Biology subjects

White, M. F.

Publications and source records attributed to White, M. F..

At least 19 recordsLinked to original sources

bioRxiv2026

Dual ATPase-activated DNA hemimethylation and cleavage by the MANTIS defence system

Bacterial defence systems are diverse and form major barriers to horizontal gene transfer and bacteriophage (phage) infection. Though restriction-modification systems were the first such defences discovered and remain the most widespread, the basis of self/non-self discrimination in many newly identified methylation-based systems remains unclear. Here, we define the mechanism of a widely distributed BREX-related defence family, previously termed Gao_RL, which we rename MANTIS (Methyltransferase, ATPases and Nuclease Targeting Inverted-repeat Sequences). Using phage infection and plasmid transformation assays in Pseudomonas aeruginosa, biochemical reconstitution, and structural biology, we show that MANTIS establishes self-identity through adenine hemimethylation of short asymmetric DNA sequences. Unexpectedly, a AAA+ ATPase (MtsA) and a co-regulator (MtsB) are both required to recruit and activate the methyltransferase MtsC, revealing a previously unknown ATP-dependent mode of epigenetic modification. DNA recognition by the MtsABC complex then triggers restriction by a dimeric helicase-nuclease effector (MtsD), with activity dependent on target-site architecture: inverted pairs of unmodified sites support robust restriction, whereas direct repeats or single sites are restricted less efficiently. Consistent with this model, virulent phages show strand-biased depletion of MANTIS target sites, indicating evolutionary pressure to evade restriction. Together, these findings reveal how dual ATPases couple self-modification to non-self restriction and provide a framework for understanding the expanding landscape of methylation-based defence systems.

molecular biology↗

bioRxiv2026

A self-limiting dimeric TIR effector specialised for type III CRISPR-mediated immunity

Antiviral defence systems frequently utilise cyclic nucleotide second messengers. A prominent example is the type III CRISPR-Cas system, which generates cyclic oligoadenylates (cOA) on detecting viral RNA. cOA molecules can bind and activate a wide range of effectors to provide antiviral defence. In both prokaryotes and higher plants, activation of a catalytic Toll/Interleukin Receptor (TIR) domain by multimerization results in degradation of NAD+, limiting cell metabolism and thus viral replication. Here, we describe a CRISPR-associated TIR-containing effector that includes a SAVED (SMODS-associated and fused to various effector domains) domain for nucleotide sensing and a cOA-degrading ring nuclease Crn4 domain. We demonstrate that the TIR-SAVED-Ring nuclease (TSR1) effector binds cA3, resulting in activation of the TIR NADase activity. The Crn4 domain, which imposes an unusual dimeric quaternary structure on the effector, degrades cA3, providing a mechanism to auto-deactivate the effector. TSR1 is thus a highly unusual example of a dimeric and self-limiting TIR effector in antiviral immunity.

microbiology↗

bioRxiv2026

Cyclic tri-adenylate signalling by a Panoptes anti-phage guard system with a CARF-TM effector

Cyclic nucleotide second messengers are used in all domains of life to amplify viral infection signals and activate cellular defences. In prokaryotes, CBASS (cyclic nucleotide based antiphage signalling system) and type III CRISPR-Cas systems generate a range of cyclic nucleotides which bind and allosterically activate effector proteins to mount an anti-viral response. Viruses have evolved counter-measures to antagonise these signalling pathways in the form of cyclic nucleotide sponges and phosphodiesterases that sequester or degrade these molecules to subvert immunity. Recently, the Panoptes system was shown to function as a guard against these viral tactics. The type I Panoptes polymerase, mCpol, generates cyclic dinucleotides as decoy molecules that, when sequestered by phage proteins, results in the activation of the membrane-permeabilising effector 2TM{beta} to halt the phage infection cycle. Here, we investigate the type II Panoptes system, demonstrating that it generates cyclic tri-adenylate (cA3) to maintain a CRISPR-associated Rossmann fold-transmembrane (CARF-TM) effector in an inactive, dimeric state. When cA3 is sequestered or degraded, the CARF-TM protein oligomerises, resulting in increased outer membrane permeability and growth arrest. These findings expand our understanding of the guard systems that constitute a fascinating component of the bacterial immune system.

biochemistry↗

bioRxiv2026

Active virus-host system in a Lokiarchaeon culture

Asgard archaea are considered the closest prokaryotic relatives of eukaryotes and having a crucial role in eukaryogenesis. Only few organisms have been cultivated from this group and their viruses were previously described only through metagenomic reconstructions. Here, we report the first successful cultivation of an Asgard archaeal virus infecting a novel strain of Ca. Lokiarchaeum ossiferum B36. The 16 kbp integrated provirus is capable to excise and replicate independently leading to the formation of virus particles. Network analysis of shared protein clusters with other archaeal viruses places it in a new family, Fylgjaviridae. The host encodes a distinctive repertoire of antiviral defense systems, including Septu, Wadjet, and type II CBASS system, all different from the defense systems of the related strain Ca. L. ossiferum B35. Our cultures provide the first opportunity to study interactions of a virus-host system in Asgards and hold significant potential for developing genetic tools.

microbiology↗

bioRxiv2026

The CRISPR ring nuclease Csx15 oligomerises on cyclic nucleotide binding to regulate antiviral defence

Prokaryotic type III CRISPR systems signal infection by generating cyclic oligoadenylate (cOA) second messengers, which activate defence proteins allosterically, providing immunity. cOA molecules are typically degraded by extrinsic, stand-alone ring nuclease (RN) enzymes with phosphodiesterase activity, or by the intrinsic RN activity of the effectors themselves. Viruses and plasmids also encode RNs, which can function as anti-CRISPRs (Acr). Eight different families of extrinsic RNs are currently known. Here, we report the structural and biochemical analysis of one of these families: Csx15. We show that Csx15 is a dimeric protein with the ability to bind cyclic tetra-adenylate (cA4) molecules in a shared binding site formed by the head-to-tail stacking of dimers in a filament conformation. Some family members are non-enzymatic, relying on the sequestration (sponging) of cA4 to regulate the host immune response, while others act as canonical RNs, slowly degrading cA4.

biochemistry↗

bioRxiv2025

Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4

Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary e\ectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.

biochemistry↗

bioRxiv2025

A viral SAVED protein with ring nuclease activity degrades the CRISPR second messenger cA4

Type III CRISPR systems typically generate cyclic oligoadenylate (cOA) second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA, activating ancillary effector proteins which elicit a diverse range of immune responses. The CalpLTS system elicits a transcriptional response to infection when CalpL binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS Sigma factor for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2, which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally-encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.

biochemistry↗

bioRxiv2025

SAM-AMP lyases in CRISPR defence and anti-defence

Type III CRISPR systems detect non-self RNA and activate the enzymatic Cas10 subunit, which generates nucleotide second messengers for activation of ancillary effectors. Although most signal via cyclic oligoadenylate (cOA), an alternative class of signalling molecule SAM-AMP, formed by conjugating ATP and S-adenosyl methionine, was described recently. SAM-AMP activates a trans-membrane effector of the CorA magnesium transporter family to provide anti-phage defence. Intriguingly, immunity also requires SAM-AMP degradation by means of a specialised CRISPR-encoded NrN family phosphodiesterase in Bacteroides fragilis. In Clostridium botulinum, the nrn gene is replaced by a gene encoding a SAM-AMP lyase. Here, we investigate the structure and activity of C. botulinum SAM-AMP lyase, which can substitute for the nrn gene to provide CorA-mediated immunity in Escherichia coli. The structure of SAM-AMP lyase bound to its reaction product methylthioadenosine-AMP (MTA-AMP) reveals key details of substrate binding and turnover by this PII superfamily protein. Bioinformatic analyses reveal candidate phage-encoded SAM-AMP lyases and we demonstrate that one, hereafter named AcrIIIB4, degrades SAM-AMP efficiently in vitro.

biochemistry↗

bioRxiv2024

Diversity and abundance of ring nucleases in type III CRISPR-Cas loci

Most type III CRISPR-Cas systems facilitate immune responses against invading mobile genetic elements such as phages by generating cyclic oligoadenylates (cOAs). Downstream effectors activated by cOAs are typically non-specific proteins that induce damage to essential cellular components, thereby preventing phage epidemics. Due to these toxic effects, it is crucial that the production and concentration of cOAs remain under tight regulatory control during infection-free periods or when deactivating the immune response after clearing an infection. Type III CRISPR loci often encode enzymes known as ring nucleases (RNs) that bind and degrade specific cOAs, while some effectors are auto-deactivating. Despite the discovery of several classes of RNs, a comprehensive bioinformatic analysis of type III CRISPR-Cas loci in this context is lacking. Here, we examined 38,742 prokaryotic genomes to provide a global overview of type III CRISPR loci, focussing on the known and predicted RNs. The candidate RNs Csx16 and Csx20 are confirmed as active enzymes, joining Crn1-3. Distributions and patterns of co-occurrence of RNs and associated effectors are explored, allowing the conclusion that a sizeable majority of type III CRISPR systems regulate cOA levels by degrading the signalling molecules, which has implications for cell fate following viral infection.

bioinformatics↗

bioRxiv2024

Cas10 based 7SL-sRNA diagnostic for the detection of active trypanosomiasis

Animal Trypanosomosis (AT) is a significant disease affecting cattle across sub-Saharan Africa, Latin America, and Asia, posing a major threat to economic productivity and animal welfare. The absence of reliable diagnostic tests has led to an over-reliance on widespread pre-emptive drug treatments, which not only compromise animal health but also heighten the risk of drug resistance. The chronic nature of AT, characterized by cyclical low or undetectable parasite levels, and the necessity for field-applicable tests that can distinguish between active infection and prior exposure, present considerable challenges in developing effective diagnostics. In previous work, we identified a parasite-specific small RNA, 7SL-sRNA, which is detectable in the serum of infected cattle, even during the chronic stages of infection. However, existing methods for detecting sRNA require specialized equipment, making them unsuitable for field use. In this study, we have developed a lateral flow diagnostic test utilizing Cas10 technology for the detection of 7SL-sRNA. This assay demonstrates high sensitivity and specificity, effectively identifying active infections in cattle, including during chronic phases (with positive signals observed up to the 63 days post-infection end point). This is the first instance of Cas10 being used for small RNA detection, paving the way for a cost-effective, user-friendly, and field-deployable diagnostic test for AT, while establishing Cas10 technology for the detection of small RNAs in general. Author summaryAnimal Trypanosomosis (AT) is a parasitic disease that affects livestock, causing major economic losses and harm to animal health. Cheap effective diagnostics that can be easily used in the field would have a major impact on the disease burden by enabling better treatment practices. However, parasites causing the disease - trypanosomes - are often undetectable via conventional methods, especially during chronic disease stages. More accurate diagnostics are not suitable for use in the field and are therefore unavailable to farmers. This has led to the overuse of drug treatments that can harm animals and lead to the emergence of drug resistance. We previously discovered a trypanosome-specific small RNA, called 7SL-sRNA, that can be detected in the blood of infected cattle, even during the chronic stages of AT when parasites are not visible via microscopy. However, detecting this RNA has required complex equipment, limiting its use in field settings. Building on this work, we have now developed a field applicable test for trypanosomosis that uses Cas10 technology to detect 7SL-sRNA via a simple lateral flow assay. This test is easy to use and accurately identifies active infections, including during the chronic phase of the disease. This is the first time Cas10 technology has been used to detect small RNAs, offering a promising, cost-effective tool for diagnosing AT and potentially other neglected parasitic diseases.

microbiology↗

bioRxiv2024

RNA processing by the CRISPR-associated NYN ribonuclease

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

molecular biology↗

bioRxiv2023

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23

CRISPR provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using a N-terminal transmembrane -helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

microbiology↗

bioRxiv2023

Reversible conjugation of a CBASS nucleotide cyclase regulates immune response to phage infection

Antiviral defence systems build the prokaryotic immune system and their proper regulation is vital for survival and fitness. While it is important that they are readily available in case of infection, they need to be tightly controlled to prevent activation of an unnecessary cellular response. Here we describe how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a Type II CBASS system from Bacillus cereus into the cognate host Bacillus subtilis, we show that the Cap2 protein conjugates the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. This cyclase-PspA conjugation is reversed after infection by the isopeptidase Cap3. Finally, we propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage and that this conjugation is released upon infection, priming the cyclase for activation.

microbiology↗

bioRxiv2023

Antiviral Type III CRISPR signalling via conjugation of ATP and AdoMet

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements (MGE) 1, 2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA (crRNA) to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against MGE either directly, via the integral HD nuclease domain 3-5 or indirectly, via synthesis of cyclic oligonucleotide (cOA) second messengers to activate diverse ancillary effectors 6-9. A subset of type III CRISPR systems encode an uncharacterised CorA-family membrane protein and an associated NrN family phosphodiesterase predicted to function in antiviral defence. Here, we demonstrate that the CorA associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against MGE when expressed inE. coli. However, B. fragilis Cmr does not synthesise cOA species on activation, instead generating a previously undescribed sigalling molecule, SAM-AMP (3-adenylyl-AdoMet) by conjugating ATP to S-adenosyl methionine via a phosphodiester bond. Once synthesised, SAM-AMP binds to the CorA effector, presumably leading to cell death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR associated phosphodiesterases or a SAM-AMP lyase, providing an "off switch" analogous to cOA specific ring nucleases 10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

microbiology↗

bioRxiv2023

Repurposing the atypical Type I-G CRISPR system for bacterial genome engineering

The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilise a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4- subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.

microbiology↗

bioRxiv2022

Structure and mechanism of the type I-G CRISPR effector

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3 end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. Type I-G Cascade is one of the most streamlined Class 1 CRISPR effectors. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

microbiology↗

bioRxiv2022

Cyclic nucleotide-induced superhelical structure activates a bacterial TIR immune effector

Cyclic nucleotide signalling is a key component of anti-viral defence in all domains of life, from bacteria to humans. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway 1, which originated in bacteria 2. These defence systems require a sensor domain such as STING or SAVED to bind the cyclic nucleotide, coupled with an effector domain that causes cell death when activated by destroying essential biomolecules 3. One example is the TIR (Toll/interleukin-1 receptor) domain, which degrades the essential cofactor NAD+ when activated in response to pathogen invasion in plants and bacteria 2,4,5 or during nerve cell programmed death 6. Here, we show that a bacterial anti-viral defence system generates a cyclic tri-adenylate (cA3) signal which binds to a TIR-SAVED effector, acting as the "glue" to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organise and complete a composite active site, allowing NAD+ degradation. Our study illuminates a striking example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

biochemistry↗

bioRxiv2021

SAVED by a toxin: Structure and function of the CRISPR Lon protease.

CRISPR antiviral defense systems such as the well-known DNA-targeting Cas9- and the more complex RNA-targeting type III systems are widespread in bacteria and archea 1, 2. The type III systems can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition 3-5. These second messenger molecules bind to the CARF (CRISPR associated Rossmann-fold) domains of dedicated effector proteins that are often DNAses, RNAses, or putative transcription factors 6. The activated effectors interfere with cellular pathways of the host, inducing cell death or a dormant state of the cell that is better suited to avoid propagation of the viral attack 7, 8. Among a large set of proteins that were predicted to be linked to the type III systems 9, 10, the CRISPR-Lon protein caught our attention. The protein was predicted to be an integral membrane protein containing a SAVED-instead of a CARF-domain as well as a Lon protease effector domain. Here, we report the crystal structure of CRISPR-Lon. The protein is a soluble monomer and indeed contains a SAVED domain that accommodates cA4. Further, we show that CRISPR-Lon forms a stable complex with the 34 kDa CRISPR-T protein. Upon activation by cA4, CRISPR-Lon specifically cleaves CRISRP-T, releasing CRISPR-T23, a 23 kDa fragment that is structurally very similar to MazF toxins and is likely a sequence specific nuclease. Our results describe the first cOA activated proteolytic enzyme and provide the first example of a SAVED domain connected to a type III CRISPR defense system. The use of a protease as a means to unleash a fast response against a threat has intriguing parallels to eukaryotic innate immunity.

biochemistry↗