Biological Context

Lead Research Organisation: John Innes Centre
Department Name: Contracts Office

Abstract

The vast wealth of natural products discovered so far, and the far greater numbers predicted from genome sequences, potentially provide entirely new opportunities for biological discoveries. Many of these molecules are likely to have roles in the environment – for example in deterring or killing competitors, facilitating symbioses, acquiring nutrients, and attracting dispersal agents. By shedding light on the roles and targets of natural products and the contexts in which they are produced, we will provide new understanding of the biology of the producing organisms and the ecological niches they occupy and also accelerate the discovery of new bioactive molecules as antibiotics, therapeutics, agrichemicals, food ingredients and industrial raw materials. This research both draws on and feeds into product and pathway discovery research in the Project “Products and pathways”. Research in the Biological context Project stems in part from new discoveries in established areas of strength: bacterial growth and differentiation and the biology of proteins that are targets for antibiotics. The Project also takes a major new direction: the exploration of small-molecule interactions between microbes and other organisms in the soil. This is an area of strong synergy with other JIC research in Plant Health, and with the University of East Anglia.
The outcomes of this Project are relevant for both the development and discovery of antibiotics and the search for new agrichemicals and interventions. Better understanding of the role of natural products in microbial interactions will underpin the identification of new antimicrobial molecules and biocontrol strains for the control of crop pests and diseases: a subject of particular concern given increasing legislative restrictions on the use of previously-allowed agrichemicals.

Publications

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Aron AT (2020) Reproducible molecular networking of untargeted mass spectrometry data using GNPS. in Nature protocols

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Bax BD (2019) DNA Topoisomerase Inhibitors: Trapping a DNA-Cleaving Machine in Motion. in Journal of molecular biology

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Becher PG (2020) Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal. in Nature microbiology

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Bush MJ (2019) BldC Delays Entry into Development To Produce a Sustained Period of Vegetative Growth in Streptomyces venezuelae. in mBio

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Bush MJ (2017) Multi-layered inhibition of Streptomyces development: BldO is a dedicated repressor of whiB. in Molecular microbiology

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Bush, N.G. (2018) DNA in a twist? How topoisomerases solve topological problems in DNA in The Biochemist

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Buttner MJ (2018) Structural insights into simocyclinone as an antibiotic, effector ligand and substrate. in FEMS microbiology reviews

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Buttner MJ (2017) Actinoplanes Swims into the Molecular Age. in Journal of bacteriology

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Casas-Pastor D (2021) Expansion and re-classification of the extracytoplasmic function (ECF) s factor family. in Nucleic acids research

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Chan PF (2017) Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase. in Proceedings of the National Academy of Sciences of the United States of America

 
Description KEY FINDINGS APRIL 2017 - MARCH 2018

OBJECTIVE 1: BACTERIAL DIFFERENTIATION AND CHROMOSOME ORGANISATION.

CONTROL OF THE INITIATION OF STREPTOMYCES DEVELOPMENT. (Schumacher et al., 2017. Nucleic Acids Res. 45: 6923-6933. doi: 10.1093/nar/gkx287; Bush et al., 2017. Mol. Microbiol. 104: 700-711. doi: 10.1111/mmi.13663). Streptomyces are ubiquitous soil bacteria that undergo a complex developmental transition coinciding with their production of antibiotics. This transition is controlled by binding of a novel tetrameric form of the second messenger, 3'-5' cyclic diguanylic acid (c-di-GMP) to the master repressor, BldD. In all domains of life, nucleotide-based second messengers allow a rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions. c-di-GMP can assume alternative oligomeric states to effect different functions, binding to effector proteins as monomers, intercalated dimers, or, uniquely in the case of BldD, as a tetramer. However, at physiological concentrations c-di-GMP is a monomer and little is known about how higher oligomeric complexes assemble on effector proteins and if intermediates in assembly pathways have regulatory significance. We have now demonstrated that c-di-GMP binds BldD using an ordered, sequential mechanism and that BldD function necessitates the assembly of the tetrameric c-di-GMP-BldD complex.

BldD-(c-di-GMP) sits on top of the regulatory network that controls differentiation in Streptomyces, repressing a large regulon of developmental genes when the bacteria are growing vegetatively. In this way, BldD functions as an inhibitor that blocks the initiation of sporulation. We have reported the identification and characterization of BldO, an additional developmental repressor that acts to sustain vegetative growth and prevent entry into sporulation. However, unlike the pleiotropic regulator BldD, we have shown that BldO functions as the dedicated repressor of a single key target gene, whiB, and that deletion of bldO or constitutive expression of whiB is sufficient to induce precocious hypersporulation.

THE ROLE OF DYNAMIN MEMBRANE-REMODELLING PROTEINS IN BACTERIA. (Schlimpert et al., 2017. PNAS 114: E6176-E6183. doi/10.1073/pnas.1704612114). Bacterial dynamins were discovered ~10 years ago and the explosion in genome sequencing has shown that they radiate throughout the bacteria, being present in >1000 species. In eukaryotes, dynamins play critical roles in the detachment of endocytic vesicles from the plasma membrane, the division of chloroplasts and peroxisomes, and both the fusion and fission of mitochondria. However, in evolutionary terms, dynamins are of bacterial origin, and yet the biological functions of bacterial dynamins remain poorly understood. We have now demonstrated a novel and critical role for dynamins in developmentally regulated cell division in Streptomyces, reminiscent of the essential role of eukaryotic dynamins in the division of chloroplasts and mitochondria.

CONTROL OF THE OXIDATIVE STRESS RESPONSE IN STREPTOMYCES. Feeney et al., 2017. mBio 8: e00815-17. doi:10.1128/mBio.00815-17). The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and post-translational levels. Here we show that sigR has a highly unusual GTC start codon, and that this leads to another level of SigR regulation, in which SigR translation is repressed by Translation Initiation Factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overexpressed relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the non-canonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper function of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Instead, we have shown that RsrA can be transcribed and translated independently of SigR, that RsrA is normally produced in excess of SigR, and we have characterized the factors that determine SigR:RsrA stoichiometry.

CHROMOSOME ORGANIZATION. (Tran et al., 2017. Cell Rep. 20: 2057-2071. doi: 10.1016/j.celrep.2017.08.026). SMC proteins, also known as cohesin and condensin in eukaryotes, is crucial for chromosome organization in all living organisms. But how SMC translocates on a protein-laden chromosome is poorly investigated. Whether SMC impacts DNA-translocating proteins such as RNA polymerase and is, in turn, influenced by such proteins, is not well understood. How different cellular processes share the same DNA but avoid, or resolve, conflicts is a question that arises in all cells and all domains of life. Our study provided the first concrete experimental evidence that the translocation of bacterial SMC on the chromosome is strongly influenced by RNA polymerases. We showed that highly-transcribed genes oriented to collide head-on with translocating SMC slow down and potentially stop SMC translocation. This might have contributed to the selection for highly-transcribed genes to be co-oriented with the direction of SMC movement. Broadly, our work demonstrated a tight interdependence of bacterial chromosome organization and the highly non-random global pattern of transcription. Further, we demonstrate that the translocation of bacterial SMC is directional, from the bacterial centromere site parS progressively to the replication terminus, extruding out DNA in a loop as a result of this directional movement. The finding that bacterial SMC translocation is directional has significant implications for how eukaryotic cohesin and CTCF may work together to extrude DNA loops (Rao et al 2015 Cell, Sanborn et al 2015 PNAS, Fudenberg et al 2016 Cell Rep).

CAULOBACTER AS A MODEL SYSTEM. Our findings were greatly aided by the use of Caulobacter as a model system. Caulobacter is easily synchronized, enabling us to generate genome-wide data for a homogenous population of G1-phase cells that each contain a single chromosome. As there is no active DNA replication in these cells, we were able to isolate and specifically study the effect of transcription on SMC translocation and global chromosome organization, without confounding effects from replication-transcription conflicts. Such a "clean" arrangement cannot be achieved in other traditional model bacteria such as Escherichia coli, Bacillus subtilis, or Streptomyces. Our work took advantage of the genetic tractability of Caulobacter but produced findings that should be of broad relevance to researchers studying chromosome organization and chromosome segregation in a wide range of bacterial species.

OBJECTIVE 2: SMALL-MOLECULE TARGET PROTEINS.

DNA GYRASE AS AN ANTIBIOTIC TARGET. (Webber et al., 2017. J Antimicrob Chemother. 72: 2755-2763. doi: 10.1093/jac/dkx201; Ghilarov et al., 2017. Structure 25: 1549-1561.e5. doi: 10.1016/j.str.2017.08.006; Chan et al., 2017. PNAS. 114: E4492-E4500. doi: 10.1073/pnas.1700721114; Wallace et al., 2018. Chem Commun. 54: 1869-1872. doi: 10.1039/c7cc09518j; Colgan et al., 2018. Mol Microbiol. Jan 20. doi: 10.1111/mmi.13911). This work concerns the physiological roles of bacterial DNA gyrase and its interactions with inhibitors, towards the aim of developing novel antibiotics and herbicides. We showed in Salmonella enterica serovar Typhimurium, which exists at low pH in macrophage vacuoles, that the physiological effects can be attributed, at least in part, to the effect of low pH on gyrase's supercoiling activity. Further, in Escherichia coli, we showed that mutations in gyrase conferring quinolone resistance have a fitness cost that is manifested by a decrease in susceptibility to triclosan. In collaboration with GSK and Sanofi we have identified a novel drug-binding pocket in gyrase that accommodates thiophene compounds, which can be exploited in the development of new antibacterial agents. With Arabidopsis thaliana gyrase we have found that, like its bacterial counterparts, it can be inhibited by quinolone antibiotics, such as ciprofloxacin, and that these compounds also have herbicidal activity. Analogues of ciprofloxacin have been prepared with increased herbicidal activity and diminished antibacterial activity. We have been developing the bacterial toxin microcin B17 as a potential substitute for quinolones and have discovered how it is proteolytically processed during its biosynthesis. To aid drug discovery in the gyrase (and topoisomerase) area we have developed a rapid high-resolution method for resolving DNA topoisomers, using microcapillary electrophoresis.

OBJECTIVE 3: SMALL-MOLECULE MEDIATED INTERACTIONS BETWEEN MICROBES AND OTHER ORGANISMS.

USING CHEMICAL ECOLOGY AND GENOMICS TO DISCOVER NEW ANTIBIOTICS. (Qin et al., 2017. Chem Sci. 8(4):3218-3227. doi: 10.1039/c6sc04265a; Holmes et al., 2018. J Biotechnol. 265: 116-118. doi: 10.1016/j.jbiotec.2017.11.011; Scott et al., 2017. Nat Commun. 8: 15935. doi: 10.1038/ncomms15935). Using a chemical ecology/genomics guided approach we discovered a new class of polyketide antibiotics that we named the formicamycins. The formicamycins are active against drug resistant pathogens, including clinical isolates of MRSA and VRE, and display an extremely high barrier for the selection of resistant isolates. We identified the BGC encoding formicamycin production and were able to interrogate the biosynthetic pathway. Future work will extend these findings: our key aims are to produce new analogues with improved properties, and identify the molecular target/mode of action as part of our efforts to generate next generation antibiotics active against drug resistant organisms. In parallel work we delineated the entire biosynthetic pathway to the unusual antibiotic obafluorin produced by Pseudomonas fluorescens and identified a new family of l-threonine transaldolase enzymes which have potential synthetic utility (10.1038/ncomms15935). More recently we uncovered the likely molecular target of obafluorin and have preliminary data suggesting a novel mode of action which has value for the design of next generation versions of obafluorin with activity against drug resistant pathogens.

THE WAX MOTH AS A MODEL SYSTEM. (Ignasiak and Maxwell, 2017a. BMC Microbiol. 17: 223. doi: 10.1186/s12866-017-1133-0; Ignasiak and Maxwell, 2017b. BMC Res Notes. 10: 428. doi: 10.1186/s13104-017-2757-8). We have been developing the greater wax moth larva (Galleria mellonella) as a system that can be used for antibacterial drug discovery, and shown that it can be used as a surrogate for rodents in infectivity trials and toxicity testing. Moreover, we have shown that the Galleria gut microbiome can be used in the discovery process for new, plant-based, antibiotics.

IDENTIFICATION OF THE BIOSYNTHETIC GENE CLUSTER FOR BICYCLOMYCIN. (Vior et al. 2018. Appl. Environ. Microbiol., in the press). Bicyclomycin is an antibiotic that is a promising candidate for treating multi-drug resistant bacterial infections. Our identification of the biosynthetic gene cluster for bicyclomycin revealed that this gene cluster is found in the genomes of a wide variety of bacteria, including hundreds of sequenced isolates of the human pathogen Pseudomonas aeruginosa. This represents a highly unusual transfer of an intact gene cluster between Gram-positive and Gram-negative bacteria, and we showed that the P. aeruginosa gene cluster produces authentic bicyclomycin. This discovery provides the basis for further investigations into how and why this antibiotic pathway has become distributed across such diverse bacteria, and whether it has a role in P. aeruginosa infection.

ACTIVATION OF SILENT ANTIBIOTIC BIOSYNTHETIC GENE CLUSTERS. (Som et al., 2017. Front Microbiol. 8:1145. doi: 10.3389/fmicb.2017.01145; Som et al., 2017. Microbiology 163: 1415-1419. doi: 10.1099/mic.0.000524). A major aim of our research is to develop methods for the activation of silent or cryptic biosynthetic gene clusters (BGCs) in order to illuminate the natural products chemical 'dark matter' encoded in actinobacterial genomes, and to increase the production of compounds otherwise produced at trace levels. We have now shown that the highly conserved actinobacterial two component system mtrAB is important in coordinating antibiotic production with development, and that mtrAB manipulation leads to the significant increase in antibiotic production and a shift in the global metabolome. We will exploit these findings for the discovery of new molecules from environmental isolates, and the anticipate it will form part of a toolkit for cryptic BGC activation useful to researchers generally.


KEY FINDINGS APRIL 2018 - MARCH 2019

OBJECTIVE 1: BACTERIAL DIFFERENTIATION AND CHROMOSOME ORGANISATION.

CONTROL OF THE INITIATION OF DEVELOPMENT IN ANTIBIOTIC-PRODUCING STREPTOMYCES. (Schumacher et al., 2018. Nat. Commun. 9: 1139. doi: 10.1038/s41467-018-03576-3; Schumacher et al., 2018. Nucleic Acids Res. 46: 7405-7417. doi: 10.1093/nar/gky493; Bush et al., 2019. mBio 10: e02812-18. doi.org/10.1128/mBio.02812-18). The complex life cycle of the antibiotic-producing bacteria Streptomyces involves two distinct filamentous cell forms: the growing or vegetative hyphae and the reproductive or aerial hyphae, which differentiate into long chains of spores. Genetic studies identified the transcription factors that control entry into development, which are called Bld (bald) regulators because mutations in the corresponding genes prevent formation of the hair-like aerial hyphae.

One of these regulators, BldC, is a small, 68-residue protein with a winged Helix-Turn-Helix (wHTH) motif, related to those found in MerR-family proteins. The basic structure of classical MerR proteins is a dimer consisting of two identical subunits, each composed of an N-terminal wHTH DNA-binding domain, a C-terminal effector-recognition domain and an interconnecting linker region that consists of a long a-helix that interacts with the same helix in the other subunit, forming an antiparallel coiled-coil responsible for homodimerization. MerR transcription factors bind to palindromic DNA sequences as homodimers. However, unlike classical members of the MerR family, BldC has neither an effector domain nor the dimerization helix, and BldC behaves as a monomer in free solution. As a consequence, how BldC might bind DNA was unclear. To address this question, we carried out biochemical and structural studies to characterize the binding of BldC to the promoters of two known target genes. These studies showed that BldC binds DNA in a completely different way to classical MerR regulators, instead involving asymmetric, cooperative, head-to-tail oligomerization on DNA direct repeats with concomitant pronounced DNA distortion. The number of direct repeats present in BldC-binding sites is variable, thus allowing cooperative, head-to-tail binding of additional BldC monomers. Since BldC-like proteins radiate throughout the bacteria, this study identified BldC as the founding member of a new structural family of transcription factors. Although this work provided a clear mechanistic understanding of how BldC binds DNA, there was less insight into its biological role and impact on Streptomyces development. Accordingly, we exploited the benefits of the model species, Streptomyces venezuelae, which sporulates in liquid culture, to study the biological role of BldC. Using ChIP-seq coupled with RNA-seq, we identified the genes under BldC control and showed that BldC can function both as a repressor and as an activator of transcription. We showed that bldC mutants are bald because they enter development prematurely, bypassing the formation of aerial hyphae. This correlates with the premature expression of BldC target genes with key roles in development, chromosome condensation and segregation, and sporulation-specific cell division, suggesting that BldC-mediated repression is critical to maintain a sustained period of vegetative growth prior to sporulation.

A second Bld regulator, BldN, is an extracytoplasmic function (ECF) s factor that plays a pivotal role in the onset of development by directing transcription of the rodlin and chaplin genes, which encode the proteins required to form an external hydrophobic sheath that permits the reproductive aerial hyphae to escape surface tension and grow into the air. BldN activity is in turn controlled by an anti-s factor, RsbN. RsbN shows no sequence similarity to known anti-s factors and binds and inhibits BldN in an unknown manner. We determined the 2.23 Å structure of the RsbN-BldN complex. The structure shows that BldN harbours s2 and s4 domains that are individually similar to other ECF s domains, which bind -10 and -35 promoter regions, respectively. The anti-s RsbN consists of three helices, with a3 forming a long helix embraced between BldN s2 and s4 while RsbN a1-a2 dock against s4 in a manner that would block -35 DNA binding. RsbN binding also freezes BldN in a conformation inactive for simultaneous -10 and -35 promoter interaction and RNAP binding. Strikingly, RsbN is structurally distinct from previously solved anti-s proteins. Thus, we have characterized the molecular determinants controlling a central Streptomyces developmental switch and shown that RsbN is the founding member of a new structural class of anti-s factor.

OBJECTIVE 2: SMALL-MOLECULE TARGET PROTEINS.

DNA GYRASE FUNCTION AND ANTIBIOTIC TARGETING. (Stracy, et al., 2019. Nucleic Acids Res, 47: 210-220; doi: 10.1093/nar/gky1143; Bush, et al., 2018. The Biochemist, 40; 26-31; Jeannot, et al., 2018. J Med Chem, 61: 3565-3581; doi: 10.1021/acs.jmedchem.7b01892; Germe, et al., 2018. Nucleic Acids Res, 46; doi: 4114-412810.1093/nar/gky181; Mori, et al., 2018. Tuberculosis, 112: 98-109; doi: 10.1016/j.tube.2018.08.004; Maxwell, et al., 2018. In Fong, I. W., Shlaes, D. and Drlica, K. (eds.), Antimicrobial resistance and implications for the 21st century. Springer, Switzerland; doi: 10.1007/978-3-319-78538-7). Working with groups in York, Oxford and Poznan (Poland) we have used high-speed single-molecule imaging in live Escherichia coli to show preferential association of gyrase at the DNA replication fork and demonstrated that the enzyme also performs a significant role in vivo in maintaining the level of negative supercoiling. In work partly funded by the EU in two consortia (mm4tb & ENABLE), we have investigated novel compounds that target gyrase as potential antibiotics. Imidazopyrazinones (IPYs), developed by Sanofi, were found to bind to gyrase at the quinolone pocket but to make different contacts, such that cross-resistance was only partial. The antimalarial compound, pyronaridine, showed modest activity against DNA gyrase from Mycobacterium tuberculosis, but higher activity versus RNA polymerase.

OBJECTIVE 3: SMALL-MOLECULE MEDIATED INTERACTIONS BETWEEN MICROBES AND OTHER ORGANISMS.

CHEMICAL WARFARE BETWEEN LEAFCUTTER ANT SYMBIONTS AND A CO-EVOLVED PATHOGEN. (Heine et al., 2018. Nat. Commun. 9:2208; doi: 10.1038/s41467-018-04520-1). Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the cultivar fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus garden and lead to colony collapse. Our interdisciplinary approach showed that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioural defences and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioural defences such that colony collapse is unavoidable once Escovopsis infections escalate.

ANTIBIOTIC AND TOXIN ACTION AND DISCOVERY. (Ignasiak & Maxwell, 2018. BMC Microbiol, 18: 228. doi: 10.1186/s12866-018-1377-3; Farrell, et al., 2018. J Antimicrob Chemother, 73; 2284-2297. doi: 10.1093/jac/dky208; Iyer, et al., 2018. Cell, 173: 1123-1134. doi: 10.1016/j.cell.2018.04.037). To further develop Galleria mellonella (greater wax moth) as a model system for studying the effects of antibiotics on the human infant gut microbiome, we have assessed the effects of treating larvae with oxytetracycline and found that it affects the composition and diversity of the insects' gut microbiome. We have investigated antibiotic compounds that have been reported in the literature and not developed as clinical antibiotics, generating an antibiotic database (Antibiotic DB) towards the goal of revitalising the drug pipeline. Investigations of the natural product toxin Microcin B17, produced by Escherichia coli in the human gut, have shown that it can trigger an intestinal inflammation response that is associated with inflammatory bowel disease.

DISCOVERY OF NOVEL RIBOSOMALLY SYNTHESISED AND POST-TRANSLATIONALLY MODIFIED PEPTIDES. (Santos-Aberturas et al., 2018. bioRxiv 494286. doi: 10.1101/494286). To enable the discovery of novel ribosomally synthesised and post-translationally modified peptides (RiPPs), we have developed a genome mining tool, RiPPER (https://github.com/streptomyces/ripper), which enables the family-independent identification of RiPP precursor peptides from genomic data. This will support our efforts to understand the natural products made by bacteria during their interactions with other organisms. We used this tool to show that thioamidated RiPP natural products are likely to be widely distributed and diverse, despite currently being incredibly rare. To illustrate this, we characterise the structure and biosynthetic pathway to a new thioamidated natural product family, the thiovarsolins. RiPPER has enabled the bioinformatic identification of many novel gene clusters that are in the process of being investigated.

KEY FINDINGS APRIL 2019 - MARCH 2020

OBJECTIVE 1: BACTERIAL DIFFERENTIATION AND CHROMOSOME ORGANISATION.

c-di-GMP ARMS AN ANTI-s TO CONTROL THE DIFFERENTIATION OF THE REPRODUCTIVE AERIAL HYPHAE INTO SPORES IN STREPTOMYCES (Gallagher et al., 2020. Molecular Cell 77: 586-599. doi.org/10.1016/j.molcel.2019.11.006). The dramatic phenotypic consequences of altered c-di-GMP levels in Streptomyces, where high c-di-GMP levels trap the bacteria in vegetative growth and low c-di-GMP levels cause precocious hypersporulation suggested that this signaling molecule must directly interact with the regulatory network controlling the life cycle. We previously showed that showed that c-di-GMP controls the activity of the master regulator BldD to control entry into development and the formation of the reproductive aerial hyphae (Tschowri et al., 2014). By contrast, what controls the final stage of development, the differentiation of the reproductive aerial hyphae into spores, has been unknown. The sporulation-specific s factor, sWhiG, is known to play a key role in this stage of development, but its function has not been subjected to systematic analysis. We have now identified a novel anti-s factor, RsiG, which controls the activity of sWhiG. Strikingly, we show that the interaction between RsiG and sWhiG is mediated by c-di-GMP. Specifically, when 'armed' with a dimer of c-di-GMP, RsiG sequesters sWhiG, blocking the differentiation of the reproductive hyphae into spores. This is the first known instance of c-di-GMP targeting a s factor. Thus, like BldD, the RsiG-sWhiG complex senses changes in c-di-GMP levels to control a specific stage of development. In this complex, RsiG primarily binding the cyclic dinucleotide via two E(X)3S(X)2R(X)3Q(X)3D signatures, one on each helix of its antiparallel coiled coil. This signature is distinct from any previously observed c-di-GMP binding motif. Notably, the anti-s factor RsiG can bind c-di-GMP in the absence of sWhiG, and that binding is specific for this nucleotide. We also show that RsiG must bind c-di-GMP to carry out its function of inhibiting sWhiG activity during vegetative growth, thereby preventing premature expression of the sWhiG regulon. Conservation of the signature c-di-GMP binding motifs in all Streptomyces RsiG homologs suggests that regulation of RsiG-sWhiG complex formation by this second messenger is a general mechanism of developmental control across the genus. As a result of this discovery, it is now clear that c-di-GMP signals through BldD and sWhiG, respectively, to control the two most dramatic transitions of the Streptomyces life cycle, the formation of the reproductive aerial hyphae, and their differentiation into spore chains.

CHROMOSOME ORGANIZATION. (Jalal et al., 2020. eLife 9: e53515. doi: 10.7554/eLife.53515). In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighbouring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Recently, we reconstituted a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and showed that CTP is required for spreading. We further showed that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB in the past 35 years and open up avenues to investigate the interactions between ParB-parS with ParA and SMC.

OBJECTIVE 2: SMALL-MOLECULE TARGET PROTEINS.

IMMUNITY-GUIDED IDENTIFICATION OF THREONYL-tRNA SYNTHETASE AS THE MOLECULAR TARGET OF OBAFLUORIN, A ?ETA LACTONE ANTIBIOTIC. (Scott et al., 2019. ACS Chem. Biol. 14:2663-2671; DOI: 10.1021/acschembio.9b00590). To meet the ever-growing demands of antibiotic discovery new molecules that work in new ways are urgently needed. Many potent natural products with antibacterial activity that were previously discarded by the pharmaceutical industry might provide new opportunities. One such example is the structurally unique molecule obafluorin that is produced by a soil bacterium. Obafluorin is active against many types of bacteria, including important human pathogens, but how it works was unknown. In this paper we reported that obafluorin targets threonyl-tRNA synthetase, an essential enzyme required for protein synthesis in bacteria. Enzymes of this type are the targets of clinically validated drugs, but threonyl-tRNA synthetase has thus far not been exploited. This means obafluorin does not show cross resistance with other clinically used antibiotics. We also reported how the obafluorin producing bacteria remains immune to obafluorin, and it was this knowledge that helped us to identify threonyl-tRNA synthetase as the target in sensitive cells. Our results will aid in the development of improved obafluorin variants that might be useful as antibiotic drugs.

DNA GYRASE FUNCTION AND ANTIBIOTIC TARGETING. (Bax et al. 2019. J. Mol. Biol. 431: 3427-3449; doi: 10.1016/j.jmb.2019.07.008; Li et al., 2019. Mol. Microbiol. 111: 1529-1543; doi: 10.1111/mmi.14235; McKie et al. 2020. Genes 11: 92; 10.3390/genes11010092). Drawing on extensive structural and mechanistic data, we have established a model for the topoisomerase catalytic cycle and proposed how metal ions are involved in the DNA cleavage-religation cycle; this has important implications for drug interaction, particularly quinolones. Working with colleagues in China, we have found that QnrB, a protein associated with quinolone resistance, can activate DNA replication initiation via binding to dnaA. A key question with gyrases and other topoisomerases is how to determine cleavage- and binding-site specificities; we have reviewed NGS-based methodologies for doing this.

OBJECTIVE 3: SMALL-MOLECULE MEDIATED INTERACTIONS BETWEEN MICROBES AND OTHER ORGANISMS.

ANTIBIOTIC AND TOXIN ACTION AND DISCOVERY. (Maxwell et al., 2019. J. Mol. Biol. 431: 3367-3369; doi: 10.1016/j.jmb.2019.06.018; Collin & Maxwell, 2019. J. Mol. Biol. 431: 3427-3449; doi: 10.1016/j.jmb.2019.05.050; Ghilarov, et al., 2019. Mol. Cell 73: 749-762; doi: 10.1016/j.molcel.2018.11.032; McCauley, et al., 2019. Cell Chem. Biol. 26: 1274-1282; doi: 10.1016/j.chembiol.2019.06.002; Narramore, et al., 2019. Bioorg. Med. Chem. 27: 3546-3550; doi: 10.1016/j.bmc.2019.06.015). We have evaluated the current status of our knowledge of the molecular basis of antibiotic action and resistance, in particular focussing on the current state of our understanding of the action of the Escherichia coli toxin microcin B17 (MccB17) and its prospects for a role in the development of new antibiotics going forward. Working with labs from Russia and Poland we have gained molecular-level understanding of the biosynthesis of MccB17 through structural work. In terms of novel antibiotics, we have been working with colleagues in Canada and identified a novel antibiotic that is a gyrase inhibitor that appears to have a unique mode of action. With colleagues in Leeds using computational medicinal chemistry, we have established the molecular mechanism of action of a series of novel gyrase ATPase inhibitors.

KEY FINDINGS APRIL 2020 - MARCH 2021

OBJECTIVE 1: BACTERIAL DIFFERENTIATION AND CHROMOSOME ORGANIZATION

BACTERIAL DIFFERENTIATION
Ramos-Léon et al. 2020. BioRxiv 10.01.322578. doi:10.1101/2020.10.01.322578

While cell division in model bacterial species like E. coli and B. subtilis is well understood, it is less well characterised in actinobacteria, including industrial and medical important bacteria of the genus Streptomyces and Mycobacterium. We have identified a highly conserved protein that plays an important role in the assembly and function of the bacterial cell division machinery in actinobacteria (doi: https://doi.org/10.1101/2020.10.01.322578). This protein, SepH, directly regulates the function of the key cell division protein FtsZ, thereby promoting the assembly of the cell division machinery and the formation of a division septum. Using a combination of live cell imaging and comparative in vitro approaches, we demonstrate that SepH plays a similar role in morphological diverse actinobacteria including filamentous growing bacteria of the genus Streptomyces and unicellular bacteria such as Mycobacterium smegmatis. Importantly, our results significantly advance the understanding of an essential biological process in this important class of bacteria, which includes major pathogens like the causative agent of TB Mycobacterium tuberculosis.

CHROMOSOME ORGANIZATION
Jalal et al., 2020. eLife 9: e53515. doi: 10.7554/eLife.53515
Szafran et al., 2020 BioRxiv doi: 10.1101/2020.12.09.403915
Jalal et al., 2021 BioRxiv doi: 10.1101/2021.02.11.430593

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParA-ParB-parS system is widely employed for chromosome organization and segregation in bacteria. We, in collaboration with Prof. Jakimowicz, investigated the spatial rearrangement of the Streptomyces venezuelae linear chromosome during its sporogenic development. We showed that at entry to sporulation, arms of S. venezuelae chromosomes are spatially separated, but they are closely aligned within the core region during sporogenic cell division. Chromosomal arm alignment is imposed by the segregation protein ParB and SMC. This work is an extension from our previous work on Caulobacter crescentus SMC and we provided further evidence for the conserved roles of SMC in bacterial chromosome organization.

A DNA-binding protein ParB nucleates on parS sites and must associate with neighbouring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Recently, we reconstituted a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and showed that CTP is required for spreading. We further showed that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB in the past 35 years and open up avenues to investigate the interactions between ParB-parS with ParA and SMC.

Next, we expanded our research to investigate the roles of CTP in nucleoid occlusion and bacterial cell division. We unexpectedly found that CTP couples membrane-binding activity of the nucleoid occlusion factor (Noc) to its spreading activity to ensure a productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity. Our study provided the first evidence for a CTP switch that controls membrane activity, adding the control of membrane association in Noc to the role of ParB-CTP in bacterial chromosome segregation. The implication of our finding is that CTP switches are likely pervasive in biology but have so far been underappreciated. We believe that our work here will motivate a systematic discovery and characterization of other CTPase proteins that will potentially open many new and unexpected horizons in biology.

OBJECTIVE 2: SMALL-MOLECULE TARGET PROTEINS

DNA TOPOISOMERASE STRUCTURE/FUNCTION AND ANTIBIOTIC TARGETING. Waraich et al. 2020. Biotechniques, 69, 356-362; doi: 10.2144/btn-2020-0059
Bush et al., 2019. Molecules, 25; doi: 10.3390/molecules25235662
Henderson et al. 2020. J Antimicrob Chemother, 75, 2835-2842; doi: 10.1093/jac/dkaa286
Skok et al. 2020. ACS Med. Chem. Letts., 11, 2433-2440; doi: 10.1021/acsmedchemlett.0c00416
Kolaric et al. 2021. Nature Comms., 12, 150; doi: 10.1038/s41467-020-20405-8
Pyne et al. 2021. Nature Comms., 12, 1053; doi: 10.1038/s41467-021-21243-y

Although the structure of B-form DNA is well-established, the structure of long, plasmid-sized DNA molecules and their dynamics is challenging to establish. Using atomic-force microscopy, molecular dynamics and SPR, we have observed kinks and defects in supercoiled DNA and how these might influence interactions with enzymes, such as DNA topoisomerases. A key log-jam in antibiotic/anticancer drug screening with topoisomerase targets has been the lack of a useful assay for DNA decatenation. We have addressed this by developing a novel decatenation assay using a singly-linked catenated substrate. Although fluoroquinolones (FQs) are highly successful antibiotics targeted to DNA gyrase, there is an urgent need to develop new agents on account of increasing resistance to FQ compounds. Working with colleagues in industry and academia (in Europe), we have developed several new series of compounds that have efficacy against pathogenic bacteria, in particular Mycobacterium tuberculosis.

OBJECTIVE 3: SMALL-MOLECULE MEDIATED INTERACTIONS BETWEEN MICROBES AND OTHER ORGANISMS

DISCOVERY THAT SIGNALLING BY VOLATILES ATTRACTS SPRINGTAILS AS VECTORS FOR STREPTOMYCES SPORE DISPERSAL
Becher et al., 2020. Nature Microbiology 5: 821-829.

Streptomyces bacteria are responsible for the characteristic earthy odour of soil. This odour is caused by production of the volatile terpenoids geosmin, which is made by all Streptomyces species, and 2-methylisoborneol (2-MIB), which is made by most species in the genus. This ubiquity suggested that geosmin and 2-MIB must provide a selective advantage for these soil microbes, but the nature of this advantage was unknown. We showed that, unlike other Streptomyces natural products, production of geosmin and 2-MIB is directly under the control of sporulation-specific transcription factors and that emission of these volatiles is confined to immature spores. In a collaboration with Paul Becher and Klas Flärdh (Lund, Sweden), we went on to show that these volatiles attract springtails (Collembola) that feed on Streptomyces colonies and disseminate spores both via their faecal pellets and through adherence to their hydrophobic cuticles. Thus, geosmin and 2-MIB production in immature spores is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods.

SUPPRESSION OF PLANT PATHOGENIC BACTERIA
Stefanato et al., 2019, bioRxiv, doi: 10.1101/783258
Moffat et al., 2021, Synthetic Biology, doi: 0.1093/synbio/ysab004.

Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. We therefore genetically and phenotypically characterized the Pseudomonas population in a commercial potato field, where we identified strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. This revealed that hydrogen cyanide and cyclic lipopeptides are key specialized metabolites associated with pathogen inhibition. However, our analysis demonstrated that some strains feature no characterised genetic loci associated with pathogen inhibition, which highlighted the potential for novel determinants of biological activity. We therefore developed an automated transposon mutagenesis screen (in collaboration with the Earlham Institute Biofoundry), which led to the discovery of a new biosynthetic gene cluster associated with S. scabies inhibition. We are currently determining the structure of this molecule and we are applying this methodology to identify strains and natural products that suppress other agriculturally important pathogens.

REWIRING THE REGULATION OF THE FORMICAMYCIN BIOSYNTHETIC GENE CLUSTER TO ENABLE THE DEVELOPMENT OF PROMISING NEW ANTIBACTERIAL COMPOUNDS
Devine et al., 2021 Cell Chem Biol. 28:1-9.

Formicamycins are a new structural class of antibiotics that inhibit the growth of MRSA and these bacteria do not become resistant to formicamycins, even after growing in the presence of sub-inhibitory concentrations. In this work we undertook a detailed analysis of the regulation of formicamycin biosynthesis and used this knowledge to engineer strains which produce formicamycins in liquid culture, and which produce 10-fold more than the wild-type strain. We also made strains which biosynthesise three new formicamycin congeners. Formicamycins are not toxic to mammalian cells and have a naturally high barrier to resistance so they are promising anti-infectives.

STREPTOMYCES ENDOPHYTES PROMOTE HOST HEALTH AND ENHANCE GROWTH ACROSS PLANT SPECIES
Worsley et al. 2020. Appl Env Microbiol. 86:e01053-20.

We isolated and genome sequenced five Streptomyces endophyte strains from the roots of Arabidopsis thaliana and demonstrated that three of them enhance the growth of A. thaliana and another protected the common bread wheat Triticum aestivum against Take-all disease. We also isolated the antifungal compounds that protect wheat seedlings against the Take-all fungus, which is the causative agent of the most economically important disease of wheat in the UK.
Exploitation Route This section will be competed at a later stage.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
 
Description NARRATIVE IMPACT APRIL 2018 - MARCH 2019 MICROCAPILLARY ELECTROPHORESIS AS A TOOL FOR DRUG DISCOVERY (Mitchenall et al., 2018. BMC Res Notes. 11: 37. doi: 10.1186/s13104-018-3147-6). We have shown that the QIAxcel microcapillary electrophoresis system can be used as a drug-discovery tool and has advantages in terms of speed, resolution and cost over conventional gel-based systems. It can be applied to DNA circles of various sizes and can readily be adapted for use in compound screening against topoisomerase targets. Currently unpublished work between Dr Andy Truman (Molecules from Nature ISP), Dr Jake Malone (Plant Health ISP) and VCS Potatoes, an East Anglian agronomy company, has led to numerous successful funding applications. Zespri International Limited (New Zealand) have awarded £76,000 over three years to employ a PhD student to identify biocontrol strains and bioactive NPs from bacterial isolates from kiwi vines that will help combat PsaV, a economically damaging bacterial pathogen of kiwi orchards. Truman has also been awarded a Royal Society Enhancement Award (£170,640) to expand his work on the potato pathogen Streptomyces scabies. This will assess the transcriptional response and metabolism of S. scabies during plant infection. Antibiotics Action - in relation to his work in the area of antibiotic research and development, Maxwell serves on the Scientific Advisory Board of the charity Antibiotic Action (http://antibiotic-action.com/ ), a global public awareness initiative to inform everyone about drug resistance, working with a wide variety of professionals as well as the general public. NARRATIVE IMPACT APRIL 2019 - MARCH 2020 Prof. Tony Maxwell attended the ABX Antibiotic Discovery Accelerator Network meeting at the Eden Project (Cornwall, UK) where, amongst other issues, solutions to the current AMR (antimicrobial resistance) problem were discussed. This led to the submission of a Big Ideas Pipeline proposal to BBSRC: 'A Publicly-funded Research & Development Pipeline for New Antibiotics', which is under discussion by the cross-UKRI team involved in antimicrobial resistance (AMR) research. Dr Andy Truman attended the China-UK AMR Workshop (Beijing, China), where he presented his work on the antibiotic bicyclomycin (collaboration with Xiamen University) and was involved in discussions around strategies for future research into AMR in the UK and China. Dr Andy Truman also participated in the International Research Alliance for Antibiotic Discovery and Development (IRAADD) Workshop Meeting, (Saarbruecken, Germany), where a European strategy for Future AMR research and funding concepts was discussed. A white paper resulting from this network meeting is currently in preparation. Collaborative research between Dr Andy Truman (Molecules from Nature ISP), Dr Jake Malone (Plant Health ISP) and VCS Potatoes (Stefanato et al, 2019, bioRxiv, doi: 10.1101/783258) has led to a follow-on partnership with a UK-based biotechnology company to develop bacterial strains to control plant pathogens. NARRATIVE IMPACT APRIL 2020 - MARCH 2021 Professor Barrie Wilkinson and Dr Andrew Truman organised the Dubrovnik Summer School in Applied Molecular Microbiology. Because of COVID-19, this was run virtually. Details can be found here: https://www.jic.ac.uk/training-careers/summer-schools/applied-molecular-microbiology/2020-applied-molecular-microbiology-summer-school-report/ Prof. Tony Maxwell participated in the UKRI AMR (antimicrobial resistance) consultation and attended the ABX Antibiotic Discovery Accelerator Network meeting (online) where, amongst other issues, solutions to the current AMR (antimicrobial resistance) problem were discussed. Information from these meetings were fed into the AMR on the NRP (Norwich Research Park) group, which is chaired by Maxwell.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description Contribution to SAW Antibiotics book
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
URL http://www.sawtrust.org/buy-the-books/saw-antibiotics/
 
Description Exploring novel binding pockets in DNA gyrase and DNA topoisomerase IV to address antibiotic resistance
Amount £642,171 (GBP)
Funding ID BB/V006983/ 
Organisation University of Leeds 
Sector Academic/University
Country United Kingdom
Start 07/2021 
End 06/2024
 
Description Royal Society Enhancement Award
Amount £170,640 (GBP)
Funding ID RGF\EA\181083 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 03/2021
 
Description Royal Society University Research Fellowship Renewal
Amount £483,734 (GBP)
Funding ID URF\R\201020 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2021 
End 12/2023
 
Description Royal Society University Research Fellowships Renewal
Amount £363,410 (GBP)
Funding ID URF\R\180007 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2021
 
Description Tackling tricky twists - how does DNA gyrase function inside living cells?
Amount £379,684 (GBP)
Funding ID BB/R001235/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Description Wellcome Trust Investigator Awards
Amount £1,271,158 (GBP)
Funding ID 221776/Z/20/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2021 
End 06/2026
 
Title QIAxcel 
Description We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact The QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets. 
 
Title RIPPER genome mining 
Description RiPPER is a command line computational tool that assists in the identification of biosynthetic gene clusters and associated precursor peptides for RiPPs, a large and important class of natural product. 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact This has assisted in research projects in my group and I have had contact with researchers in other groups who have benefited from the tool and the results we have reported from its use. It assisted in the identification of a new family of natural products, the thiovarsolins, which were characterised by my group. The identification of these natural products and the development of the tool were reported in a BioRxiv pre-print (https://www.biorxiv.org/content/10.1101/494286v1) and have just been accepted for publication in Nucleic Acids Research. 
URL https://github.com/streptomyces/ripper
 
Title AntibioticDB 
Description Repository of antibiotics developed since 1961. Open-access and searchable 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Provision of database that can be used by other researchers working on antibiotics 
URL http://www.antibioticdb.com/
 
Description A pipeline for antibiotic disovery 
Organisation University of East Anglia
Department School of Biological Sciences UEA
Country United Kingdom 
Sector Academic/University 
PI Contribution Natural products chemistry and biosynthesis; actinomycete genetics; genome mining; bioinformatics; anti-infective assays
Collaborator Contribution Entomology; actinomycete biology; genetic regulation; bioinformatics; anti-infective assays
Impact Publications and subseqeunt grant applications
Start Year 2013
 
Description Agnes Noy 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Synthesis of small DNA circles
Collaborator Contribution Simulations
Impact Two papers published so far: 1. Noy, A., Maxwell, A. and Harris, S.A. (2017) Interference between Triplex and Protein Binding to Distal Sites on Supercoiled DNA. Biophys J, 112, 523-531. 2. Bates, A.D., Noy, A., Piperakis, M.M., Harris, S.A. and Maxwell, A. (2013) Small DNA circles as probes of DNA topology. Biochem Soc Trans, 41, 565-570. Multidisciplinary: Maxwell - biochemistry; Noy - simulations
Start Year 2011
 
Description Bax 
Organisation Cardiff University
Department Cardiff Synthetic Biology Initiative
Country United Kingdom 
Sector Academic/University 
PI Contribution We have performed mechanistic enzymology work to complement the structural biology performed by Bax and others.
Collaborator Contribution They have established a crystallography platform to analyse structures of topoisomerase-DNA-drug complexes. That expertise has now been transferred to JIC.
Impact Research publications. Multi-disciplinary: biochemistry, crystallography, molecular biology
Start Year 2014
 
Description Benesch 
Organisation University of Oxford
Department Wolfson College
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We have provided samples to be analysed by mass spec
Collaborator Contribution They have employed high-resolution non-covalent mass spec methodology to investigate subunit exchange in DNA gyrase
Impact None so far
Start Year 2017
 
Description Blumberg 
Organisation Harvard University
Department Department of Gastroenterology Harvard
Country United States 
Sector Academic/University 
PI Contribution We have provided materials, toxins and DNA constructs to the Harvard team
Collaborator Contribution They discovered that the toxin MccB17 was associated with IBD and carried out the immunology and animal experiments
Impact Cell paper 2018
Start Year 2017
 
Description CSIRO collaboration 
Organisation Commonwealth Scientific and Industrial Research Organisation
Department CSIRO Black Mountain Laboratories
Country Australia 
Sector Private 
PI Contribution My research group hosted a senior researcher for a collaborative research project associated with microbe-plant and microbe-microbe interactions, with a particular focus on Streptomyces strains with inhibitory activity towards fungal pathogens of plants. We provided expertise in Streptomyces genetics, natural product biosynthesis and mass spectrometry.
Collaborator Contribution The CSIRO researcher provided Streptomyces strains with biocontrol activity, as well as extensive expertise in fungal pathogens of crop plants. The bench work for this collaboration was carried out by the CSIRO researcher while they visited JIC. The research visit was for 4 months, but the collaboration is still ongoing.
Impact No outputs yet. Multi-disciplinary colaboration: microbiology, fungal biology, natural products chemistry, mass spectrometry, plant pathology.
Start Year 2019
 
Description Calabria thioviridamides 
Organisation University of Calabria
Country Italy 
Sector Academic/University 
PI Contribution We established a project to discovery new thioviridamide-like molecules (TLMs) by the use of a genome mining method. This involved pathway identification, strain fermentation, pathway cloning and mutagenesis, and then purification and chemical analysis of the products of these pathways.
Collaborator Contribution The group of Anna Rita Cappello determined the biological activity of our purified compounds against bacteria, fungi and human cell lines.
Impact Publication: https://pubs.acs.org/doi/10.1021/acschembio.7b00677 This collaboration is multi-disciplinary. We carry out microbiology, genetics and chemistry and the partners carry out cell biology assays.
Start Year 2016
 
Description Dmitry Ghilarov 
Organisation Jagiellonian University
Country Poland 
Sector Academic/University 
PI Contribution We hosted to Ghilarov and proved resources for X-ray crystallography
Collaborator Contribution Ghilarov purified and characterized the proteins and carried out the crystallography
Impact Publications
Start Year 2017
 
Description Fishwick 
Organisation University of Leeds
Department University of Leeds Special Collections
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been testing compounds synthesised by the med chem research team in Leeds
Collaborator Contribution Using information provided by us, they have been using computational and medicinal chemistry methods to synthesise novel compounds that have antibacterial potential
Impact Grant application to the Wellcome Trust currently under consideration
Start Year 2017
 
Description HIPS Bottromycin Biosynthesis 
Organisation Helmholtz Association of German Research Centres
Department Helmholtz Institute for Pharmaceutical Research, Saarbrucken
Country Germany 
Sector Academic/University 
PI Contribution Using tandem mass spectrometry, we characterised enzymatic transformations to the bottromycin precursor peptide. In addition, we generated mutant forms of the bottromycin producing organism (Streptomyces scabies) to determine the importance of a number of enzyme residues for catalysis. This was assessed by looking at the metabolites produced by these mutants using LC-MS.
Collaborator Contribution They expressed and purified enzymes involved in bottromycin biosynthesis, and then carried out enzymatic assays. The products of these assays were then sent to us for analysis. They also carried out mutagenesis of these enzymes, which guided our mutations in Streptomyces scabies.
Impact Publication: https://pubs.acs.org/doi/10.1021/jacs.7b09898
Start Year 2017
 
Description Isomerase PPIase inhibitors 
Organisation Isomerase Therapeutics
Country United Kingdom 
Sector Private 
PI Contribution Study of putative virulence factors in Gram-negative bacteria. Molecular genetics and in vitro & in vivo bioassays.
Collaborator Contribution Provision of proprietary inhibitor molecules.
Impact None so far
Start Year 2017
 
Description Janez Ilas 
Organisation University of Ljubljana
Country Slovenia 
Sector Academic/University 
PI Contribution Assessing novel compounds synthesised by Ilas' team
Collaborator Contribution Synthesis of model compounds; computational design
Impact Paper in preparation
Start Year 2019
 
Description Josh Mylne 
Organisation University of Western Australia
Department School of Molecular Sciences
Country Australia 
Sector Academic/University 
PI Contribution We assayed compounds provided by UWA against Arabidopsis gyrase
Collaborator Contribution UWA synthesised new quinolone compounds and tested them in planta
Impact Publication
Start Year 2017
 
Description Leake 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of materials and expertise
Collaborator Contribution They are carrying out high-resolution microscopy experiments using material generated in my lab.
Impact One published paper
Start Year 2017
 
Description Lindsay 
Organisation Quadram Institute Bioscience
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been analysing the gut microbiome of Galleria mellonella as a surrogate for the human infant gut microbiome.
Collaborator Contribution Provision of human infant fecal samples and bacteria. Assistance with sequencing.
Impact None so far
Start Year 2017
 
Description Luis Aragon 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been assessing novel nybomycin analogues made by the ICL team
Collaborator Contribution They are using synthetic chemistry to make new analogues of the drug nybomycin
Impact No
Start Year 2019
 
Description Macquarie University collaboration 
Organisation Macquarie University
Department Department of Chemistry and Biomolecular Sciences
Country Australia 
Sector Academic/University 
PI Contribution Biosynthetic chemistry and molecular microbiology
Collaborator Contribution Natural products chemistry, structural chemistry
Impact Manuscript in preparation
Start Year 2019
 
Description Mark Leake 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Making proteins with fluorescent tags
Collaborator Contribution High-resolution microscopy of bacteria
Impact One paper so far: 1. Stracy, M., Wollman, A.J.M., Kaja, E., Gapinski, J., Lee, J.E., Leek, V.A., McKie, S.J., Mitchenall, L.A., Maxwell, A., Sherratt, D.J. et al. (2019) Single-molecule imaging of DNA gyrase activity in living Escherichia coli. Nucleic Acids Res, 47, 210-220. Multi-disciplinary: Maxwell - biochemistry; Leake - physics
Start Year 2017
 
Description Matt Piggott 
Organisation University of Perth
Country Australia 
Sector Academic/University 
PI Contribution Assessing activity of compounds made by collaborator
Collaborator Contribution Drug synthesis
Impact Not yet
Start Year 2016
 
Description Nagaraja 
Organisation Indian Institute of Science Bangalore
Country India 
Sector Academic/University 
PI Contribution Collaborative experiments and provision of materials. Detailed discussions on projects
Collaborator Contribution Collaborative experiments and provision of materials. Detailed discussions on projects
Impact Several research papers
 
Description Neuman 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution We have carried out ensemble biochemical experiments on M mazei topo VI, and provided materials to the NIH team
Collaborator Contribution They have been carrying out single-molecule experiments with M mazei topo VI
Impact Not yet
Start Year 2016
 
Description Nodwell 
Organisation University of Toronto
Department Toronto Western Research Institute
Country Canada 
Sector Hospitals 
PI Contribution We have performed assays using novel compounds provided by the Canadian team.
Collaborator Contribution They have screened for an characterised novel inhibitors of gyrase
Impact Paper in preparation
Start Year 2018
 
Description Pyne 
Organisation University College London
Department UCL Energy Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided DNA minicircles for analysis
Collaborator Contribution She has been analysing DNA sample using atomic force microscopy.
Impact Paper in preparation
Start Year 2015
 
Description Sarah Harris 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been designing and making small DNA circle substrates
Collaborator Contribution Sarah's group have carried out atomistic simulations
Impact Two published papers so far: 1. Bates, A.D., Noy, A., Piperakis, M.M., Harris, S.A. and Maxwell, A. (2013) Small DNA circles as probes of DNA topology. Biochem Soc Trans, 41, 565-570. 2. Noy, A., Maxwell, A. and Harris, S.A. (2017) Interference between Triplex and Protein Binding to Distal Sites on Supercoiled DNA. Biophys J, 112, 523-531. Multidisciplinary: Maxwell - biochemistry; Harris - simulations
Start Year 2011
 
Description University of León Collaboration 
Organisation Fundacion MEDINA
Country Spain 
Sector Charity/Non Profit 
PI Contribution This collaboration was started to identify the natural products that were responsible for antifungal activity in Streptomyces clavuligerus following introduction of a regulatory gene into this strain. We also mapped the global changes to metabolism in this strain too using LC-MS. This led to the identification of a wide variety of tunicamycins, where a few specific versions of this molecule exhibited antifungal activity. We helped write the resulting research paper.
Collaborator Contribution The partners led this research project, where they genetically manipulated Streptomyces clavuligerus and assessed changes to gene expression using microarrays. They also assessed the changes in production of other natural products known to be produced by Streptomyces clavuligerus , including the beta-lactam antibiotic cephamycin and the beta-lactamase inhibitor clavulanic acid. They led the writing of the research paper.
Impact A paper was published in Frontiers in Microbiology that reports this work: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00580/abstract
Start Year 2016
 
Description University of León Collaboration 
Organisation University of Leon
Country Spain 
Sector Academic/University 
PI Contribution This collaboration was started to identify the natural products that were responsible for antifungal activity in Streptomyces clavuligerus following introduction of a regulatory gene into this strain. We also mapped the global changes to metabolism in this strain too using LC-MS. This led to the identification of a wide variety of tunicamycins, where a few specific versions of this molecule exhibited antifungal activity. We helped write the resulting research paper.
Collaborator Contribution The partners led this research project, where they genetically manipulated Streptomyces clavuligerus and assessed changes to gene expression using microarrays. They also assessed the changes in production of other natural products known to be produced by Streptomyces clavuligerus , including the beta-lactam antibiotic cephamycin and the beta-lactamase inhibitor clavulanic acid. They led the writing of the research paper.
Impact A paper was published in Frontiers in Microbiology that reports this work: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00580/abstract
Start Year 2016
 
Description University of Tübingen Actinonin Biosynthesis 
Organisation Eberhard Karls University of Tübingen
Country Germany 
Sector Academic/University 
PI Contribution We sequenced the genome of the actinonin producing organism to identify its biosynthetic gene cluster, as well as identifying the gene cluster of the related compound matlystatin in a different organism. We carried out chemical feeding experiments to determine the biosynthesis of actinonin and also carried out feeding experiments to make novel matlystatin derivatives.
Collaborator Contribution They independently identified the same gene clusters as ourselves. Following this, they cloning the matlystatin gene cluster and investigated its biosynthesis by mutating most genes in the pathway. They also carried out different feeding experiments to ourselves to support biosynthetic proposals.
Impact Publication: http://europepmc.org/abstract/med/29213087
Start Year 2017
 
Description VCS Potatoes 
Organisation VCS Potatoes
Country United Kingdom 
Sector Private 
PI Contribution This is a collaboration to identify Pseudomonas strains that can control potato pathogens. Working with the group of Dr Jake Malone (JIC), we have carried out all scientific aspects of this project, including sampling and genome sequencing of isolated strains, as well as phenotyping, genetics and natural product analysis of these strains.
Collaborator Contribution VCS Potatoes have provided sites for sampling and field trials. Their input also includes technical expertise relating to potato disease and field trials.
Impact Manuscript under review and currently available as a pre-print at bioRxiv (https://doi.org/10.1101/783258).
Start Year 2015
 
Description Chair and Organiser for RSC Directing Biosynthesis V Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chaired the organising committee and conference.
Year(s) Of Engagement Activity 2017
URL http://www.rsc.org/events/detail/22912/Directing%20Biosynthesis%20V
 
Description Dubrovnik Summer School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I provided a seminar and led multiple small group discussions as part of the Dubrovnik Summer School in Applied Molecular Microbiology. This was attended by 45 postgraduate and post-doctoral researchers from around the world to learn about concepts and methods in natural product biosynthesis. Following the summer school, I have been contacted by multiple attendees regarding various aspects of the work I discussed.
Year(s) Of Engagement Activity 2018
URL https://www.jic.ac.uk/training-careers/summer-schools/applied-molecular-microbiology/
 
Description International Research Alliance for Antibiotic Discovery and Development (IRAADD) Workshop Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attendance and presentation as part of the International Research Alliance for Antibiotic Discovery and Development (IRAADD) Workshop Meeting (Saarbruecken, Germany), which is a Europe-wide network of researchers and industry partners involved in antibiotic discovery and development.
Year(s) Of Engagement Activity 2019
 
Description Interview with BBC Radio Five Live for a podcast 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interview with BBC Radio Five Live for a podcast relating to the science of weather. In particular, the smell and role of bacterial metabolites.
Year(s) Of Engagement Activity 2017
 
Description Interview with science writer (Kat Arney) about antibiotic discovery and combating antimicrobial resistance. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview with science writer (Kat Arney) about antibiotic discovery and combating antimicrobial resistance. In particular a focus on the re-purposing of old antibiotics for treating multi-drug resistant infections. This was in relation to an in-depth article she is preparing on AMR and antibiotic discovery, which was later published in the Daily Mail.
Year(s) Of Engagement Activity 2018
URL https://www.dailymail.co.uk/health/article-5623417/Could-antibiotics-mens-beards-soil-weapons-battle...
 
Description Invited talk at China-UK AMR Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A UKRI-funded workshop held in Beijing to discuss ongoing and recent UK-China research projects on AMR (antimicrobial resistance), as well as future directions and prospects for this research area.
Year(s) Of Engagement Activity 2019
 
Description Invited talk at Environmental Genomics and Advanced Microbiological Techniques Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Presented a talk about using mass spectrometry-based metabolomics in microbiology to PhD students attending an Environmental Genomics and Advanced Microbiological Techniques Workshop at the University of East Anglia. I received multiple questions after the seminar.
Year(s) Of Engagement Activity 2019
 
Description Invited talk at Plant-Microbe Interactions Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Seminar provided on Streptomyces-Pseudomonas interactions at a Plant-Microbe Interactions Workshop organised at the John Innes Centre.
Year(s) Of Engagement Activity 2019
 
Description Invited talk at the University of Leeds 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited talk for the seminar series in the School of Molecular and Cellular Biology, University of Leeds. Participation in a discussion about my research with a group of MSc students.
Year(s) Of Engagement Activity 2018
 
Description JIC 50 Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Outreach stand for Department of Molecular Microbiology as part of an Open Day in relation to the 50th anniversary of the John Innes Centre in Norwich.
Year(s) Of Engagement Activity 2017
URL https://www.jic.ac.uk/news-and-events/blog-copy/2017/09/open-day/
 
Description London International Youth Forum 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact • Aug 2017 - lecture to students visiting as part of London International Youth Science Form: 'Where will the new antibiotics come from?'
Year(s) Of Engagement Activity 2017
 
Description Presentation at Science for Innovation Showcase Event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Provided presentation entitled "Discovery and biosynthesis of bacterial peptides with antibacterial and anticancer activities" to an audience of industry and research council representatives invited to the John Innes Centre. Participated in a follow-up panel discussion.
Year(s) Of Engagement Activity 2018
 
Description Presentation to Bayer Agronomy team 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation and discussion with the agronomy team at Bayer about biocontrol products
Year(s) Of Engagement Activity 2019
 
Description School Science week presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Gave a one hour talk on the role that microbes and insects play in the carbon cycle and bioremediation.
Year(s) Of Engagement Activity 2018
 
Description School Science week presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Policymakers/politicians
Results and Impact School science week - gave one hour presentations on the role of microbes in bioremediation
Year(s) Of Engagement Activity 2019
 
Description School visit (Cambourne Cambs) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Deliver a Science, Arts and Writing workshop event on the topic 'Antibiotics: what they are and where they come from'. Delivered a one day event, to two Year 6 classes (one each day). The event generated significant discussion, and involved hands on learning.
Year(s) Of Engagement Activity 2020
URL http://sawtrust.org/buy-the-books/saw-antibiotics/
 
Description School visit (Cambourne Cambs) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Deliver a Science, Arts and Writing workshop event on the topic 'Antibiotics: what they are and where they come from'. Delivered a one day event, to two Year 6 classes (one each day).

The event generated significant discussion, and involved hands on learning.
Year(s) Of Engagement Activity 2019
URL http://sawtrust.org/buy-the-books/saw-antibiotics/
 
Description School visit (Cambourne Cambs) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Spent two days presenting a cross curriculum presentation to two classes of Year 6 pupils at a primary school in collaboration with the SAW Trust (http://sawtrust.org/ ). Much debate and discussion about the topics of microbes, antibiotics, ecology and antimicrobial resistance.
Year(s) Of Engagement Activity 2018
URL http://sawtrust.org/buy-the-books/saw-antibiotics/
 
Description Science Museum Superbugs Late Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact "Antibiotic Hunters" exhibit at a Science Museum Superbug Late Event. This was an adult-only event held in the evening at the Science Museum in London and attended by thousands of people. The exhibit was organised jointly between members of my research group and members of the Wilkinson group (also at JIC).
Year(s) Of Engagement Activity 2018
 
Description Year 10 Science Camp Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A talk on natural product discovery to attendees of the year 10 Science Camp at the John Innes Centre. This was followed by questions on antibiotic and anticancer compound discovery.
Year(s) Of Engagement Activity 2018
URL https://www.jic.ac.uk/training-careers/work-experience/year-10-science-camp/