Genomic insights into high exopolysaccharide-producing dairy starter. bacterium Streptococcus thermophilus ASCC 1275

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1 Supplementary information of Genomic insights into high exopolysaccharide-producing dairy starter bacterium Streptococcus thermophilus ASCC 1275 Qinglong Wu, Hein Min Tun, Frederick Chi-Ching Leung, Nagendra P. Shah* Food and Nutritional Science, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong Correspondence should be addressed to Professor Nagendra P. Shah

2 Supplementary information Supplementary Table 1. Carbohydrate utilization system in S. thermophilus ASCC 1275 Supplementary Table 2. Proteolytic system in S. thermophilus ASCC 1275 Supplementary Table 3. Two-component regulatory system (TCRS) in S. thermophilus ASCC 1275 Supplementary Table 4. Stress response systems in S. thermophilus ASCC 1275 Supplementary Table 5. Defense systems in S. thermophilus ASCC 1275 Supplementary Table 6. EPS production from selected S. thermophilus strains under identical cultivation conditions from previous reports.

3 Supplementary Table 1. Carbohydrate utilization system in S. thermophilus ASCC 1275 Encoded protein Phosphotransferase system (PTS) PTS sucrose transporter subunit IIABC PTS mannose transporter subunit IIAB PTS mannose transporter subunit IIC PTS mannose transporter subunit IID PTS fructose transporter subunit IIA PTS fructose transporter subunit IIC PTS glucose transporter subunit IIABC ABC transporter Sugar ABC transporter ATP-binding protein Sugar ABC transporter permease Permease Glycerol uptake permease Lactose/galactose permease Glucose/ribose permease Hydrolase α-amylase Glycogen debranching protein Alkaline amylopullulanase Dextranase 1,6-α-Glucanhydrolase β-galactosidase, LacZ 6-Phospho-β-glucosidase Locus tag(s) T303_00375 (truncated), T303_09505 T303_02805 T303_02800 T303_02795 T303_07665 T303_03160 (truncated) T303_02185 (truncated) T303_04995 T303_05270 T303_09175 T303_07870 T303_05455 T303_00360, T303_08530 T303_07940, T303_07945, T303_07950 T303_06145, T303_06150 T303_01995 (truncated) T303_02000 T303_07865 T303_06310 (truncated), T303_08980

4 Supplementary Table 2. Proteolytic system in S. thermophilus ASCC 1275 Encoded protein Peptidase Peptidase Oligopeptidase PepB Aminopeptidase Methionine aminopeptidase X-prolyl-dipeptidyl aminopeptidase Glutamyl aminopeptidases Endopeptidase O-sialoglycoprotein endopeptidase Oligoendopeptidase F Metalloendopeptidase Dipeptidase xaa-pro dipeptidase Protease Protease ATP-dependent protease ATP-dependent Zn protease C3-degrading proteinase Serine protease Cell envelope serine proteinase CAAX amino terminal protease Glycoprotease Metalloprotease Zinc metalloprotease Transporter Amino acid ABC transporter ATP-binding protein Amino acid ABC transporter substrate-binding protein Locus tag(s) T303_02160, T303_03980, T303_09195, T303_09070, T303_04605, T303_04830, T303_05365 (truncated), T303_02665 (truncated), T303_03905, T303_06540, T303_01015, T303_01020, T303_01610 (truncated) T303_03370 T303_01570, T303_06060, T303_02345 T303_08615 T303_09180 T303_00190 T303_00350 T303_02660 T303_07535 (truncated) T303_07905 T303_04315, T303_06485 T303_02965 T303_04610, T303_08490 (truncated) T303_04060 T303_07690 T303_09165 T303_01085, T303_05340, T303_06285 T303_05205 T303_00200, T303_00205, T303_00210, T303_09170 T303_09680 T303_02220, T303_04255 T303_07910 T303_09095 T303_02630, T303_02655, T303_08290, T303_08300, T303_08305, T303_08720, T303_09105

5 Amino acid ABC transporter permease Polar amino acid ABC transporter permease Amino acid ABC transporter ATPase Amino acid transporter Amino acid permease Amino acid specific permease Branched-chain amino acid ABC transporter ATPbinding protein Branched-chain amino acid ABC transporter substrate-binding protein Branched-chain amino acid ABC transporter permease Branched-chain amino acid transporter Peptide ABC transporter ATP-binding protein Peptide ABC transporter substrate-binding protein Peptide ABC transporter permease Glutamine ABC transporter substrate-binding protein Glutamine ABC transporter permease Glutamine ABC transporter ATP-binding protein Spermidine/putrescine ABC transporter substratebinding protein Spermidine/purescine ABC transporter permease Spermidine/putrescine ABC transporter ATPbinding protein Arginine ABC transporter ATP-binding protein Methionine ABC transporter ATP-binding protein Methionine ABC transporter permease Serine/threonine transporter SstT Threonine transporter RhtB T303_05525, T303_05920 (truncated), T303_08335, T303_08730, T303_09100, T303_08735 T303_02950, T303_05530 T303_03645, T303_04070 T303_07640 T303_08545 T303_02945 T303_02930, T303_07625 T303_02940, T303_05265, T303_06795, T303_09675 T303_09670 T303_02050, T303_02130, T303_02140 (truncated), T303_02555 (truncated), T303_03795, T303_04185, T303_07775, T303_08070, T303_08075, T303_08090, T303_08100, T303_08340 T303_00350, T303_06800 T303_02125, T303_05915, T303_07580, T303_08080, T303_08085 T303_02055, T303_05535 (truncated), T303_06655 T303_04180, T303_06660, T303_06665 T303_08725 T303_05195, T303_08495 T303_05190, T303_08500, T303_08505 T303_08510, T303_09150 T303_06650 T303_02670 T303_02675 T303_02680 T303_07750

6 Supplementary Table 3. Two-component regulatory system (TCRS) in S. thermophilus ASCC 1275 Number Encoded protein Locus tag(s) TCRS_1 Histidine kinase T303_01140 TCRS_2 Sensor histidine kinase T303_02735 Transcriptional regulator T303_02730, T303_02740 TCRS_3 Histidine kinase T303_03290 (truncated) TCRS_4 Response regulator T303_03855 Sensor histidine kinase T303_03850 TCRS_5 Transcriptional regulator T303_05165 (truncated) TCS sensor kinase ciah T303_05170 (truncated) TCRS_6 Sensor histidine kinase T303_05645 TCRS_7 Sensor kinase T303_06640 Transcriptional regulator T303_06645 TCRS_8 Transcriptional regulator T303_07495 Histidine kinase T303_07500 TCRS_9 Sensor histidine kinase T303_07590 Response regulator T303_07595 TCRS_10 Response regulator T303_07985 Sensor kinase T303_07990 TCRS_11 Histidine kinase T303_08280 TCRS_12 Response regulator T303_09260 Sensor histidine kinase T303_09265 TCRS_13 Histidine kinase T303_09435

7 Supplementary Table 4. Stress response systems in S. thermophilus ASCC 1275 Encoded protein General stress regulator Universal stress protein UspA Acid resistance F 0 F 1 -ATPase system F 0 F 1 -ATP synthase gene cluster Urease Urease gene cluster Ammonia permease Cold & heat response Heat shock protein GrpE (conserved chaperone complexes: GrpE-DnaK-DnaJ) Heat shock protein Hsp33 Molecular chaperone GroES-GroEL Heat shock protein HtpX Molecular chaperone GroES CtsR family transcriptional regulator of class III stress genes (heat shock response) ATP-dependent Clp protease ATP-binding subunit ATP-dependent Clp protease proteolytic subunit Cold-shock protein Salt resistance Salt-stress induced protein Sodium transporter Cation transporter Oxidative stress response Glutamate-cysteine ligase Glutathione reductase Thioredoxin Thioredoxin reductase NADH oxidase Superoxide dismutase Locus tag(s) T303_00880 T303_09015 T303_03495, T303_03500, T303_03505, T303_03510, T303_03515, T303_03520, T303_03525, T303_03530 T303_02560, T303_02565, T303_02570, T303_02575, T303_02580, T303_02585, T303_02590, T303_02595, T303_02600, T303_02605, T303_02610 T303_03575 T303_01845, T303_01850, T303_01855 T303_02150 T303_02240, T303_02245 T303_04800 T303_05765 T303_01635 T303_01640, T303_04165, T303_08905 T303_02915 T303_05375, T303_05380 T303_08380 T303_04705, T303_07795 T303_05575, T303_06275 T303_07930 T303_03165 T303_00180, T303_09090, T303_09840 T303_09085 T303_07350 T303_00265, T303_04825

8 Peptide methionine sulfoxide reductase Msr B Methionine sulfoxide reductase A Peroxidase T303_07620 T303_08990 T303_06115

9 Supplementary Table 5. Defense systems in S. thermophilus ASCC 1275 Encoded protein Bacteriocin production Bacteriocin biosynthesis protein Bacteriocin Lantibiotic biosynthesis protein Bacteriocin class II with double-glycine leader peptide Bacteriocin immunity protein Putative bacteriocin self-immunity protein Immunity/modification protein Lantibiotic gene cluster Bacteriocin ABC transporter ATP-binding protein Lantibiotic transporter Bacteriocin transport accessory protein Multidrug resistance Penicillin-binding proteins β-lactamase Glycopeptide antibiotics resistance protein Multidrug transporter Multidrug-efflux transporter Multidrug ABC transporter Multidrug ABC transporter ATP-binding protein Multidrug ABC transporter ATPase Multidrug ABC transporter permease Drug/metabolite transporter permease Small multi-drug export Competence proteins Competence protein Late competence protein required for DNA binding and uptake Locus tag(s) T303_00670 T303_01755, T303_05370 T303_01760 (truncated), T303_01765 T303_09330 (truncated) T303_04480 T303_09185 T303_09200 T303_09220 ~ T303_09280 T303_07585 (truncated), T303_09550 (truncated) T303_01770 T303_09855 T303_00280, T303_02285, T303_02350, T303_04240, T303_09345 T303_01140, T303_09160 T303_02030, T303_09310 T303_04280 (truncated), T303_07615 T303_00665 T303_05640 T303_03240, T303_03830, T303_04990, T303_07235, T303_07510, T303_09295, T303_09300, T303_08060, T303_08875 T303_07925 T303_07475, T303_08950 T303_08390 T303_01630 T303_00230, T303_00235, T303_00245, T303_02970, T303_02975, T303_00250, T303_00255, T303_00260, T303_03365, T303_04440, T303_08635 T303_08640

10 Supplementary Table 6. EPS production from selected S. thermophilus strains under identical cultivation conditions from previous reports. Strain Cultivation conditions Medium EPS production S. thermophilus LY , uncontrolled 32 mg (polymer S. thermophilus ST , uncontrolled 42, ph 5.5 & 24 h 42, ph 6.2 & 24 h 42, ph 6.2 & 24 h 4.4% lactose 2% peptone 1% yeast extract S. thermophilus CH , uncontrolled ph & 12 h S. thermophilus SY , uncontrolled S. thermophilus D , uncontrolled S. thermophilus ST , uncontrolled S. thermophilus 01/ , uncontrolled 352 mg (polymer 491 mg (polymer 546 mg (polymer 284 mg (polymer 185 mg (polymer 286 mg (polymer 260 mg (polymer 236 mg (polymer 290 mg (polymer S. thermophilus SY 4 36, ph 6.4 & 36 h whey-based media 4 g/l yeast extract 152 mg (polymer S. thermophilus ST , ph 5.8 & 24 h 70 mg (polymer 37, ph 5.8 & 24 h 1.6% tryptone 507 mg (polymer 37, ph 5.8 & 24 h 1.6% WPC 330 mg (polymer S. thermophilus ASCC ; Fermentation was terminated at ph % skim milk 769 mg (polymer dry mass) per 1 kg of yoghurt

11 S. thermophilus ASCC 42 ; Fermentation ,7 was terminated at ph , uncontrolled ph & 24 h 14% skim milk 860 mg (polymer dry mass) per 1 kg of yoghurt 406 mg (glucose as standard) per liter 40, ph 5.5 & 24 h 622 mg (glucose as standard) per liter 37, ph 5.5 & 12 h 0.5% WPC 37, ph 5.5 & 24 h 0.5% WPC WPC, whey protein concentrate, is a by-product during the manufacture of cheese. References 745 mg (glucose as standard) per liter 1029 mg (glucose as standard) per liter 1 De Vuyst, L., Vanderveken, F., Van de Ven, S. & Degeest, B. Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis. J Appl Microbiol 84, (1998). 2 De Vuyst, L. et al. Exopolysaccharide-producing Streptococcus thermophilus strains as functional starter cultures in the production of fermented milks. Int Dairy J 13, (2003). 3 Vaningelgem, F. et al. Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics. Appl Environ Microb 70, (2004). 4 Ricciardi, A. et al. Exopolysaccharide production by Streptococcus thermophilus SY: production and preliminary characterization of the polymer. J Appl Microbiol 92, (2002). 5 Vaningelgem, F., Zamfir, M., Adriany, T. & De Vuyst, L. Fermentation conditions affecting the bacterial growth and exopolysaccharide production by Streptococcus thermophilus ST 111 in milk-based medium. J Appl Microbiol 97, (2004). 6 Purwandari, U., Shah, N. P. & Vasiljevic, T. Effects of exopolysaccharide-producing strains of Streptococcus thermophilus on technological and rheological properties of set-type yoghurt. Int Dairy J 17, (2007). 7 Zisu, B. & Shah, N. P. Effects of ph, temperature, supplementation with whey protein concentrate, and adjunct cultures on the production of exopolysaccharides by Streptococcus thermophilus J Dairy Sci 86, (2003).