GoKawiilStatistics and data reproducibility
Where applicable, statistical analyses were conducted using unpaired, two-sided Student’s t-tests in GraphPad Prism. Relevant statistical results, including P values and standard deviations, are reported in the figure legends alongside the data. No statistical methods were used to predetermine sample size, and blinding and randomization were not used.
Patient stool samples and isolation of bacteria and phage
Cholera patient stool samples were collected and screened for V. cholerae and phages as described previously34. Stool samples were collected from patients with suspected cholera at the icddr,b Dhaka Hospital and the Government Health Complex in Mathbaria, Pirojpur, under protocol number PR-16083 approved by the icddr,b Ethical Review Committee, with written consent obtained from participants or their guardians. Samples positive for V. cholerae serogroup O1 and/or O139 on VC RapidDipStick test (Span Diagnostics) were de-identified and stored at −80 °C with w/v 20% glycerol. For on-site isolation of V. cholerae, positive samples were enriched in alkaline peptone water (APW, pH 8.4, Difco) at 37 °C for 6–8 h and then cultured overnight on taurocholate tellurite-gelatin agar (Difco). V. cholerae appearing colonies were further confirmed using previously described biochemical and serological methods35. Further purification of V. cholerae and isolation of phages from stool was performed at the University of California, Berkeley. V. cholerae isolates were further purified twice on Luria-Bertani (LB) agar plates and analysed by PCR for PLE and/or whole-genome sequencing (see Supplementary Table 7 for primers). For phage isolation, a panel of V. cholerae hosts (including PLE(−) E7946 and PLE11(+) BFS783) was used to probe for phages from stool. Where possible, phages were isolated and purified on the cognate V. cholerae strain isolated from the stool sample. Bacterial hosts were grown to the mid-log phase, incubated with a small amount of frozen stool sample collected on a pipette tip (and dilutions thereof) and the mixture was plated in 0.5% LB top agar. Single plaques were picked and purified twice on the same host before being analysed by PCR for adi, CRISPR–cas/odn or TMP mutations using the primers listed in Supplementary Table 7 and/or whole-genome sequencing.
Whole-genome sequencing
Genomic DNA from phages and bacteria was purified using Monarch Genomic DNA Purification Kit (New England BioLabs). Phage samples were initially treated with DNase I to remove non-encapsidated DNA. Illumina sequencing (150-base pair by 150-base pair paired end) was performed by the Microbial Genome Sequencing Center or SeqCenter (for all bacteria and most phage), and Nanopore sequencing was performed by the Barker Sequencing Core at the University of California, Berkeley (for a subset of phage isolates). Genomes were assembled using SPAdes36, and for escape phages selected on PLE11(+) V. cholerae, genomes were analysed using BreSeq (v.0.33)37.
Bioinformatic analysis
The PLE genomes were aligned on the basis of gene product identity using clinker38 at a 30% identity cut-off. We performed BLASTn searches against ICP1 genomes using odn, adi and CRISPR–cas from ICP1_2001_Dha_0, ICP1_2006_Dha_E or ICP1_2011_Dha_A as queries. TMP substitutions were called if the sequence differed from ICP1_2006_Dha_E or ICP1_2011_Dha_A. ICP1 CRISPR spacers were manually annotated between direct repeats in the CRISPR arrays. The phage phylogeny was built by comparing whole-genome sequences of 29 phages isolated from this study and 67 isolates from previous work6 using tBLASTx analysis from ViPTree39. The intergenomic similarities between phages sequenced in this study were determined at VIRIDICweb using BLASTn parameters ‘-word_size 7 -reward 2 -penalty -3 gapopen 5-gapextended 2’ (ref. 40). ICP1 and PLE11 gene products identified in proteomics were analysed for functional predictions using HHPred41 or extracted from previous work (for ICP1)6.
The phylogeny of bacterial genomes was calculated as described previously15. Briefly, fastp v.0.23.2 (ref. 42) was used to evaluate the quality of the raw shotgun paired-end sequences. Genetic variants were identified by mapping the raw reads to the V. cholerae N16961 reference genome (National Center for Biotechnology Information (NCBI) accession IDs NC_002505.1 and NC_002506.1) using snippy v.4.6.0 (ref. 43). Phylogenetic analysis was performed using IQ-TREE v.2.2.0 (ref. 44) with 1,000 bootstrap and the best fitted evolutionary model was selected using ModelFinder45. Spades v.3.15.4 genome assembler was used to generate contigs. Each of the ten previously known PLEs13 and PLE11 were used as BLASTn queries against the V. cholerae genomes and annotated in the phylogeny. Lists of the single-nucleotide polymorphisms in the core genome and strains used to build the phylogeny are in Supplementary Tables 8 and 9, respectively.
The structural predictions for TACPLE4 and Rta were made using ColabFold46 on COSMIC2 (ref. 47) and GoogleColab Structural alignments TACHK97 (Protein Data Bank (PDB) ID 2OB9), TACPLE1 (5IR0) and predicted TACPLE4 were done on ChimeraX48 using ‘Smith-Watermann ssFraction 0.8008 matrix BLOSUM-45 hgap 10 sgap 10 ogap 4′ parameters. The root mean-squared deviation values for aligned pruned amino acid residues are reported. Putative satellite genomes from non-cholera Vibrio spp.34 and cf-PICIs49 were analysed for the presence of TMPs and integrases using BLASTp and HHPred41. Genome visualizations were generated with R, using the gggenes package.
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