A phage parasite deploys a nicking nuclease effector to inhibit viral host replication.

PLEs (phage-inducible chromosomal island-like elements) are phage parasites integrated into the chromosome of epidemic Vibrio cholerae. In response to infection by its viral host ICP1, PLE excises, replicates and hijacks ICP1 structural components for transduction. Through an unknown mechanism, PLE prevents ICP1 from transitioning to rolling circle replication (RCR), a prerequisite for efficient packaging of the viral genome. Here, we characterize a PLE-encoded nuclease, NixI, that blocks phage development likely by nicking ICP1's genome as it transitions to RCR. NixI-dependent cleavage sites appear in ICP1's genome during infection of PLE(+) V. cholerae. Purified NixI demonstrates in vitro nuclease activity specifically for sites in ICP1's genome and we identify a motif that is necessary for NixI-mediated cleavage. Importantly, NixI is sufficient to limit ICP1 genome replication and eliminate progeny production, representing the most inhibitory PLE-encoded mechanism revealed to date. We identify distant NixI homologs in an expanded family of putative phage parasites in vibrios that lack nucleotide homology to PLEs but nonetheless share genomic synteny with PLEs. More generally, our results reveal a previously unknown mechanism deployed by phage parasites to limit packaging of their viral hosts' genome and highlight the prominent role of nuclease effectors as weapons in the arms race between antagonizing genomes.

Parasites of viruses, often referred to as satellites, are found in all domains of life and have been co-opted for host defense across diverse virus-host systems multiple independent times. This study describes the mechanism by which such an element prevents a bacterial virus (a 'phage') from otherwise infecting Vibrio cholera and related bacteria. The study is of broad interest to investigators with interests in phage-host interactions and microbial genetics.

Temporal shifts in antibiotic resistance elements govern phage-pathogen conflicts.

Bacteriophage predation selects for diverse antiphage systems that frequently cluster on mobilizable defense islands in bacterial genomes. However, molecular insight into the reciprocal dynamics of phage-bacterial adaptations in nature is lacking, particularly in clinical contexts where there is need to inform phage therapy efforts and to understand how phages drive pathogen evolution. Using time-shift experiments, we uncovered fluctuations in Vibrio cholerae's resistance to phages in clinical samples. We mapped phage resistance determinants to SXT integrative and conjugative elements (ICEs), which notoriously also confer antibiotic resistance. We found that SXT ICEs, which are widespread in γ-proteobacteria, invariably encode phage defense systems localized to a single hotspot of genetic exchange. We identified mechanisms that allow phage to counter SXT-mediated defense in clinical samples, and document the selection of a novel phage-encoded defense inhibitor. Phage infection stimulates high-frequency SXT ICE conjugation, leading to the concurrent dissemination of phage and antibiotic resistances.

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Genome Editing Method for the Anaerobic Magnetotactic Bacterium Desulfovibrio magneticus RS-1.

Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacteria of the genus Magnetospirillum that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.