Biology subjects

Hoikkala, V.

Publications and source records attributed to Hoikkala, V..

9 recordsLinked to original sources

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

A chromosomal inversion accumulates adaptive and barrier loci in a cold-adapted insect species

Chromosomal inversions can play a crucial role in population adaptation and divergence by reducing gene flow and preserving adaptive allelic combinations between populations with different arrangements. However, demonstrating this empirically is challenging due to numerous interacting processes with similar genomic signatures affecting inversion evolution. In this study, we characterized a large (9.5Mb) polymorphic inversion in the cold-adapted and widely distributed species, Drosophila montana, using long- and short-read sequencing across several populations. The origin of this inversion predates the divergence of North American (NA) and Fennoscandian (North European) populations, suggesting it emerged in the ancestral D. montana population in the Rocky Mountains of NA. Despite the species expansion across the northern hemisphere, this inversion has remained exclusive to the Rocky Mountains populations, where it is fixed in the southernmost high-elevation population and appears at lower frequencies in the more northern and lower-elevation populations. By independently mapping SNPs linked to climate adaptation and barriers to gene flow (barrier loci; identified through reduced migration rates), we found enrichment of both within the inversion, with barrier and adaptive regions partially overlapping. However, the inversion was not enriched for SNPs related to cold tolerance. These findings suggest that inversions may maintain associations between multiple adaptive and barrier loci, effectively coupling them, and that locally adaptive regions may act as barriers to gene flow. Our study provides empirical evidence that inversions can contribute to population adaptation and divergence by reducing gene flow, maintaining adaptive allelic combinations, and facilitating the coupling of different barriers to gene flow. SignificanceChromosomal inversions, genomic rearrangements with reversed gene order, are important drivers of population adaptation and divergence, but demonstrating this empirically is challenging due to complex interacting processes that produce similar genomic signatures. We characterized and analysed a large polymorphic inversion in Drosophila montana, a cold-adapted and widely distributed insect, using whole-genome sequencing data, phenotypic experiments, and recent methodological developments. This inversion is found only in mountain populations and is enriched with SNPs potentially associated with climate adaptation, as well as regions that act as barriers to gene flow, with these regions partially overlapping. These results suggest that adaptive loci can act as barriers to gene flow and that inversions help maintain adaptive allelic combinations and associations between multiple barrier loci, thereby contributing to population adaptation and divergence.

evolutionary biology↗

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↗

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↗

bioRxiv2021

Mucin induces CRISPR-Cas defence in an opportunistic pathogen

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we exposed the bacterial fish pathogen Flavobacterium columnare to its virulent phage V156 in the presence of a eukaryotic host signal (mucin). All tested conditions led to some level of innate immunity, but the presence of mucin led to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies had obtained at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the concentration of phages on metazoan surfaces may select for the diversification of bacterial immune systems.

microbiology↗

bioRxiv2020

Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and Type IX Secretion System

Increasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably is a multifactorial process including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections, since phage resistance is associated with decrease in bacterial virulence. ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce a development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

microbiology↗

bioRxiv2020

Cooperation between CRISPR-Cas types enables adaptation in an RNA-targeting system

CRISPR-Cas immune systems adapt to new threats by acquiring spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition, despite apparent variation in spacer content between strains. It has been suggested that such loci may use acquisition machinery from co-occurring CRISPR-Cas systems. Here, using a lytic dsDNA phage, we observe spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient subtype VI-B locus and a complete subtype II-C locus. We characterize acquisition events in both loci and show that the RNA-targeting VI-B locus acquires spacers in trans using acquisition machinery from the DNA-targeting II-C locus. Our observations reinforce the concept of modularity in CRISPR-Cas systems and raise further questions regarding plasticity of adaptation modules.

microbiology↗

bioRxiv2017

Long-term genomic coevolution of host-parasite interaction in natural environment

The antagonistic coevolution of parasite infectivity and host resistance alters the biological functionality of species, with effects spanning to communities and ecosystems. Still, studies describing long-term host-parasite coevolutionary dynamics in nature are largely missing. Furthermore, the role of host resistance mechanisms for parasite evolution is poorly understood, necessitating for the molecular and phenotypic characterization of both coevolving parasites and their hosts. We combined long-term field sampling (2007-2014), in vitro cross-infections and time-shift experiments with bacteriophage whole genome sequencing and bacterial (Flavobacterium columnare) CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) profiling to show the molecular details of the phage-bacterium arms race in the environment. Bacteria were generally resistant to phages from the past and susceptible to phages in the future. The bacterial resistance selected for increased phage infectivity and host range, correlating directly with the expansion of phage genome size by 2656 bp. In the bacterial host, two CRISPR loci were identified: a type II-C locus and an RNA-targeting type VI-B locus. While maintaining a core set of conserved spacers, phage-matching spacers appeared in the variable end of both CRISPR loci over time. The appearance of these CRISPR spacers in the bacterial host often corresponded with arms race -manner molecular changes in the protospacers of the coevolving phage population. However, the phenotypic data indicated that the relative role of constitutive defence may be more important in high phage pressure, highlighting the importance of our findings for understanding microbial community ecology and in the development of phage therapy applications.

microbiology↗