Supplementary figures and datasets of the manuscript entitled “Prophage border curation reveals horizontal transfer of lysogeny-related elements between filamentous and double jelly-roll phages”

Supporting information captionsSupplementary Data 1. RefSeq accession codesof 688 Vibriospp. genomes analyzed in this study.Supplementary Data 2. Annotation of genomic coordinates and attL/R junctions of 515 inoviruses from Vibrio.Supplementary Data 3. Annotation of genomic coordinates and attL/R junctions of 258 DJR prophages from Vibrio.Supplementary Data 4. Genomic length and cross-species relative abundance data for each phage subtypes and results derived by statistical analyses.Supplementary Figure 1 Sequencing gaps within non-tailed prophage genomes. Sequencing gaps are highly likely to occur during de novo genome assembly using short-read Illumina data when dif1- and dif2-targeting IMEXs (inoviruses, a; DJR phages, b) with similar content are present together.Supplementary Figure 2 Marker genes specific to inoviruses (upper) and DJR phages (lower) in Vibrio spp. genomes. Entries tothe InterPro database(IPR) or NCBI conserved domain database(PHA/cd) are labeled. Marker genes conserved in all inoviral subtypes or DJR subtypes are provided. Note, subtype-specific marker genes arehighlighted in different colors.Supplementary Figure 3 Int-encoding inoviral genomes curated from α-Proteobacteria (a) and Archaea (b). Comparative genomic analysis of differences between pairs of the parental genome (highlighted in red) and its isogenic prophage-free genome (blue). Homologous host fragments are visualized through gray-shaded connectors. For each reference genome, the precise attB integration site is annotated, and for each parental bacterial genome, the attL/R junction of integrated inoviral genome is displayed. To aid readers, accession numbersof all analyzed genomes are indicated, with genomic coordinates explicitly labeled.Supplementary Figure 4 HGT represents a critical driver shaping pHVR composition. The three pHVR-associated genes (highlighted in yellow) from V. fluvialis- and V. alginolyticus-targeting inoviruses exhibiting significant sequence homology (80%–90% identity and 100% coverage via BLASTp analysis) to counterparts in a V. cholerae genomic contig (RefSeq, NZ_JAEMFS010000003.1). Homologous proteins are interconnected by gradient blue shadings, with transparency levels reflecting pairwise sequence similarity. Genome accessions and fragment coordinates are annotated. OB-fold, oligonucleotide/oligosaccharide binding motif; CobQ, cobyric acid synthase; ModA, molybdate ABC transporter; ModB, molybdenum transport system permease; ModC, molybdenum transport system ATP-binding protein; C5 Mtase, C-5 cytosine methyltransferase; dGTPase, deoxyguanosine triphosphatase; NT_sf, nucleotidyltransferase superfamily; His_Pase_superF, histidine phosphatase superfamily.Supplementary Figure 5 Thermostable direct hemolysin (TDH)-encoding genes identified in pHVRs. (a) Annotation of pHVR genes (yellow) from inoviruses targeting Vibrio mimicus revealing the presence of TDH-encoding genes (striped yellow patterns). (b) Superimposed structures of thecrystallographically determined TDH in V. parahaemolyticus (#PDB: 3A57) and AlphaFold-predicted model of pHVR TDH (protein id, ENO8416004.1), with the conserved region outlined in orange. The resulting RMSD value (0.254) indicated a high degree of conservation in the two three-dimensional folding structures.Supplementary Figure 6 Characterized CTX−I cryptic elements. CTX−I cryptic elements displayed the same terminal gene pair (RstR/XafT) as CTX−Iphages but lackedcanonical phage features whileretaining pHVR genes (filled in yellow). Accession numbers of parental bacterial genomes are indicated, and the genomic coordinates of each element are provided.Supplementary Figure 7 Organizational complexity of IMEX arrays identified in Vibrio spp. genomes. Each prophage region is distinguished by the attsite shared by the two adjacent prophages, while the prophage subtypes are characterized by the corresponding phage marker genes. The accession number of the parental bacterial genome is indicated on the left, and the genomic coordinates of these IMEXs are provided.