1

Supplementary material

TABLE S1 Sequencing and analysis of genomes of strain PAO1 and KPP22-resistant PAO1

Genomic

DNA

Data obtained by Illumina

sequencing technology Genome mapping analysis

No. of reads

(read)

Total read

length (base)

No. of

mapped

reads (read)

Mapped read

length (base)

Mapped

rate

Average

sequence

depth

PAO1

5,831,268 874,690,200

5,168,992 775,348,800 88.6% 123.8

PAO1R1

7,004,078 1,050,611,700

6,112,638 916,895,700 87.3% 146.4

PAO1R2

452,412 67,861,800

389,731 58,459,650 86.1% 9.3

PAO1R3 2,001,536 300,230,400 1,796,004 269,400,600 89.7% 43.0

2

Supplementary material

TABLE S2 Annotation table of the phage KPP22 genome TABLE S2-p.1

ORF Directiona

Location (nt) Length of protein

(aa)

Identified as virion protein in this studyb

Bioinfomatical analysis

Function Start End InterProScan

Conserved domain search

ORF001 + 720 1121 133

Hypothetical protein ORF002 + 1153 1476 107

Hypothetical protein

ORF003 + 1473 1676 67

Hypothetical protein ORF004 + 1682 1993 103

Hypothetical protein

ORF005 + 2242 2589 115

Hypothetical protein

ORF006 + 2693 3625 310

NADH dehydrogenase

subunit C; Validated

Hypothetical protein

ORF007 + 3727 4314 195

Hypothetical protein ORF008 + 4331 4768 145

Hypothetical protein

ORF009 + 4855 5634 259

Protein of unknown function

(DUF2786)

Protein of unknown function

(DUF2786) Hypothetical protein

ORF010 + 5637 6035 132

Hypothetical protein ORF011 + 6080 6412 110

Hypothetical protein

ORF012 + 6412 6630 72

Hypothetical protein ORF013 + 6627 7010 127

Hypothetical protein

ORF014 - 7047 8429 460

Phage terminase

large subunit

Phage terminase large subunit

Putative phage terminase

ORF015 + 8597 8791 64

Hypothetical protein ORF016 + 8837 8998 53

Hypothetical protein

ORF017 + 9194 9445 83

Hypothetical protein ORF018 + 9442 9642 66

Hypothetical protein

ORF019 + 9639 9854 71

Hypothetical protein ORF020 + 9851 10051 66

Hypothetical protein

ORF021 + 10048 10260 70

Hypothetical protein

ORF022 + 10292 10936 214

FGAM-synthase; Provisional

Hypothetical protein

ORF023 + 11328 11552 74

Hypothetical protein ORF024 + 11622 11861 79

Hypothetical protein

ORF025 + 11871 12092 73

Hypothetical protein ORF026 + 12142 12453 103

Hypothetical protein

ORF027 + 12463 13068 201

Hypothetical protein ORF028 + 13258 13872 204

Hypothetical protein

ORF029 - 14042 14611 189

Hypothetical protein

ORF030 - 15563 17206 547

PF05272 Virulence-associated protein

E

Virulence-associated protein E

Hypothetical protein

ORF041 + 25242 25460 72 Hypothetical protein ORF042 + 25444 25662 72 Hypothetical protein ORF043 + 25695 25919 74 Hypothetical protein ORF044 + 25927 26133 68 Hypothetical protein

ORF045 + 26133 27050 305

Flavin- dependent

thymidylate synthase

(thyX) domain profile

FAD-dependent thymidylate

synthase

Putative thymidylate synthase

ORF046 + 27052 27243 63

Hypothetical protein

ORF047 + 27246 28286 346

SUPERFAMILY SSF56784

HAD superfamily Hypothetical protein

ORF048 + 28362 28916 184

Exonuclease, RNase T/DNA polymerase III

DnaQ-like exonuclease superfamily

Putative DnaQ

a "+" and "-" indicate forward and reverse directions, respectively, of the orf. b V, virion protein identified by LC–MS/MS (see Table S4 in the supplementary material); MVP, major virion protein identified by N-terminal protein sequencing (see Fig. S1 in the supplementary material).

3

Supplementary material

TABLE S2 Annotation table of the phage KPP22 genome TABLE S2-p.2

ORF Directiona

Location (nt) Length of

protein

(aa)

Identified as

virion protein

in this studyb

Bioinfomatical analysis

Function Start End InterProScan

Conserved

domain search

ORF049 + 28916 32023 1035

Bacterial DNA

polymerase III alpha subunit

Bacterial DNA polymerase III alpha subunit

Putative DNA polymerase III alpha

subunit

ORF050 + 32016 32426 136

Hypothetical protein

ORF051 + 32423 33982 519

Helicase conserved C-terminal

domain

Superfamily II DNA or RNA

helicase Putative helicase

ORF052 + 34077 34697 206

Hypothetical protein

ORF053 + 34786 35685 299

Hypothetical protein

ORF054 + 35740 36345 201

Type I restriction enzyme EcoKI

subunit R Hypothetical protein

ORF055 + 36342 36896 184

Phosphoribosyl-ATP

pyrophosphohydrolase

Nucleoside Triphosphate

Pyrophosphohydrolase

Hypothetical protein

ORF056 + 36951 37862 303

ATP dependent

DNA ligase domain

Adenylation_DNA_ligase_like;

OBF_DNA_ligase_family

Putative ligase

ORF057 + 38142 38393 83

Regulatory vacuolar ATP

synthase subunit H

Hypothetical protein

ORF058 - 38418 39080 220

Glycoside hydrolase, family 19, catalytic

Lysozyme_like domain

Putative endolysin

ORF059 - 39080 39508 142

Hypothetical protein

ORF060 - 39510 42404 964 V

Virion protein

ORF061 - 42409 43923 504 V

Protein of unknown function

(DUF2612)

Protein of unknown function

(DUF2612) Virion protein

ORF062 - 43920 45173 417 V

Uncharacterized phage protein

gp47/JayE Virion protein

ORF063 - 45230 45793 187 V

Bacteriophage Mu Gp45 protein

Virion protein

ORF064 - 45951 46484 177

Hypothetical protein

ORF065 - 46484 47314 276

Hypothetical protein

ORF066 - 47347 49923 858 V Prokaryotic

transglycosylases signature

Lytic Transglycosylase (LT) and Goose

Egg White Lysozyme

(GEWL) domain

Virion protein

ORF067 - 49927 50355 142

Hypothetical protein

ORF068 - 50365 50958 197

Hypothetical protein

ORF069 - 50967 51506 179

Hypothetical protein

ORF070 - 51506 52009 167 Hypothetical protein a "+" and "-" indicate forward and reverse directions, respectively, of the orf. b V, virion protein identified by LC–MS/MS (see Table S4 in the supplementary material); MVP, major virion protein identified by N-terminal protein sequencing (see Fig. S1 in the supplementary material).

4

Supplementary material

TABLE S2 Annotation table of the phage KPP22 genome TABLE S2-p.3

ORF Directiona

Location (nt) Length of

protein

(aa)

Identified as

virion protein

in this studyb

Bioinfomatical analysis

Function Start End InterProScan

Conserved

domain search

ORF071 - 52019 52450 143 V Prolipoprotein diacylglyceryl

transferase Virion protein

ORF072 - 52452 52802 116 G3DSA:3.10.

450.40 Hypothetical protein

ORF073 - 52799 53122 107

Hypothetical protein

ORF074 - 53122 53574 150 V

Virion protein

ORF075 - 53633 55147 504 V

Protein of

unknown

function

(DUF3383)

Protein of

unknown function

(DUF3383)

Virion protein

ORF076 - 55163 55744 193

Hypothetical protein

ORF077 - 55741 56292 183

Hypothetical protein

ORF078 - 56300 56698 132 V

hypothetical

protein Virion protein

ORF079 - 56695 57162 155 V

Protein of

unknown

function

(DUF4054)

Protein of

unknown function

(DUF4054)

Virion protein

ORF080 - 57177 57614 145

Hypothetical protein

ORF081 - 57716 58864 382 V, MVP

Major virion protein

ORF082 - 58874 59509 211 V

Virion protein

ORF083 - 59513 60907 464 V

Uncharacteriz

ed protein

conserved in

bacteria

(DUF2213)

Uncharacterized

protein conserved

in bacteria

(DUF2213)

Virion protein

ORF084 - 61596 61802 68 V

4-aminobutyrate

transaminase Virion protein

ORF085 - 61821 62657 278

Phage Mu

protein F like

protein

Phage Mu protein

F like protein Hypothetical protein

ORF086 - 62657 539 765 V

Protein of

unknown

function

(DUF1073)

Protein of

unknown function

(DUF1073)

Virion protein

a "+" and "-" indicate forward and reverse directions, respectively, of the orf. b V, virion protein identified by LC–MS/MS (see Table S4 in the supplementary material); MVP, major virion protein identified by N-terminal protein sequencing (see Fig. S1 in the supplementary material).

5

Supplementary material

TABLE S3 Analysis of phage KPP22 genome sequecne by BLASTn at the NCBI

Phage genome

BLASTn

Max

score

Total

score

Query

cover

E

value Ident Accession

Pseudomonas phage KPP12 75030 104900 98% 0 97% AB560486

Pseudomonas phage

vB_PaeM_PAO1_Ab27 74608 105900 98% 0 97% LN610579

Pseudomonas phage

vB_PaeM_CEB_DP1 73741 104900 98% 0 97% KR869157

Pseudomonas phage SN 72228 100500 98% 0 96% FM887021

Pseudomonas phage DL68 72049 100400 97% 0 96% KR054033

Pseudomonas phage NH-4 71030 103300 99% 0 95% JN254800

Pseudomonas phage

vB_Pae_PS44 69229 101100 98% 0 94% KM434184

Pseudomonas phage LBL3 67057 94578 98% 0 93% FM201281

Bacteriophage F8 61956 85597 98% 0 91% DQ163917

Pseudomonas phage DL60 59177 82254 98% 0 89% KR054030

Pseudomonas phage

vB_PaeM_C1-14_Ab28 58980 83295 98% 0 89% LN610589

Pseudomonas phage PB1 58814 83678 97% 0 90% EU716414

Pseudomonas phage DL52 58205 82821 98% 0 89% KR054028

Pseudomonas phage LMA2 53240 104900 98% 0 97% FM201282

Pseudomonas phage SPM-1 51361 85407 97% 0 91% KF981875

Pseudomonas phage

vB_PaeM_PAO1_Ab29 49950 107900 99% 0 99% LN610588

Pseudomonas phage JG024 39107 98847 97% 0 96% GU815091

Pseudomonas phage 14-1 38930 99359 97% 0 96% FM897211

6

Supplementary material

TABEL S4 Shotgun protein analysis of KPP22 virion proteins by LC–MS/MS

Na

ProtScore Sequence coverage

(%)

No. of peptides with

95% confidence

Identified ORF of

phage KPP22 Unused Total

1 32.81 32.81 51.3 41 ORF081

2 19.58 19.58 29.2 11 ORF060

3 16.27 16.27 44.1 29 ORF082

4 13.7 13.7 17.5 12 ORF086

5 12 12 29.3 11 ORF062

6 10.11 10.11 19.4 9 ORF066

7 10 10 20.6 11 ORF075

8 10 10 27.3 6 ORF071

9 7.1 7.1 26.8 6 ORF083

10 5.35 5.35 12.3 3 ORF061

11 5.24 5.24 42.7 6 ORF074

12 4 4 18.7 3 ORF079

13 2 2 16.2 4 ORF084

14 2 2 6.4 2 ORF063

15 1.4 1.4 9.1 1 ORF078

a MS/MS data were analyzed using ProteinPilot (AB Sciex).

7

Supplementary material

TABLE S5 In silico function prediction of ORFs 060, 065 and 086 in phage KPP22

ORF

BLASTp

InterProScana

HHpred

ORF060

Some ORFs in the PB1-like phages are

assumed to be tail fiber proteins. N/D

Similar to short and long tail

fiber of phage T4

ORF065

Homologous ORFs in the PB1-like

phages are assumed to be putative

structural proteins.

N/D

Similar to bacteriophage MU,

baseplate

ORF086

Homologous ORFs in the PB1-like

phages are assumed to be putative minor

head proteins.

Protein of

unknown function

DUF1073

Similar to HK97 family phage

portal protein

a N/D, not detected.

8

Supplementary material

FIG. S1 Phage virion protein analysis using SDS–PAGE. (A) Image of SDS–PAGE of virion

proteins of phage KPP22. The virion proteins of phage KPP22 together with an (unstained)

XL-Ladder Broad marker (APRO Life Science Institute, Tokushima, Japan) were separated in

a 12.5% SDS–PAGE gel, and were stained with Coomassie brilliant blue R-250. In the left

column, a molecular standard is shown. An arrow indicates the major virion protein subjected

to N-terminal amino acid analysis by the Edam degradation method. The protein sequence

was SNFTAPVTTPSIPTPIQF. (B) Location of N-terminal amino acid sequence of major

virion protein, ORF081. The protein sequence was matched from amino acids 65 to 82 of

ORF081, which is shown in red. The result suggested that the N-terminal portion of the major

virion protein was post-translationally cleaved.

9

Supplementary material

FIG. S2-p.1

10

Supplementary material

FIG. S2-p.2

FIG. S2 Comparison of (A) ORF060 of phage KPP22 with ORF43 of phage KPP12, (B)

ORF065 with ORF38 of phage KPP12, and (C) ORF086 of phage KPP22 with ORF17 of

phage KPP12. The protein sequences were compared by BLASTp. Letters in red in the phage

KPP22 are the amino acid changes that occurred due to the missense mutation in the KPP22

mutant phages.

11

Supplementary material

FIG. S3 Western blot analysis of various P. aeruginosa strains to detect O5 antigen. Bands

were detected in the lanes for strains PA4, PA10, PA14, PA15, PA16, PA23, PA27, PA28,

PA29 and PAO1.