Characterization of a Novel N4-Methylcytosine Restriction-Modification System in Deinococcus radiodurans.

Deinococcus radiodurans is an extremophilic microorganism that possesses a unique DNA damage repair system, conferring a strong resistance to radiation, desiccation, oxidative stress, and chemical damage. Recently, we discovered that D. radiodurans possesses an N4-methylation (m4C) methyltransferase called M.DraR1, which recognizes the 5'-CCGCGG-3' sequence and methylates the second cytosine. Here, we revealed its cognate restriction endonuclease R.DraR1 and recognized that it is the only endonuclease specially for non-4C-methylated 5'-CCGCGG-3' sequence so far. We designated the particular m4C R.DraR1-M.DraR1 as the DraI R-M system. Bioinformatics searches displayed the rarity of the DraI R-M homologous system. Meanwhile, recombination and transformation efficiency experiments demonstrated the important role of the DraI R-M system in response to oxidative stress. In addition, in vitro activity experiments showed that R.DraR1 could exceptionally cleave DNA substrates with a m5C-methlated 5'-CCGCGG-3' sequence instead of its routine activity, suggesting that this particular R-M component possesses a broader substrate choice. Furthermore, an imbalance of the DraI R-M system led to cell death through regulating genes involved in the maintenance of cell survival such as genome stability, transporter, and energy production. Thus, our research revealed a novel m4C R-M system that plays key roles in maintaining cell viability and defending foreign DNA in D. radiodurans.

DNA methylases for site-selective inhibition of type IIS restriction enzyme activity.

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques. KEY POINTS: • Non-switchable methylases always inhibit the relevant type IIS endonuclease activity • Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site • Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assembly.

Reappraisal of the DNA phosphorothioate modification machinery: uncovering neglected functional modalities and identification of new counter-invader defense systems.

The DndABCDE systems catalysing the unusual phosphorothioate (PT) DNA backbone modification, and the DndFGH systems, which restrict invasive DNA, have enigmatic and paradoxical features. Using comparative genomics and sequence-structure analyses, we show that the DndABCDE module is commonly functionally decoupled from the DndFGH module. However, the modification gene-neighborhoods encode other nucleases, potentially acting as the actual restriction components or suicide effectors limiting propagation of the selfish elements. The modification module's core consists of a coevolving gene-pair encoding the DNA-scanning apparatus - a DndD/CxC-clade ABC ATPase and DndE with two ribbon-helix-helix (MetJ/Arc) DNA-binding domains. Diversification of DndE's DNA-binding interface suggests a multiplicity of target specificities. Additionally, many systems feature DNA cytosine methylase genes instead of PT modification, indicating the DndDE core can recruit other nucleobase modifications. We show that DndFGH is a distinct counter-invader system with several previously uncharacterized domains, including a nucleotide kinase. These likely trigger its restriction endonuclease domain in response to multiple stimuli, like nucleotides, while blocking protective modifications by invader methylases. Remarkably, different DndH variants contain a HerA/FtsK ATPase domain acquired from multiple sources, including cellular genome-segregation systems and mobile elements. Thus, we uncovered novel HerA/FtsK-dependent defense systems that might intercept invasive DNA during replication, conjugation, or packaging.

Bacteria defend against selfish genetic elements by distinguishing their genetic material through special chemical modifications and using specific enzymes to break down viral DNA. This study explores the Dnd defense system, revealing several of its poorly understood facets. The Dnd modification system, utilizing sulfur to distinguish bacterial from viral DNA, cooperates with various anti-viral and cell-suicide nuclease enzymes to limit viral infection. While previously considered its restriction component, DndFGH emerges as an independent defense system, recognizing signals like nucleotides and DNA to thwart protective modifications of invader DNA. DndH, featuring diverse versions of the HerA/FtsK ATPase domain, helped unveil several unrecognized bacterial defense systems. This discovery illuminates sophisticated bacterial defenses against viral threats during crucial cellular processes.

dbAPIS: a database of anti-prokaryotic immune system genes.

Anti-prokaryotic immune system (APIS) proteins, typically encoded by phages, prophages, and plasmids, inhibit prokaryotic immune systems (e.g. restriction modification, toxin-antitoxin, CRISPR-Cas). A growing number of APIS genes have been characterized and dispersed in the literature. Here we developed dbAPIS (https://bcb.unl.edu/dbAPIS), as the first literature curated data repository for experimentally verified APIS genes and their associated protein families. The key features of dbAPIS include: (i) experimentally verified APIS genes with their protein sequences, functional annotation, PDB or AlphaFold predicted structures, genomic context, sequence and structural homologs from different microbiome/virome databases; (ii) classification of APIS proteins into sequence-based families and construction of hidden Markov models (HMMs); (iii) user-friendly web interface for data browsing by the inhibited immune system types or by the hosts, and functions for searching and batch downloading of pre-computed data; (iv) Inclusion of all types of APIS proteins (except for anti-CRISPRs) that inhibit a variety of prokaryotic defense systems (e.g. RM, TA, CBASS, Thoeris, Gabija). The current release of dbAPIS contains 41 verified APIS proteins and ∼4400 sequence homologs of 92 families and 38 clans. dbAPIS will facilitate the discovery of novel anti-defense genes and genomic islands in phages, by providing a user-friendly data repository and a web resource for an easy homology search against known APIS proteins.

Toxin/antitoxin systems induce persistence and work in concert with restriction/modification systems to inhibit phage.

IMPORTANCE: To date, there are no reports of phage infection-inducing persistence. Therefore, our results are important since we show for the first time that a phage-defense system, the MqsRAC toxin/antitoxin system, allows the host to survive infection by forming persister cells, rather than inducing cell suicide. Moreover, we demonstrate that the MqsRAC system works in concert with restriction/modification systems. These results imply that if phage therapy is to be successful, anti-persister compounds need to be administered along with phages.

The streptococcal phase-variable type I restriction modification system SsuCC20p dictates the methylome of Streptococcus suis impacting the transcriptome and virulence in a zebrafish larvae infection model.

IMPORTANCE: Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.

Lethal perturbation of an Escherichia coli regulatory network is triggered by a restriction-modification system's regulator and can be mitigated by excision of the cryptic prophage Rac.

Bacterial gene regulatory networks orchestrate responses to environmental challenges. Horizontal gene transfer can bring in genes with regulatory potential, such as new transcription factors (TFs), and this can disrupt existing networks. Serious regulatory perturbations may even result in cell death. Here, we show the impact on Escherichia coli of importing a promiscuous TF that has adventitious transcriptional effects within the cryptic Rac prophage. A cascade of regulatory network perturbations occurred on a global level. The TF, a C regulatory protein, normally controls a Type II restriction-modification system, but in E. coli K-12 interferes with expression of the RacR repressor gene, resulting in de-repression of the normally-silent Rac ydaT gene. YdaT is a prophage-encoded TF with pleiotropic effects on E. coli physiology. In turn, YdaT alters expression of a variety of bacterial regulons normally controlled by the RcsA TF, resulting in deficient lipopolysaccharide biosynthesis and cell division. At the same time, insufficient RacR repressor results in Rac DNA excision, halting Rac gene expression due to loss of the replication-defective Rac prophage. Overall, Rac induction appears to counteract the lethal toxicity of YdaT. We show here that E. coli rewires its regulatory network, so as to minimize the adverse regulatory effects of the imported C TF. This complex set of interactions may reflect the ability of bacteria to protect themselves by having robust mechanisms to maintain their regulatory networks, and/or suggest that regulatory C proteins from mobile operons are under selection to manipulate their host's regulatory networks for their own benefit.

PacBio sequencing of human fecal samples uncovers the DNA methylation landscape of 22 673 gut phages.

Gut phages have an important impact on human health. Methylation plays key roles in DNA recognition, gene expression regulation and replication for phages. However, the DNA methylation landscape of gut phages is largely unknown. Here, with PacBio sequencing (2120×, 4785 Gb), we detected gut phage methylation landscape based on 22 673 gut phage genomes, and presented diverse methylation motifs and methylation differences in genomic elements. Moreover, the methylation rate of phages was associated with taxonomy and host, and N6-methyladenine methylation rate was higher in temperate phages than in virulent phages, suggesting an important role for methylation in phage-host interaction. In particular, 3543 (15.63%) phage genomes contained restriction-modification system, which could aid in evading clearance by the host. This study revealed the DNA methylation landscape of gut phage and its potential roles, which will advance the understanding of gut phage survival and human health.

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems.

IMPORTANCE: The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.

Efficient Genome Editing in Most Staphylococcus aureus by Using the Restriction-Modification System Silent CRISPR-Cas9 Toolkit.

Staphylococcus aureus is a clinically important pathogen that threatens human health due to its strong pathogenicity and drug resistance, leading to meningitis, endocarditis, and skin and soft tissue infections. Genetic manipulation in S. aureus is a powerful approach for characterizing the molecular mechanisms of bacterial drug resistance, pathogenicity, and virulence. However, a strong restriction barrier presents a major obstacle to the extensive utilization of genetic manipulation tools in clinical isolates of S. aureus. Here, we constructed a restriction-modification (RM) system silent CRISPR-Cas9 toolkit that synonymously eliminated the type I RM targets of S. aureus from plasmids, downsized plasmids using minicircle technology, and combined with a plasmid artificial modification (PAM) method to circumvent the type II RM system. The RM-silent CRISPR-Cas9 toolkit enables a significant improvement in transformation (105-106 transformants per microgram plasmid in strains we tested) and high-success efficiency editing for gene deletion (knockout strain obtained in one-round electroporation) in a wide range of S. aureus species including clinical isolates of unknown genetic background. The RM-silent CRISPR-Cas9 toolkits could expedite the process of mutant construction in most S. aureus strains, and this approach could be applied to the design of other genetic toolkit plasmids for utilization in a wider range of S. aureus strains.

Methylome-dependent transformation of emm1 group A streptococci.

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A Streptococcus (GAS), a lethal human pathogen currently associated with an unprecedented surge of infections worldwide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this "restriction barrier" thus presents a major technical challenge. Here, we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Furthermore, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRDAG, encoded by all sequenced strains of the dominant and upsurge-associated emm1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol, whereby the restriction barrier is overcome by the addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRDAG strains including clinical isolates representing all emm1 lineages and will expedite critical research interrogating the genetics of emm1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation and the importance of defining lineage-specific mechanisms of genetic recalcitrance. IMPORTANCE Understanding the mechanisms by which bacterial pathogens are able to cause disease is essential to enable the targeted development of novel therapeutics. A key experimental approach to facilitate this research is the generation of bacterial mutants, through either specific gene deletions or sequence manipulation. This process relies on the ability to transform bacteria with exogenous DNA designed to generate the desired sequence changes. Bacteria have naturally developed protective mechanisms to detect and destroy invading DNA, and these systems severely impede the genetic manipulation of many important pathogens, including the lethal human pathogen group A Streptococcus (GAS). Many GAS lineages exist, of which emm1 is dominant among clinical isolates. Based on new experimental evidence, we identify the mechanism by which transformation is impaired in the emm1 lineage and establish an improved and highly efficient transformation protocol to expedite the generation of mutants.

Landscape of New Nuclease-Containing Antiphage Systems in Escherichia coli and the Counterdefense Roles of Bacteriophage T4 Genome Modifications.

Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, raising the question of the role of phage genome modifications in countering these systems. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli and demonstrated the roles of T4 genome modifications in countering these systems. Our analysis identified at least 17 nuclease-containing defense systems in E. coli, with type III Druantia being the most abundant system, followed by Zorya, Septu, Gabija, AVAST type 4, and qatABCD. Of these, 8 nuclease-containing systems were found to be active against phage T4 infection. During T4 replication in E. coli, 5-hydroxymethyl dCTP is incorporated into the newly synthesized DNA instead of dCTP. The 5-hydroxymethylcytosines (hmCs) are further modified by glycosylation to form glucosyl-5-hydroxymethylcytosine (ghmC). Our data showed that the ghmC modification of the T4 genome abolished the defense activities of Gabija, Shedu, Restriction-like, type III Druantia, and qatABCD systems. The anti-phage T4 activities of the last two systems can also be counteracted by hmC modification. Interestingly, the Restriction-like system specifically restricts phage T4 containing an hmC-modified genome. The ghmC modification cannot abolish the anti-phage T4 activities of Septu, SspBCDE, and mzaABCDE, although it reduces their efficiency. Our study reveals the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of T4 genomic modification in countering these defense systems. IMPORTANCE Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms. However, phages have evolved different strategies to modify their genomes to prevent cleavage. Recent studies have revealed many novel nuclease-containing antiphage systems from various bacteria and archaea. However, no studies have systematically investigated the nuclease-containing antiphage systems of a specific bacterial species. In addition, the role of phage genome modifications in countering these systems remains unknown. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli using all 2,289 genomes available in NCBI. Our studies reveal the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of genomic modification of phage T4 in countering these defense systems.

Base-excision restriction enzymes: expanding the world of epigenetic immune systems.

The restriction enzymes examined so far are phosphodiesterases, which cleave DNA strands by hydrolysing phosphodiester bonds. Based on the mobility of restriction-modification systems, recent studies have identified a family of restriction enzymes that excise a base in their recognition sequence to generate an abasic (AP) site unless the base is properly methylated. These restriction glycosylases also show intrinsic but uncoupled AP lyase activity at the AP site, generating an atypical strand break. Action of an AP endonuclease at the AP site may generate another atypical break, rejoining/repairing of which is difficult. This PabI family of restriction enzymes contain a novel fold (HALFPIPE) and show unusual properties, such as non-requirement of divalent cations for cleavage. These enzymes are present in Helicobacteraceae/Campylobacteraceae and in few hyperthermophilic archaeal species. In Helicobacter genomes, their recognition sites are strongly avoided, and the encoding genes are often inactivated by mutations or replacement, indicating that their expression is toxic for the cells. The discovery of restriction glycosylases generalizes the concept of restriction-modification systems to epigenetic immune systems, which may use any mode of damage to DNA that are considered 'non-self' based on epigenetic modifications. This concept will add to our understanding of immunity and epigenetics.

Restriction-modification systems have shaped the evolution and distribution of plasmids across bacteria.

Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M systems) are by far the most abundant bacterial defense system and therefore represent one of the key barriers to plasmid spread. However, their effect on plasmid evolution and host range has been neglected. Here we analyse the avoidance of targets of the most abundant R-M systems (Type II) for complete genomes and plasmids across bacterial diversity. For the most common target length (6 bp) we show that target avoidance is strongly correlated with the taxonomic distribution of R-M systems and is greater in plasmid genes than core genes. We find stronger avoidance of R-M targets in plasmids which are smaller and have a broader host range. Our results suggest two different evolutionary strategies for plasmids: small plasmids primarily adapt to R-M systems by tuning their sequence composition, and large plasmids primarily adapt through the carriage of additional genes protecting from restriction. Our work provides systematic evidence that R-M systems are important barriers to plasmid transfer and have left their mark on plasmids over long evolutionary time.

DNA modifications impact natural transformation of Acinetobacter baumannii.

Acinetobacter baumannii is a dangerous nosocomial pathogen, especially due to its ability to rapidly acquire new genetic traits, including antibiotic resistance genes (ARG). In A. baumannii, natural competence for transformation, one of the primary modes of horizontal gene transfer (HGT), is thought to contribute to ARG acquisition and has therefore been intensively studied. However, knowledge regarding the potential role of epigenetic DNA modification(s) on this process remains lacking. Here, we demonstrate that the methylome pattern of diverse A. baumannii strains differs substantially and that these epigenetic marks influence the fate of transforming DNA. Specifically, we describe a methylome-dependent phenomenon that impacts intra- and inter-species DNA exchange by the competent A. baumannii strain A118. We go on to identify and characterize an A118-specific restriction-modification (RM) system that impairs transformation when the incoming DNA lacks a specific methylation signature. Collectively, our work contributes towards a more holistic understanding of HGT in this organism and may also aid future endeavors towards tackling the spread of novel ARGs. In particular, our results suggest that DNA exchanges between bacteria that share similar epigenomes are favored and could therefore guide future research into identifying the reservoir(s) of dangerous genetic traits for this multi-drug resistant pathogen.

Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates.

Streptococcus pneumoniae is capable of randomly switching their genomic DNA methylation pattern between six distinct bacterial subpopulations (A-F) via recombination of a type 1 restriction-modification locus, spnIII. These pneumococcal subpopulations exhibit phenotypic changes which favor carriage or invasive disease. In particular, the spnIIIB allele has been associated with increased nasopharyngeal carriage and the downregulation of the luxS gene. The LuxS/AI-2 QS system represent a universal language for bacteria and has been linked to virulence and biofilm formation in S. pneumoniae. In this work, we have explored the link between spnIII alleles, the luxS gene and virulence in two clinical pneumococcal isolates from the blood and cerebrospinal fluid (CSF) of one pediatric meningitis patient. The blood and CSF strains showed different virulence profiles in mice. Analysis of the spnIII system of these strains recovered from the murine nasopharynx showed that the system switched to different alleles commensurate with the initial source of the isolate. Of note, the blood strain showed high expression of spnIIIB allele, previously linked with less LuxS protein production. Importantly, strains with deleted luxS displayed different phenotypic profiles compared to the wildtype, but similar to the strains recovered from the nasopharynx of infected mice. This study used clinically relevant S. pneumoniae strains to demonstrate that the regulatory network between luxS and the type 1 restriction-modification system play a key role in infections and may support different adaptation to specific host niches.

Evolution of Restriction-Modification Systems Consisting of One Restriction Endonuclease and Two DNA Methyltransferases.

Some restriction-modification systems contain two DNA methyltransferases. In the present work, we have classified such systems according to the families of catalytic domains present in the restriction endonucleases and both DNA methyltransferases. Evolution of the restriction-modification systems containing an endonuclease with a NOV_C family domain and two DNA methyltransferases, both with DNA_methylase family domains, was investigated in detail. Phylogenetic tree of DNA methyltransferases from the systems of this class consists of two clades of the same size. Two DNA methyltransferases of each restriction-modification system of this class belong to the different clades. This indicates independent evolution of the two methyltransferases. We detected multiple cross-species horizontal transfers of the systems as a whole, as well as the cases of gene transfer between the systems.

Delineation of a lactococcal conjugation system reveals a restriction-modification evasion system.

Plasmid pUC11B is a 49.3-kb plasmid harboured by the fermented meat isolate Lactococcus lactis subsp. lactis UC11. Among other features, pUC11B encodes a pMRC01-like conjugation system and tetracycline-resistance. In this study, we demonstrate that this plasmid can be conjugated at high frequencies to recipient strains. Mutational analysis of the 22 genes encompassing the presumed pUC11B conjugation cluster revealed the presence of several genes with essential conjugation functions, as well as a gene, trsR, encoding a putative transcriptional repressor of this conjugation cluster. Furthermore, plasmid pUC11B encodes an anti-restriction protein, TrsAR, which facilitates higher conjugation frequencies when pUC11B is transferred into recipient strains containing Type II or Type III RM systems. These findings demonstrate how RM mechanisms can be circumvented when they act as a biological barrier for conjugation events.

Restriction endonuclease cleavage of phage DNA enables resuscitation from Cas13-induced bacterial dormancy.

Type VI CRISPR systems protect against phage infection using the RNA-guided nuclease Cas13 to recognize viral messenger RNA. Upon target recognition, Cas13 cleaves phage and host transcripts non-specifically, leading to cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is unclear. Here we show that type VI CRISPR and DNA-cleaving restriction-modification (RM) systems frequently co-occur and synergize to clear phage infections and resuscitate cells. In the natural type VI CRISPR host Listeria seeligeri, we show that RM cleaves the phage genome, thus removing the source of phage transcripts and enabling cells to recover from Cas13-induced cellular dormancy. We find that phage infections are neutralized more effectively when Cas13 and RM systems operate together. Our work reveals that type VI CRISPR immunity is cell-autonomous and non-abortive when paired with RM, and hints at other synergistic roles for the diverse host-directed immune systems in bacteria.

Systematic identification of gene-altering programmed inversions across the bacterial domain.

Programmed chromosomal inversions allow bacteria to generate intra-population genotypic and functional heterogeneity, a bet-hedging strategy important in changing environments. Some programmed inversions modify coding sequences, producing different alleles in several gene families, most notably in specificity-determining genes such as Type I restriction-modification systems, where systematic searches revealed cross phylum abundance. Yet, a broad, gene-independent, systematic search for gene-altering programmed inversions has been absent, and little is known about their genomic sequence attributes and prevalence across gene families. Here, identifying intra-species variation in genomes of over 35 000 species, we develop a predictive model of gene-altering inversions, revealing key attributes of their genomic sequence attributes, including gene-pseudogene size asymmetry and orientation bias. The model predicted over 11,000 gene-altering loci covering known targeted gene families, as well as novel targeted families including Type II restriction-modification systems, a protein of unknown function, and a fusion-protein containing conjugative-pilus and phage tail domains. Publicly available long-read sequencing datasets validated representatives of these newly predicted inversion-targeted gene families, confirming intra-population genetic heterogeneity. Together, these results reveal gene-altering programmed inversions as a key strategy adopted across the bacterial domain, and highlight programmed inversions that modify Type II restriction-modification systems as a possible new mechanism for maintaining intra-population heterogeneity.