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Mahony, Douwe van Sinderen. 8. 9. 10. Supplementary Information. 11. 12. 13 ... UCC2003, and B. breve 017W4-39 cultures, grown for 8 h, and treated with a ...

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Characterization and induction of prophages in human gut-associated Bifidobacterium hosts

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Travis N. Mavrich, Eoghan Casey, Joana Oliveira, Francesca Bottacini, Kieran James, Charles

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M.A.P. Franz, Gabriele A. Lugli, Horst Neve, Marco Ventura, Graham F. Hatfull, Jennifer

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Mahony, Douwe van Sinderen

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Supplementary Information

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Supplementary Materials and Methods

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Optimization of mitomycin C induction. Mitomycin C concentration was optimized using 96-

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well microtiter plates. Wells with 500 µl RCM were inoculated from B. breve JCM 7017, B. breve

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UCC2003, and B. breve 017W4-39 cultures, grown for 8 h, and treated with a 10-fold serial

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titration of mitomycin C (ranging from 0.0003 µg/ml to 3 µg/ml) for 14 h. Growth inhibition was

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observed for concentrations at and above 0.03 µg/ml. Similar inhibitory profiles were observed

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with 0.03 µg/ml mitomycin C for 2.5 ml, but not 50 ml, cultures. Therefore, for 50 ml cultures, 0.3

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µg/ml mitomycin C was used.

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Induction verification using PCR. Prophage induction was confirmed by PCR amplification

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across the attP site that forms after excision and circularization. One pair of primers was

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designed to test dnaJ2-integrated phages (5’-TCCGTAAAAACAGGTTAAAAACCG-3’, 5’-

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AAACGTTGGAATCACGCCATTCC-3’). Separate primer pairs were designed for Bb447phi1 (5’-

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GTCACACCCACCAGAATCATGAATC-3’,5’-GTTAAGAAGACTTGCTGATGGAGTTG-3’) and

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Bb423phi2 (5’-CGAACCACTGTGTCATCATCTC-3’, 5’-AGCGAGATAACTTGGACGATCAAC-

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3’). Induction of previously described prophages in strains B. choerinum LMG 10510 and B.

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moukalabense DSM 27321 was confirmed using previously described primers19. Amplification

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proceeded in 25 µl reactions containing 1 µl filtered supernatant with Taq polymerase according

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to manufacturer’s instructions, using a thermocycler protocol of 25-30 cycles of denaturation at

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94oC for 30 s, annealing at 55oC for 30 s, and extension at 72oC for 1 min.

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Plaque assays. Plaque generation was attempted using a variety of phage samples, indicator

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strains, and growth media. To test for spontaneous phage release, filtered supernatants of

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saturated cultures were used. To test for mitomycin C-induced phage release, filtered

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supernatants or PEG precipitated samples from mitomycin C-treated cultures (as generated for



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flow cytometry) were used. Confluent lawns were prepared by mixing 4.5 ml Reinforced

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Clostridial Top Agar (30 ml Reinforced Clostridial Agar + 60 ml RCM, with or without 2 mM

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CaCl2) with 200-300 µl saturated bifidobacterial culture (grown overnight directly from a freezer

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stock) and allowed to solidify on RCA plates. For each phage sample, 3-5 µl was spotted onto

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the overlay and allowed to dry, and plates were incubated at 37oC in an anaerobic chamber for

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24-48 h. Phage samples were generated from several lysogens (B. choerinum LMG 10510 and

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B. moukalabense DSM 27321) and predicted lysogens (B. breve 082W4-8, B. breve 180W8-3,

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B. breve 139W4-23, B. breve 017W4-39, and B. breve 215W4-47a), and they were tested

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against all of the originating lysogens and predicted lysogens as well as several non-lysogens

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(B. breve JCM 7017, B. breve NCIMB 702258, and B. breve UCC2003). As an alternative to

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spotting, some saturated cultures were directly mixed with phage samples in a 1.5 ml tube and

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aerobically incubated on the bench at room temperature for 10-15 min prior to being mixed with

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top agar and poured as an overlay. Additionally, TOS media (Sigma) was used as an alternative

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to RCM.

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Rin shufflon inversion analysis in uninduced Bb423phi1. Genomic inversions within the Rin

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shufflon of the uninduced Bb423phi1 prophage were identified in previously reported B. breve

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139W4-23 raw whole genome sequencing reads18. Pacbio long reads (average read length >10

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kb) that map across the Rin shufflon locus (coordinates 1,307,900 to 1,310,888) with at least

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80% sequence identity were selected using BLAT aligner v36x2. Variant shufflon orientations in

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this subset of reads were identified using dotplot alignments in mummer v3.0. Sequence

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coverage of each variant was computed using the identified long reads as reference sequences

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and performing a mapping assembly using the RS_Resequencing.1 protocol implemented in

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SMRT Analysis portal v2.3. The resulting assembled reads were inspected using the Next

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Generation Sequencing (NGS) visualization tool Tablet (https://ics.hutton.ac.uk/).

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Gene content flux analysis. Changes in gene content and nucleotide sequence similarity were

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computed as previously described53. Briefly, for each bifidophage genome, pairwise nucleotide

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distances to all other actinobacteriophages were computed using Mash, and gene content

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dissimilarities were computed using pham designations in Phamerator. Each phage’s

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evolutionary mode is predicted by assessing the distribution of pairwise genomic similarities

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using previously determined mode boundaries.

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Supplementary Figure Legends

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Supplementary Figure S1. Mitomycin C treatment increases bifidoprophage excision.

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Excision and circularization of the predicted prophages were examined by PCR. a, Primers

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(arrows) were designed in all predicted prophages so that they are divergent in the integrated

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genome orientation but convergent in the excised and circularized genome to amplify across the

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attP. b, Prophage induction was tested in several strains by PCR amplification of filtered culture

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supernatants (F.S.) treated (+) or not treated (-) with mitomycin C. Three to four replicates were

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tested per strain. For each prophage of interest, a no template control (NTC) and several

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unfiltered saturated cultures (S.C.) were included as negative and positive controls. The full

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length of each lane from the loading well to leading edge is displayed. A star (*) indicates the

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expected band size corresponding to attP amplification.

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Supplementary Figure S2. Mitomycin C treatment increases dnaJ2-integrated

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bifidoprophage copy number. DNA from mitomycin C-treated culture supernatants was

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sequenced for several B. breve strains and reads were mapped to the lysogen genome (black

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line). Enlarged view of the integrated prophage (white box) locus in each strain highlights the

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increased sequencing coverage of the prophage relative to the host genome.

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Supplementary Figure S3. Flow cytometry calibration and gating strategy. a, FACSCalibur

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settings were calibrated using mitomycin C-treated L. lactis non-lysogen (strain UC509.9) and

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lysogen (strain NZ9000 with TP901-1 prophage) samples. Scatterplots comparing forward

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scatter (FSC-H) to (bottom) side scatter (SSC-H) and (top) Syto9 fluorescence (FL1-H) were

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adjusted to reproduce previously described results41. b, Flow cytometry of several negative

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controls, plotted as in panel a, to identify different types of events to gate. Samples include flow

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sample buffer (¼ strength Ringer’s solution), flow sample buffer with reference microsphere



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beads (¼ strength Ringer’s solution + Beads), and growth medium processed with the entire

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protocol (RCM). c, Flow cytometry of common bifidobacterial growth media (MRS, MMRS +

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Glucose, TOS), plotted as in panel b. d, Boxplots of individual parameters (FSC-H, SSC-H,

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FL1-H) from flow cytometry results for several strain-free controls are used to define boundaries

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of each parameter for debris (beige) and bead (blue) events. Some samples have been treated

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(+) or not treated (-) with mitomycin C (MmC), Syto9 stain, and beads. e, Boundaries defined in

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panel d were used to create three-dimensional debris and bead gates. The gating strategy for

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all flow cytometry samples utilizes these two gates for removal of debris events followed by

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removal of bead events. All non-debris and non-bead “gated” events are used for downstream

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analysis to assess levels of prophage induction.

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Supplementary Figure S4. Flow cytometric analysis of mitomycin C-treated

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bifidobacterial samples. Gated events from one representative replicate of each sample type

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are plotted. Scatterplots of FSC-H and SSC-H (left) and density plots of FL1-H (right) comparing

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events either between mitomycin C-treated L. lactis non-lysogenic UC509.9 (blue) and

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lysogenic NZ9000(TP901-1) (red) strain samples or between mitomycin C-treated (red) and

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untreated (blue) bifidobacterial growth medium (RCM) and strain samples.

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Supplementary Figure S5. Mitomycin C induced changes in supernatant composition. a,

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For all gated events from each replicate set of paired mitomycin C treated (red) and untreated

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(blue) samples (from Supplementary Fig. S4), (top) barplot of the proportion of total events and

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(bottom) boxplots of event fluorescence (FL1-H) highlight changes in supernatant composition.

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Replicate sets are numbered. Individual strain names are indicated along with whether they are

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non-lysogens, lysogens, or predicted lysogens. RCM = growth medium with no cell culture. L.

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lactis untreated = mitomycin C-treated non-lysogen (UC509.9); L. lactis treated = mitomycin C-



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treated lysogen (NZ9000 with TP901-1 prophage). b, Boxplots display the fold changes in the

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(top) abundance and (bottom) median fluorescence of events from paired samples in panel a.

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Supplementary Figure S6. Bifidoprophages contain phase variation systems. a, Enlarged

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view of the left arm genes of dnaJ2-integrated prophages from Fig. 1a highlights the genomic

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context of the Rin shufflon. Genes are colored according to pham designation, and any putative

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functions are listed (TMP = tape measure protein; DIT = distal tail protein; RBP = receptor

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binding protein). The color spectrum between genomes is the same as in Fig. 1a. b, Enlarged

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view of the left arm genes of tRNAMet-integrated phages from Fig. 1c highlights a putative phase

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variation system in these genomes.

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Supplementary Figure S7. Bb423phi1 induced phage genomes harbor multiple Rin

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shufflon variants. a, Three contigs (numbered by size) representing the entire phage genome

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are assembled by Newbler, but a 100% consensus of the complete genome is not achieved.

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Contigs can be connected in multiple arrangements due to reads mapping across more than

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one contig, and these discrepant reads occur near or within the RBP locus (colors and gene

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numbering as in Fig. 5a). b, One possible contig orientation involves reads that straddle the

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three contigs (dashed lines) with approximately equal coverage. c, Other contig orientations are

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possible, but they are represented by much lower read coverage and they do not obviously

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assemble into a single alternative genome. d, Two sequential inversion events (double arrows)

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at rix sites result in three shufflon variants that sufficiently account for all hybrid sequence reads.

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Supplementary Figure S8. Bb423phi1 uninduced prophage genomes harbor multiple Rin

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shufflon variants. Analysis of the previously reported B. breve 139W4-23 genome sequencing

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reads18 reveals three variant orientations of the Bb423phi1 prophage Rin shufflon. (Left) The

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variant nucleotide sequence orientations were assembled and all reads in the sample were



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mapped to each variant. The genome map below the histogram and the coordinates above the

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histogram reflect the predominant variant in the published genome. The points of inversion in

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each variant are indicated below (double arrows and coordinates). Average coverage across

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each variant is indicated by the dotted line, and the percentage of all reads in the sample that

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map to the variant orientation is indicated. (Right) Dotplot sequence comparison of each variant

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to the published prophage locus orientation highlights the points of inversion.

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Supplementary Figure S9. dnaJ2-integrated prophages exhibit high gene content flux.

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Pairwise comparisons (black circles) of nucleotide sequence and gene content between dnaJ2-

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integrated phages and all other actinobacteriophages as previously described53 to highlight

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gene content flux patterns, with high (HGCF, blue) and low (LGCF, green) gene content flux

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regions indicated.

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a

b

Supplementary Figure S1

230

139W4-23

0 80

Bb423phi1

082W4-8

0

60

Bb48phi1

180W8-3

0 Bb83phi1

40

017W4-39

0 30

Bb439phi1

215W4-47a

0 Bb447phi1

30

0

139W4-23

Bb423 phi2

20kb Supplementary Figure S2

a UC509.9

NZ9000 (TP901-1)

FSC-H

FSC-H

d 104 103 102 101 1 104 103 102 101 1 104 103 102 101 1 Syto9 MmC Beads -

b Ringer’s

Ringer’s + Beads

RCM

Sample

-

-

H2O

+ -

+

+ +

Ringer’s

e FSC-H

FSC-H

All

FSC-H

debris gate

c MRS

MMRS+G

TOS

non-Debris

Debris

bead gate non-Beads (“gated”)

Beads

downstream analysis FSC-H

FSC-H

FSC-H

Supplementary Figure S3

L. lactis

LMG 10510

RCM

DSM 27321

JCM 7017

082W4-8

NCIMB 702258

180W8-3

UCC2003

139W4-23

FSC-H

FL1-H

017W4-39

215W4-47a

FSC-H

FL1-H

Supplementary Figure S4

a 0.25 0.20 0.15 0.10 0.05 0 104 103 102 101

treated untreated

1 1 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 4 1 2 34 1 2 3 4 1 2 3 1 2 3 4

b

Supplementary Figure S5

a

2 kb Binf-1

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15

1135 1134

lysin

RBP DIT

1136

TMP

Bb48phi1

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1

999 998

lysin

RBP DIT

10

TMP

Bb83phi1

14

999

1 1000

lysin holin

RBP DIT

TMP

Bb439phi1

14

1014

1 1015

lysin holin

RBP DIT

TMP

14

Bb423phi1

15

1126 1125

holin lysin

112

TMP

14

Bl30698phi1

1159

115 1158

holin lysin

TMP

11

Bl157phi1 Rin

TMP

2 kb 689b-1 int

Bb423phi2

106 1069

holin lysin

b

12

1070

lysin

tail protein

portal

capsid

lysin

tail protein

portal

capsid

int inversion locus

Supplementary Figure S6

a contig 1 (38,499 bp)

Rc

rin

1117 nterm.

1112

Rv2 contig 2 1116 (1,255 bp) contig 3 (326 bp)

Rv3 1115

Rv4

Rv5

1114

1113

Rv1 1117

b

160x

1112

207x

1117

156x

1116

1115

1114

1113

1117 nterm.

1114

1113

1117 nterm.

7x

c

6x

1112

1117

1116

1115

12x 12x

d Variant 1

1

1112

1113

1114

1115

1116

1117

1117 nterm.

Variant 2

2

1112

1117 nterm.

1112

1117 nterm.

Variant 3

3

Supplementary Figure S7

1112

1113

1114

1115

1116

1117

1117 nterm.

1112

1113

1114

1115

1116

1117

1117 nterm.

1112

1113

1114

1115

1116

1117

1117 nterm.

Supplementary Figure S8

1.0 0.8 HGCF 0.6 0.4 0.2 0.0

LGCF 0.0 0.1 0.2 0.3 0.4 0.5 nucleotide distance

Supplementary Figure S9

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