Vibrio cholerae, the causative agent of the diarrheal disease cholera, is antagonized by the lytic phage ICP1 in the aquatic environment and in human hosts. Mobile genetic elements called PLEs (phage-inducible chromosomal island-like elements) protect V. cholerae from ICP1 infection and initiate their anti-phage response by excising from the chromosome. Here, we show that PLE 1 encodes a large serine recombinase, Int, that exploits an ICP1-specific protein as a recombination directionality factor (RDF) to excise PLE 1 in response to phage infection. We show that this phage-encoded protein is sufficient to direct Int-mediated recombination in vitro and that it is highly conserved in all sequenced ICP1 genomes. Our results uncover an aspect of the molecular specificity underlying the conflict between a single predatory phage and V. cholerae PLE and contribute to our understanding of long-term evolution between phage and their bacterial hosts.

Vibrio cholerae-specific bacteriophages are common features of the microbial community during cholera infection in humans. Phages impose strong selective pressure that favors the expansion of phage-resistant strains over their vulnerable counterparts. The mechanisms allowing virulent V. cholerae strains to defend against the ubiquitous threat of predatory phages have not been established. Here, we show that V. cholerae PLEs (phage-inducible chromosomal island-like elements) are widespread genomic islands dedicated to phage defense. Analysis of V. cholerae isolates spanning a 60-year collection period identified five unique PLEs. Remarkably, we found that all PLEs (regardless of geographic or temporal origin) respond to infection by a myovirus called ICP1, the most prominent V. cholerae phage found in cholera patient stool samples from Bangladesh. We found that PLE activity reduces phage genome replication and accelerates cell lysis following ICP1 infection, killing infected host cells and preventing the production of progeny phage. PLEs are mobilized by ICP1 infection and can spread to neighboring cells such that protection from phage predation can be horizontally acquired. Our results reveal that PLEs are a persistent feature of the V. cholerae mobilome that are adapted to providing protection from a single predatory phage and advance our understanding of how phages influence pathogen evolution.

Phage defense systems are often found on mobile genetic elements (MGEs), where they constitutively defend against invaders or are induced to respond to new assaults. Phage satellites, one type of MGE, are induced during phage infection to promote their own transmission, reducing phage production and protecting their hosts in the process. One such satellite in Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), sabotages the lytic phage ICP1, which triggers PLE excision from the bacterial chromosome, replication, and transduction to neighboring cells. Analysis of patient stool samples from different geographic regions revealed that ICP1 has evolved to possess one of two syntenic loci encoding an SF1B-type helicase, either of which PLE exploits to drive replication. Further, loss of PLE mobilization limits anti-phage activity because of phage-mediated degradation of the bacterial genome. Our work provides insight into the unique challenges facing parasites of lytic phages and underscores the adaptions of satellites to their ever-evolving target phage.

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'.

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.