transport mechanism might differ in various members of the Amt/Mep/Rh superfamily.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology., Competing Interests: The authors declare they have no competing interests., (Copyright: © 2023 Goodall et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plant growth-promoting endophytic (PGPE) actinomycetes have been known to enhance plant growth and mitigate plant from abiotic stresses via their PGP-traits. In this study, PGPE Streptomyces sp. GKU 895 promoted growth and alleviated salt tolerance of salt-susceptible rice cultivar IR29 by augmentation of plant weight and declined ROS after irrigation with 150 mM NaCl in a pot experiment. Transcriptome analysis of IR29 exposed to the combination of strain GKU 895 and salinity demonstrated up and downregulated differentially expressed genes (DEGs) classified by gene ontology and plant reactome. Streptomyces sp. GKU 895 induced changes in expression of rice genes including transcription factors under salt treatment which involved in growth and development, photosynthesis, plant hormones, ROS scavenging, ion transport and homeostasis, and plant-microbe interactions regarding pathogenesis- and symbiosis-related proteins. Taken together, these data demonstrate that PGPE Streptomyces sp. GKU 895 colonized and enhanced growth of rice IR29 and triggered salt tolerance phenotype. Our findings suggest that utilisation of beneficial endophytes in the saline fields could allow for the use of such marginal soils for growing rice and possibly other crops., (© 2023. The Author(s).)

Natural products are the raw material for drug discovery programmes. Bioactive natural products are used extensively in medicine and agriculture and have found utility as antibiotics, immunosuppressives, anti-cancer drugs and anthelminthics. Remarkably, the natural role and what mechanisms drive evolution of these molecules is relatively poorly understood. The exponential increase in genome and chemical data in recent years, coupled with technical advances in bioinformatics and genetics have enabled progress to be made in understanding the evolution of biosynthetic gene clusters and the products of their enzymatic machinery. Here we discuss the diversity of natural products, incorporating the mechanisms that govern evolution of metabolic pathways and how this can be applied to biosynthetic gene clusters. We build on the nomenclature of natural products in terms of primary, integrated, secondary and specialised metabolism and place this within an ecology-evolutionary-developmental biology framework. This eco-evo-devo framework we believe will help to clarify the nature and use of the term specialised metabolites in the future., (Copyright © 2023. Published by Elsevier Inc.)

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 carbon-limited biofilms compared to nitrogen-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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

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., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 The Authors.)

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 Tϋ 365. However, the genetic organization 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 supported a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis followed that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus Tϋ 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. IMPORTANCE Enterohemorrhagic 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 while 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 characterized the aurodox biosynthetic gene cluster from Streptomyces goldiniensis and 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.

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.

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 the pore of these transporters, forming the family's 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 on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, 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 specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors. IMPORTANCE Fungal infections represent a significant threat to human health and cause huge damage to crop yields worldwide. The dimorphic switch between yeast and filamentous growth is associated with the virulence of pathogenic fungi. Of note, fungal Mep2 proteins of the conserved Amt-Mep-Rh family play a transceptor role in the induction of filamentation; however, the signaling mechanism remains largely unknown. Amt-Mep-Rh proteins ensure the specific scavenging of NH 4 + through a mechanism relying on substrate deprotonation, thereby preventing competition and translocation of similar-sized K + . Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.

SARS-CoV-2 diagnostic practices broadly involve either quantitative polymerase chain reaction (qPCR)-based nucleic amplification of viral sequences or antigen-based tests such as lateral flow assays (LFAs). Reverse transcriptase-qPCR can detect viral RNA and is the gold standard for sensitivity. However, the technique is time-consuming and requires expensive laboratory infrastructure and trained staff. LFAs are lower in cost and near real time, and because they are antigen-based, they have the potential to provide a more accurate indication of a disease state. However, LFAs are reported to have low real-world sensitivity and in most cases are only qualitative. Here, an antigen-based electrochemical aptamer sensor is presented, which has the potential to address some of these shortfalls. An aptamer, raised to the SARS-CoV-2 spike protein, was immobilized on a low-cost gold-coated polyester substrate adapted from the blood glucose testing industry. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times as short as 15 min on nasopharyngeal swab samples. This assay can readily be optimized for mass manufacture and is compatible with a low-cost meter.

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 predators and parasites, to prevent desiccation, to control gaseous exchange, to buffer 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 túngara frog ( Engystomops pustulosus ) are stable ex situ with useful physiochemical and biocompatible properties and are 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 topical drug delivery systems., (© 2021 The Authors.)

Accurate and rapid diagnostic tests are critical to reducing the impact of SARS-CoV-2. This study presents early, but promising measurements of SARS-CoV-2 using the ACE2 enzyme as the recognition element to achieve clinically relevant detection. The test provides a scalable route to sensitive, specific, rapid and low cost mass testing.

Actinobacteria is 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 and made using the enzyme pathways encoded in their complex genomes. Whilst the number of sequenced genomes has increased rapidly in the last 20 years, the large size, complexity and high G+C content 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 genomic and evolutionary studies. To enable greater understanding and exploitation of actinobacterial genomes, specialized 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 re-annotation. Utilizing 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. This article contains data hosted by Microreact.

The genus Streptomyces is widely recognized for its biotechnological potential. Due to a need to improve crops, clean up the environment and produce novel antimicrobial molecules exploiting Streptomyces has become a priority. To further explore the biotechnological potential of these organisms we analyzed the genome of the strain Streptomyces sp. Z38 isolated from contaminated roots tissues. Our analysis not only confirmed the ability of the strain to produce plant growth promoting traits but also a range of mechanisms to cope with the toxic effect of heavy metals through genes involved in metal homeostasis and oxidative stress response. The production of silver nanoparticles indicated that Streptomyces sp. Z38 may find utility in Green, Grey and Red biotechnology., (Copyright © 2020. Published by Elsevier Inc.)

Antibiotic resistance has been cited by the World Health Organisation (WHO) as one of the greatest threats to public health. Mitigating the spread of antibiotic resistance requires a multipronged approach with possible interventions including faster diagnostic testing and enhanced antibiotic stewardship. This study employs a low-cost diagnostic sensor test to rapidly pinpoint the correct antibiotic for treatment of infection. The sensor comprises a screen-printed gold electrode, modified with an antibiotic-seeded hydrogel to monitor bacterial growth. Electrochemical growth profiles of the common microorganism, Escherichia coli ( E. coli ) (ATCC 25922) were measured in the presence and absence of the antibiotic streptomycin. Results show a clear distinction between the E. coli growth profiles depending on whether streptomycin is present, in a timeframe of ≈2.5 h ( p < 0.05), significantly quicker than the current gold standard of culture-based antimicrobial susceptibility testing. These results demonstrate a clear pathway to a low cost, phenotypic and reproducible antibiotic susceptibility testing technology for the rapid detection of E. coli within clinically relevant concentration ranges for conditions such as urinary tract infections.

Microbial natural products, particularly those produced by filamentous Actinobacteria , underpin the majority of clinically used antibiotics. Unfortunately, only a few new antibiotic classes have been discovered since the 1970s, which has exacerbated fears of a postapocalyptic world in which antibiotics have lost their utility. Excitingly, the genome sequencing revolution painted an entirely new picture, one in which an average strain of filamentous Actinobacteria harbors 20 to 50 natural product biosynthetic pathways but expresses very few of these under laboratory conditions. Development of methodology to access this "hidden" biochemical diversity has the potential to usher in a second Golden Era of antibiotic discovery. The proliferation of genomic data has led to inconsistent use of "cryptic" and "silent" when referring to biosynthetic gene clusters identified by bioinformatic analysis. In this Perspective, we discuss this issue and propose to formalize the use of this terminology., (Copyright © 2020 Hoskisson and Seipke.)

This Concept article describes the latest developments in the emerging area of late-stage biocatalytic alkylation. Central to these developments is the ability to efficiently prepare S-adenosyl methionine (SAM) cofactor analogues and couple this with enzymatic alkyl transfer. Recent developments in the enzymatic synthesis of SAM cofactor analogues are summarized first, followed by their application as alkyl transfer agents catalyzed by methyltransferases (MTases). Second, innovative methods to regenerate SAM cofactors by enzymatic cascades is reported. Finally, future opportunities towards establishing a generalized platform for late-stage alkylation are described., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ability of microorganisms to grow as aggregated assemblages has been known for many years, however their structure has remained largely unexplored across multiple spatial scales. The development of the Mesolens, an optical system which uniquely allows simultaneous imaging of individual bacteria over a 36 mm 2 field of view, has enabled the study of mature Escherichia coli macro-colony biofilm architecture like never before. The Mesolens enabled the discovery of intra-colony channels on the order of 10 μm in diameter, that are integral to E. coli macro-colony biofilms and form as an emergent property of biofilm growth. These channels have a characteristic structure and re-form after total mechanical disaggregation of the colony. We demonstrate that the channels are able to transport particles and play a role in the acquisition of and distribution of nutrients through the biofilm. These channels potentially offer a new route for the delivery of dispersal agents for antimicrobial drugs to biofilms, ultimately lowering their impact on public health and industry.

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH 4 + transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea . The pathway underpins a mechanism by which charged H + and neutral NH 3 are carried separately across the membrane after NH 4 + deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process., Competing Interests: GW, GT, AB, MB, GD, MB, AP, CI, ET, PH, AM, UZ, AJ No competing interests declared, (© 2020, Williamson et al.)

Covering: 2008 up to 2019The forces of biochemical adaptive evolution operate at the level of genes, manifesting in complex phenotypes and the global biodiversity of proteins and metabolites. While evolutionary histories have been deciphered for some other complex traits, the origins of natural product biosynthesis largely remain a mystery. This fundamental knowledge gap is surprising given the many decades of research probing the genetic, chemical, and biophysical mechanisms of bacterial natural product biosynthesis. Recently, evolutionary thinking has begun to permeate this otherwise mechanistically dominated field. Natural products are now sometimes referred to as 'specialized' rather than 'secondary' metabolites, reinforcing the importance of their biological and ecological functions. Here, we review known evolutionary mechanisms underlying the overwhelming chemical diversity of bacterial secondary metabolism, focusing on enzyme promiscuity and the evolution of enzymatic domains that enable metabolic traits. We discuss the mechanisms that drive the assembly of natural product biosynthetic gene clusters and propose formal definitions for 'specialized' and 'secondary' metabolism. We further explore how biosynthetic gene clusters evolve to synthesize related molecular species, and in turn how the biological and ecological roles that emerge from metabolic diversity are acted on by selection. Finally, we reconcile chemical, functional, and genetic data into an evolutionary model, the dynamic chemical matrix evolutionary hypothesis, in which the relationships between chemical distance, biomolecular activity, and relative fitness shape adaptive landscapes.

Background: Streptomycete bacteria are prolific producers of specialized 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-functionalization or sub-functionalization. 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., Results: RNA-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 specialized metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source., Conclusions: Expression 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., Competing Interests: The author declare no competing financial or non-financial interests., (© 2020 The Authors.)

Improvements in hygiene and health management have driven significant increases in human lifespan over the last 50 years. Frustratingly however, this extension of lifespan has not been matched by equivalent improvements in late-life health, not least due to the global pandemic in type-2 diabetes, obesity and cardiovascular disease, all ageing-associated conditions exacerbated and accelerated by widespread adoption of the high calorie Western diet (HCD). Recently, evidence has begun to emerge that parasitic worm infection might protect against such ageing-associated co-morbidities, as a serendipitous side-effect of their evolution of pro-survival, anti-inflammatory mechanisms. As a novel therapeutic strategy, we have therefore investigated the potential of ES-62, an anti-inflammatory secreted product of the filarial nematode Acanthocheilonema viteae, to improve healthspan (the period of life before diseases of ageing appear) by targeting the chronic inflammation that drives metabolic dysregulation underpinning ageing-induced ill-health. We administered ES-62 subcutaneously (at a dose of 1 μg/week) to C57BL/6J mice undergoing HCD-accelerated ageing throughout their lifespan, while subjecting the animals to analysis of 120 immunometabolic responses at various time-points. ES-62 improved a number of inflammatory parameters, but markedly, a range of pathophysiological, metabolic and microbiome parameters of ageing were also successfully targeted. Notably, ES-62-mediated promotion of healthspan in male and female HCD-mice was associated with different mechanisms and reflecting this, machine learning modelling identified sex-specific signatures predictive of ES-62 action against HCD-accelerated ageing. Remarkably, ES-62 substantially increased the median survival of male HCD-mice. This was not the case with female animals and unexpectedly, this difference between the two sexes could not be explained in terms of suppression of the chronic inflammation driving ageing, as ES-62 tended to be more effective in reducing this in female mice. Rather, the difference appeared to be associated with ES-62's additional ability to preferentially promote a healthier gut-metabolic tissue axis in male animals., Competing Interests: The authors have declared that no competing interests exist.

The deltaproteobacterium Myxococcus xanthus is a model for bacterial motility and has provided unprecedented insights into bacterial swarming behaviors. Fluorescence microscopy techniques have been invaluable in defining the mechanisms that are involved in gliding motility, but these have almost entirely been limited to two-dimensional (2D) studies, and there is currently no understanding of gliding motility in a three-dimensional (3D) context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, revealing aperiodic oscillatory behavior with changes in the position of the basal membrane relative to the substrate on the order of 90 nm in vitro First, we use a model planoconvex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus bacteria and show that cells undergo previously unobserved changes in their adhesion profile as they glide. We compare the wild type with mutants that have reduced motility, which also exhibit the same changes in the adhesion profile during gliding. We find that the general gliding behavior is independent of the proton motive force-generating complex AglRQS and suggest that the novel behavior that we present here may be a result of recoil and force transmission along the length of the cell body following firing of the type IV pili. IMPORTANCE 3D imaging of live bacteria with optical microscopy techniques is a challenge due to the small size of bacterial cells, meaning that previous studies have been limited to observing motility behavior in 2D. We introduce the application of confocal multiwavelength interference reflection microscopy to bacteria, which enables visualization of 3D motility behaviors in a single 2D image. Using the model organism Myxococcus xanthus , we identified novel motility behaviors that are not explained by current motility models, where gliding bacteria exhibit aperiodic changes in their adhesion to an underlying solid surface. We concluded that the 3D behavior was not linked to canonical motility mechanisms and that IRM could be applied to study a range of microbiological specimens with minimal adaptation to a commercial microscope., (Copyright © 2020 Rooney et al.)

To avoid an antibiotic resistance crisis, we need to develop antibiotics at a pace that matches the rate of evolution of resistance. However, the complex functions performed by antibiotics-combining, e.g., penetration of membranes, counteraction of resistance mechanisms, and interaction with molecular targets-have proven hard to achieve with current methods for drug development, including target-based screening and rational design. Here, we argue that we can meet the evolution of resistance in the clinic with evolution of antibiotics in the laboratory. On the basis of the results of experimental evolution studies of microbes in general and antibiotic production in Actinobacteria in particular, we propose methodology for evolving antibiotics to circumvent mechanisms of resistance. This exploits the ability of evolution to find solutions to complex problems without a need for design. We review evolutionary theory critical to this approach and argue that it is feasible and has important advantages over current methods for antibiotic discovery., (Copyright © 2019 Wollein Waldetoft et al.)

A tandem enzymatic strategy to enhance the scope of C-alkylation of small molecules via the in situ formation of S-adenosyl methionine (SAM) cofactor analogues is described. A solvent-exposed channel present in the SAM-forming enzyme SalL tolerates 5'-chloro-5'-deoxyadenosine (ClDA) analogues modified at the 2-position of the adenine nucleobase. Coupling SalL-catalyzed cofactor production with C-(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C-(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C-alkylation provides the basis to develop a late-stage enzymatic platform for the preparation of high value small molecules., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Antimicrobial resistance (AMR) is an issue of upmost global importance, with an annually increasing mortality rate and growing economic burden. Poor antimicrobial stewardship has resulted in an abundance and diverse range of antimicrobial resistance mechanisms. To tackle AMR effectively, better diagnostic tests must be developed in order to improve antibiotic stewardship and reduce the emergence of antibiotic resistant organisms. This study employs a low-cost, commercially available screen printed electrode modified with an agarose-based hydrogel deposit to monitor bacterial growth using the techniques of electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) giving rise to a new approach to measuring susceptibility. Susceptible and drug resistant Staphylococcus aureus strains were deposited onto agarose gel modified electrodes which contained clinically important antibiotics to establish growth profiles for each bacterial strain and monitor the influence of the antibiotic on bacterial growth. The results show that S. aureus is able to grow on electrodes modified with gel containing no antibiotic, but is inhibited when the gel modified electrode is seeded with antibiotic. Conversely, methicillin-resistant S. aureus (MRSA; the drug resistant strain) is able to grow on gel modified electrodes containing clinically relevant concentrations of antibiotic. Results show rapid growth profiles, with possible time to results for antibiotic susceptibility <45 min, a significant improvement on the current gold standards of at least 1-2 days., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Polar and subpolar ecosystems are highly vulnerable to global climate change with consequences for biodiversity and community composition. Bacteria are directly impacted by future environmental change and it is therefore essential to have a better understanding of microbial communities in fluctuating ecosystems. Exploration of Polar environments, specifically sediments, represents an exciting opportunity to uncover bacterial and chemical diversity and link this to ecosystem and evolutionary parameters. In terms of specialized metabolite production, the bacterial order Actinomycetales , within the phylum Actinobacteria are unsurpassed, producing 10 000 specialized metabolites accounting for over 45 % of all bioactive microbial metabolites. A selective isolation approach focused on spore-forming Actinobacteria of 12 sediment cores from the Antarctic and sub-Arctic generated a culture collection of 50 strains. This consisted of 39 strains belonging to rare A ctinomycetales genera including Microbacterium, Rhodococcus and Pseudonocardia . This study used a combination of nanopore sequencing and molecular networking to explore the community composition, culturable bacterial diversity, evolutionary relatedness and specialized metabolite potential of these strains. Metagenomic analyses using MinION sequencing was able to detect the phylum Actinobacteria across polar sediment cores at an average of 13 % of the total bacterial reads. The resulting molecular network consisted of 1652 parent ions and the lack of known metabolite identification supports the argument that Polar bacteria are likely to produce previously unreported chemistry.

Reconciling transcription and DNA replication in the growing hyphae of the filamentous bacterium Streptomyces presents several physical constraints on growth due to their apically extending and branching, multigenomic cells and chromosome replication being independent of cell division. Using a GFP translational fusion to the β '-subunit of RNA polymerase ( rpoC-egfp ), in its native chromosomal location, we observed growing Streptomyces hyphae using time-lapse microscopy throughout the lifecycle and under different growth conditions. The RpoC-eGFP fusion co-localized with DNA around 1.8 µm behind the extending tip, whereas replisomes localize around 4-5 µm behind the tip, indicating that at the growing tip, transcription and chromosome replication are to some degree spatially separated. Dual-labelled RpoC-egfp/DnaN-mCherry strains also indicate that there is limited co-localization of transcription and chromosome replication at the extending hyphal tip. This likely facilitates the use of the same DNA molecule for active transcription and chromosome replication in growing cells, independent of cell division. This represents a novel, but hitherto unknown mechanism for reconciling two fundamental processes that utilize the same macromolecular template that allows for rapid growth without compromising chromosome replication in filamentous bacteria and may have implications for evolution of filamentous growth in micro-organisms, where uncoupling of DNA replication from cell division is required.

Producing specialised metabolites such as antibiotics, immunosuppressives, anti-cancer agents and anti-helminthics draws on primary metabolism to provide the building blocks for biosynthesis. The growth phase-dependent nature of production means that producing organisms must deal with the metabolic conflicts of declining growth rate, reduced nutrient availability, specialised metabolite production and potentially morphological development. In recent years, our understanding of gene expansion events, integration of metabolic function and gene regulation events that facilitate the sensing and responding to metabolite concentrations has grown, but new data are constantly expanding our horizons. This review highlights the role evolutionary gene or pathway expansion plays in primary metabolism and examine the adoption of enzymes for specialised metabolism. We also look at recent insights into sensing and responding to metabolites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)