Phage capsid recognition triggers activation of a bacterial toxin-antitoxin defense system.

Zhang et al.1 reveal a previously unknown route to toxin activation whereby bacteriophage capsid proteins bind the antitoxin domain of the CapRel fused toxin-antitoxin system, triggering translational inhibition via pyrophosporylation of tRNAs and culminating in abortive infection-mediated phage resistance.

Inhibitors of bacterial immune systems: discovery, mechanisms and applications.

To contend with the diversity and ubiquity of bacteriophages and other mobile genetic elements, bacteria have developed an arsenal of immune defence mechanisms. Bacterial defences include CRISPR-Cas, restriction-modification and a growing list of mechanistically diverse systems, which constitute the bacterial 'immune system'. As a response, bacteriophages and mobile genetic elements have evolved direct and indirect mechanisms to circumvent or block bacterial defence pathways and ensure successful infection. Recent advances in methodological and computational approaches, as well as the increasing availability of genome sequences, have boosted the discovery of direct inhibitors of bacterial defence systems. In this Review, we discuss methods for the discovery of direct inhibitors, their diverse mechanisms of action and perspectives on their emerging applications in biotechnology and beyond.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

Turning down the (C)BASS: Phage-encoded inhibitors jam bacterial immune signaling.

Where there is defense, there is counter-defense. A paper by Hobbs et al. (2022) reconfirms that this emerging tenet of the arms race between bacteria and their predators also holds true for phage defense systems that rely on secondary messenger signals.

Type III CRISPR-Cas provides resistance against nucleus-forming jumbo phages via abortive infection.

Bacteria have diverse defenses against phages. In response, jumbo phages evade multiple DNA-targeting defenses by protecting their DNA inside a nucleus-like structure. We previously demonstrated that RNA-targeting type III CRISPR-Cas systems provide jumbo phage immunity by recognizing viral mRNA exported from the nucleus for translation. Here, we demonstrate that recognition of phage mRNA by the type III system activates a cyclic triadenylate-dependent accessory nuclease, NucC. Although unable to access phage DNA in the nucleus, NucC degrades the bacterial chromosome, triggers cell death, and disrupts phage replication and maturation. Hence, type-III-mediated jumbo phage immunity occurs via abortive infection, with suppression of the viral epidemic protecting the population. We further show that type III systems targeting jumbo phages have diverse accessory nucleases, including RNases that provide immunity. Our study demonstrates how type III CRISPR-Cas systems overcome the inaccessibility of jumbo phage DNA to provide robust immunity.

Bacteriophages suppress CRISPR-Cas immunity using RNA-based anti-CRISPRs.

Many bacteria use CRISPR-Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids1. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity2,3. Here we unveil a distinct type of CRISPR-Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units4. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR-Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR-Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes5. Functional testing of nine candidates spanning the two CRISPR-Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications6.

Diverse CRISPR-Cas Complexes Require Independent Translation of Small and Large Subunits from a Single Gene.

CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution.

The arms race between bacteria and their phage foes.

Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.

Publisher Correction: Targeting of temperate phages drives loss of type I CRISPR-Cas systems.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting of temperate phages drives loss of type I CRISPR-Cas systems.

On infection of their host, temperate viruses that infect bacteria (bacteriophages; hereafter referred to as phages) enter either a lytic or a lysogenic cycle. The former results in lysis of bacterial cells and phage release (resulting in horizontal transmission), whereas lysogeny is characterized by the integration of the phage into the host genome, and dormancy (resulting in vertical transmission)1. Previous co-culture experiments using bacteria and mutants of temperate phages that are locked in the lytic cycle have shown that CRISPR-Cas systems can efficiently eliminate the invading phages2,3. Here we show that, when challenged with wild-type temperate phages (which can become lysogenic), type I CRISPR-Cas immune systems cannot eliminate the phages from the bacterial population. Furthermore, our data suggest that, in this context, CRISPR-Cas immune systems are maladaptive to the host, owing to the severe immunopathological effects that are brought about by imperfect matching of spacers to the integrated phage sequences (prophages). These fitness costs drive the loss of CRISPR-Cas from bacterial populations, unless the phage carries anti-CRISPR (acr) genes that suppress the immune system of the host. Using bioinformatics, we show that this imperfect targeting is likely to occur frequently in nature. These findings help to explain the patchy distribution of CRISPR-Cas immune systems within and between bacterial species, and highlight the strong selective benefits of phage-encoded acr genes for both the phage and the host under these circumstances.

CRISPR-Cas immunity is repressed by the LysR-type transcriptional regulator PigU.

Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Horticultural diseases caused by bacterial pathogens provide an obstacle to crop production globally. Management of the infection of kiwifruit by the Gram-negative phytopathogen Pseudomonas syringae pv. actinidiae (Psa) currently includes copper and antibiotics. However, the emergence of bacterial resistance and a changing regulatory landscape are providing the impetus to develop environmentally sustainable antimicrobials. One potential strategy is the use of bacteriophage endolysins, which degrade peptidoglycan during normal phage replication, causing cell lysis and the release of new viral progeny. Exogenous use of endolysins as antimicrobials is impaired by the outer membrane of Gram-negative bacteria that provides an impermeable barrier and prevents endolysins from accessing their target peptidoglycan. Here, we describe the synergy between citric acid and a phage endolysin, which results in a reduction of viable Psa below detection. We show that citric acid drives the destabilization of the outer membrane via acidification and sequestration of divalent cations from the lipopolysaccharide, which is followed by the degradation of the peptidoglycan by the endolysin. Scanning electron microscopy revealed clear morphological differences, indicating cell lysis following the endolysin-citric acid treatment. These results show the potential for citric acid-endolysin combinations as a possible antimicrobial approach in agricultural applications. IMPORTANCE: The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems.

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

The biology and type I/III hybrid nature of type I-D CRISPR-Cas systems.

Prokaryotes have adaptive defence mechanisms that protect them from mobile genetic elements and viral infection. One defence mechanism is called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). There are six different types of CRISPR-Cas systems and multiple subtypes that vary in composition and mode of action. Type I and III CRISPR-Cas systems utilise multi-protein complexes, which differ in structure, nucleic acid binding and cleaving preference. The type I-D system is a chimera of type I and III systems. Recently, there has been a burst of research on the type I-D CRISPR-Cas system. Here, we review the mechanism, evolution and biotechnological applications of the type I-D CRISPR-Cas system.

A mobile restriction-modification system provides phage defence and resolves an epigenetic conflict with an antagonistic endonuclease.

Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction-modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.

Type I CRISPR-Cas provides robust immunity but incomplete attenuation of phage-induced cellular stress.

During infection, phages manipulate bacteria to redirect metabolism towards viral proliferation. To counteract phages, some bacteria employ CRISPR-Cas systems that provide adaptive immunity. While CRISPR-Cas mechanisms have been studied extensively, their effects on both the phage and the host during phage infection remains poorly understood. Here, we analysed the infection of Serratia by a siphovirus (JS26) and the transcriptomic response with, or without type I-E or I-F CRISPR-Cas immunity. In non-immune Serratia, phage infection altered bacterial metabolism by upregulating anaerobic respiration and amino acid biosynthesis genes, while flagella production was suppressed. Furthermore, phage proliferation required a late-expressed viral Cas4 homologue, which did not influence CRISPR adaptation. While type I-E and I-F immunity provided robust defence against phage infection, phage development still impacted the bacterial host. Moreover, DNA repair and SOS response pathways were upregulated during type I immunity. We also discovered that the type I-F system is controlled by a positive autoregulatory feedback loop that is activated upon phage targeting during type I-F immunity, leading to a controlled anti-phage response. Overall, our results provide new insight into phage-host dynamics and the impact of CRISPR immunity within the infected cell.

Molecular basis of anti-CRISPR operon repression by Aca10.

CRISPR-Cas systems are bacterial defense systems for fighting against invaders such as bacteriophages and mobile genetic elements. To escape destruction by these bacterial immune systems, phages have co-evolved multiple anti-CRISPR (Acr) proteins, which inhibit CRISPR-Cas function. Many acr genes form an operon with genes encoding transcriptional regulators, called anti-CRISPR-associated (Aca) proteins. Aca10 is the most recently discovered Aca family that is encoded within an operon containing acrIC7 and acrIC6 in Pseudomonas citronellolis. Here, we report the high-resolution crystal structure of an Aca10 protein to unveil the molecular basis of transcriptional repressor role of Aca10 in the acrIC7-acrIC6-aca10 operon. We identified that Aca10 forms a dimer in solution, which is critical for binding specific DNA. We also showed that Aca10 directly recognizes a 21 bp palindromic sequence in the promoter of the acr operon. Finally, we revealed that R44 of Aca10 is a critical residue involved in the DNA binding, which likely results in a high degree of DNA bending.

Widespread repression of anti-CRISPR production by anti-CRISPR-associated proteins.

Many bacteria use CRISPR-Cas systems to defend against invasive mobile genetic elements (MGEs). In response, MGEs have developed strategies to resist CRISPR-Cas, including the use of anti-CRISPR (Acr) proteins. Known acr genes may be followed in an operon by a putative regulatory Acr-associated gene (aca), suggesting the importance of regulation. Although ten families of helix-turn-helix (HTH) motif containing Aca proteins have been identified (Aca1-10), only three have been tested and shown to be transcriptional repressors of acr-aca expression. The AcrIIA1 protein (a Cas9 inhibitor) also contains a functionally similar HTH containing repressor domain. Here, we identified and analysed Aca and AcrIIA1 homologs across all bacterial genomes. Using HMM models we found aca-like genes are widely distributed in bacteria, both with and without known acr genes. The putative promoter regions of acr-aca operons were analysed and members of each family of bacterial Aca tested for regulatory function. For each Aca family, we predicted a conserved inverted repeat binding site within a core promoter. Promoters containing these sites directed reporter expression in E. coli and were repressed by the cognate Aca protein. These data demonstrate that acr repression by Aca proteins is widely conserved in nature.

PADLOC: a web server for the identification of antiviral defence systems in microbial genomes.

Most bacteria and archaea possess multiple antiviral defence systems that protect against infection by phages, archaeal viruses and mobile genetic elements. Our understanding of the diversity of defence systems has increased greatly in the last few years, and many more systems likely await discovery. To identify defence-related genes, we recently developed the Prokaryotic Antiviral Defence LOCator (PADLOC) bioinformatics tool. To increase the accessibility of PADLOC, we describe here the PADLOC web server (freely available at https://padloc.otago.ac.nz), allowing users to analyse whole genomes, metagenomic contigs, plasmids, phages and archaeal viruses. The web server includes a more than 5-fold increase in defence system types detected (since the first release) and expanded functionality enabling detection of CRISPR arrays and retron ncRNAs. Here, we provide user information such as input options, description of the multiple outputs, limitations and considerations for interpretation of the results, and guidance for subsequent analyses. The PADLOC web server also houses a precomputed database of the defence systems in > 230,000 RefSeq genomes. These data reveal two taxa, Campylobacterota and Spriochaetota, with unusual defence system diversity and abundance. Overall, the PADLOC web server provides a convenient and accessible resource for the detection of antiviral defence systems.