A cell wall‐associated polysaccharide is required for bacteriophage adsorption to the Streptococcus thermophilus cell surface

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A cell wall-associated polysaccharide is required for bacteriophage

adsorption to the Streptococcus thermophilus cell surface

Brian McDonnell

a

, Laurens Hanemaaijer

b

, Francesca Bottacini

a

, Philip Kelleher

a

, Katherine

Lavelle

a

, Irina Sadovskaya

c

, Evgeny Vinogradov

d

, Emiel Ver Loren van Themaat

b

, Thijs

Kouwen

b

_{, Jennifer Mahony}

a

_{and Douwe van Sinderen}

a†

_.

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Supplementary Figure S1. Differences in wild-type and mutant cell pellets formed after overnight incubation. Clockwise from top left: ST64987 WT; BIM3; BIM8, BIM4.

Supplementary Table S1. Efficiencies of plaquing (EOP) of phages 9871-4 on S. thermophilus parent strain ST64987 and its derived BIMs. 1_{Visible plaques appeared consistently less defined and smaller than those}

observed on the WT. Strain/BIM EOP 9871 9872 9873 9874 ST64987 WT 1 1 1 1 BIM3 ≤ 3.3 x 10-8 _{≤ 7.7 x 10}-8 _{≤ 7.6 x10}-8 _{≤ 4.3 x 10}-8 BIM4 ≤ 9.5 x 10-9 _{≤ 1.3 x 10}-10_{≤ 9.0 x 10}-9 _{≤ 1.3 x 10}-9 BIM8 ≤ 2.8 x 10-9 1_{1.27 x 10}-6 _{≤ 3.2 x 10}-9 _{≤ 6.9 x 10}-8

Supplementary Table S2. Size, direct repeat sequence, and spacer number of deduced CRISPR loci of parent S.

thermophilus strain ST64987 and derived BIMs.

Strain CRISPR1 (bp) Spacers CRISPR2 (bp) Spacers CRISPR3 (bp) Spacers

ST64987 WT 1952 29 843 11 1289 19 BIM3 1952 29 843 11 1289 19 BIM3::pNZ44 1952 29 843 11 1289 19 BIM3::pCOMP1 1489 22 843 11 1289 19 BIM3 Cured 1489 22 843 11 1289 19 BIM4 1952 29 843 11 1289 19 BIM4::pNZ44 1952 29 843 11 1289 19 BIM4::pCOMP2 1952 29 843 11 1289 19 BIM4 Cured 2018 30 843 11 1289 19 BIM8 1952 29 843 11 1289 19 BIM8::pNZ44 1952 29 843 11 1289 19 BIM8::pCOMP2 1952 29 843 11 1289 19 BIM8 Cured 1952 29 843 11 1289 19

Supplementary Table S3. A list of primers applied in this study. Restriction sites, where included, are indicated by an underline.

Primer name Sequence (5’ - 3’) Target Source

Plasmid constructs

pNZ44F CTAATGTCACTAACCTGCCCCG pNZ44 MCS This study

pNZ44R GCTTTATCAACTGCTGCT pNZ44 MCS “ 987o969F AGCAGCCTGCAGAGGAGGTAAT TATCATGAATAAAATAACCATG ACA ST64987 orf969 “ 987o969R AGCAGCTCTAGACTATGCCACCT TTTCTAT ST64987 orf969 “

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3 Acr907F AGCAGCTCTAGAAGGAGGTAAAC AAAATGAAAATAAATGACGACAT CAAAGAG S. thermophilus phage ST907-9 orf19/predicted AcrⅡa6 homolog UCC phage collection Acr907R AGCAGCAAGCTTTTATCGAATGTT TTCCAAG “ 987o968F AGCAGCCCATGGAGGAGAAGGAA AAAATATAATGGTTAAATTTTCTA TAATT

ST64987 orf968 This study

987o968R AGCAGCCTGCAGCTATTTGATTTT

CTTACC

ST64987 orf968 “

CRISPR loci

CRISPR1F TGCTGAGACAACCTAGTCTCTC CRISPR1 repeat/spacer loci (Horvath et

al., 2008)

CRISPR1R TAAACAGAGCCTCCCTATCC “ “

987C1F2 CCTAGTGACAATCTGTTCGC “ This study

CRISPR2F TTAGCCCCTACCATAGTGCTG CRISPR2 repeat/spacer loci (Horvath et

al., 2008)

CRISPR2R TTAGTCTAACACTTTCTGGAAGC “ “

CRISPR3F CTGAGATTAATAGTGCGATTACG CRISPR3 repeat/spacer loci “

CRISPR3R GCTGGATATTCGTATAACATGTC “ “

987C3F2 GCAACAGAAAGCCCACCGA “ This study

Supplementary Table S4. SNPs identified in derived BIMs of ST64987.

BIM Pos. SNP Location (by function of encoded product)

BIM3 133104 gt → gTt Intergenic

" 134160 taaa → tAaaa Intergenic

" 941782 G → A EpsP (orf0969)

" 1084280 G → A SAM-dependent methyltransferase

" 133313 attt → aTttt Intergenic

" 133957 gaaaaaaa → gaaaaaa Intergenic

" 940181 caaaa → caaa Eps7G (orf0968)

" 133957 gaaaaaaa → gaaaaaa Intergenic

" 940209 T → C Eps7G (orf0968)

Supplementary Table S5. NMR data for S. thermophilus ST64987 deaminated RGP (DPS, 600 MHz, 25 °C,  ppm). Sugar H/C 1 H/C 2 H/C 3 H/C 4 H/C 5 H/C 6 -Glc A H 5.27 3.69 3.90 3.53 4.13 3.73; 3.96 C 95.8 76.2 72.3 70.5 71.9 67.6 -RhaB H 5.20 4.13 3.91 3.55 3.78 1.31 C 100.4 77.6 70.6 73.3 70.7 17.9 -Rha C H 5.09 4.01 3.93 3.49 3.75 1.31 C 97.2 80.1 71.0 73.4 70.0 17.9 -Glc D H 4.94 3.57 3.78 3.46 3.92 3.79; 3.85

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C 100.2 72.5 74.1 70.6 73.0 61.5

Supplementary Table S6. NMR data for S. thermophilus ST64987 RGP deamination products, OS1 and OS2 (600 MHz, 25 °C,  ppm). Sugar H/C 1 H/C 2 H/C 3 H/C 4 H/C 5 H/C 6 anhMan X H 5.10 3.84 4.22 4.18 3.95 3.68; 3.76 C 90.7 84.7 85.0 76.6 84.3 61.8 -Rha E H 5.18 4.05 3.89 3.59 3.84 1.31 C 97.4 75.9 70.7 73.1 70.5 17.6 -Rha H H 5.09 4.02 3.89 3.54 3.84 1.31 C 98.2 77.0 70.7 73.2 70.5 17.6 -Gal B H 5.31 3.96 4.09 4.04 4.17 3.73; 3.73 C 94.7 73.1 68.8 70.3 72.2 62.1 -Gal G H 5.03 3.82 3.94 4.02 4.25 3.73; 3.73 C 98.8 69.4 70.3 70.3 72.1 62.1 -Glc F H 5.08 3.57 3.78 3.44 3.88 3.77; 3.86 C 96.7 72.6 74.1 70.6 73.0 61.7

Supplementary Table S7. NMR data for S. thermophilus ST64987 N-deacetylated RGP (600 MHz, 25 °C,  ppm). Sugar H/C 1 H/C 2 H/C 3 H/C 4 H/C 5 H/C 6 -Glc A H 5.25 3.65 3.87 3.68 4.22 3.83; 3.91 C 96.2 76.3 72.4 69.7 71.4 67.1 -Glc A' H 5.27 3.68 3.91 3.54 4.12 3.73; 3.98 C 96.2 76.3 72.4 70.5 71.5 67.6 -Glc C H 5.08 3.57 3.77 3.46 3.92 3.79; 3.85 C 97.3 72.4 74.1 70.5 73.0 61.5 -Rha B H 5.18 4.34 4.00 3.68 3.80 1.32 C 100.7 77.9 79.9 72.6 70.7 17.8 -Rha B' H 5.20 4.20 3.91 3.56 3.85 1.30 C 100.4 76.6 71.2 72.5 70.7 17.8 -Rha D H 4.92 4.04 3.91 3.47 3.75 1.34 C 100.7 79.5 71.1 73.4 70.0 18.2 -Rha D' H 4.93 4.03 3.91 3.47 3.75 1.34 C 100.1 79.5 71.1 73.4 70.0 18.2 -Rha E H 5.23 4.20 3.88 3.59 4.01 1.27 C 99.5 76.6 71.0 72.5 70.4 17.7 -Rha E' H 5.13 4.18 3.89 3.55 4.01 1.28 C 100.1 77.3 71.0 72.5 70.4 17.7 -Rha E" H 4.99 4.11 3.79 3.47 3.99 1.25 C 102.8 71.5 71.4 73.4 70.4 -Gal G H 5.35 3.97 4.10 4.05 4.20 3.75; 3.75 C 95.1 73.3 68.8 70.4 72.4 62.1 -Gal G' H 5.08 3.84 3.94 4.02 4.26 3.74; 3.74 C 98.8 69.4 70.3 70.4 72.2 62.1 -Glc F H 5.10 3.57 3.78 3.44 3.90 3.79; 3.85 C 97.2 72.4 74.1 70.6 73.0 61.5 -GlcN X H 4.60 2.64 3.53 3.45 3.46 3.74; 3.96

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C 106.0 58.5 86.2 69.9 77.0 62.1

Supplementary Table S8. NMR data for S. thermophilus ST64987 EPS (600 MHz, 30 °C). Residue E corresponds to a terminal -Glc, probably end of chain.

Unit H/C-1 H/C-2 H/C-3 H/C-4 H/C-5 H/C-6a;b -Glc A H 4.91 3.60 3.85 3.79 4.40 3.99; 4.16 C 100.9 72.6 72.4 79.0 70.7 68.3 -Glc B H 4.62 3.37 3.66 3.67 3.60 3.84; 3.99 C 103.3 74.0 75.4 79.6 75.9 61.2 -Glc C H 4.53 3.37 3.66 3.67 3.60 3.82; 3.98 C 103.5 74.0 75.4 79.6 75.9 61.2 -Gal D H 4.50 3.58 3.76 4.02 3.78 3.77; 3.77 C 103.6 71.9 73.1 78.5 76.5 62.2 -Glc E H 4.51 3.33 3.51 3.40 3.46 3.73; 3.92 C 104.4 74.1 76.9 70.8 76.9 61.8 -Gal F H 4.45 3.55 3.67 3.93 3.73 3.77; 3.77 C 104.2 72.1 73.7 69.7 76.5 62.2