Convergent evolution of a modified, acetate-driven TCA cycle in bacteria

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1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 2 ARTICLE NUMBER: Convergent evolution of a modified, acetate-driven TCA cycle in bacteria Waldan K. Kwong, Hao Zheng, and Nancy A. Moran Correspondence to: wk.kwong@aya.yale.edu This document includes: Supplementary Figures 1 3 Supplementary Tables 1 & 2 NATURE MICROBIOLOGY DOI: /nmicrobiol

2 Supplementary Figure 1. Growth effects of ASCT-complementation in different E. coli mutant backgrounds. Strains with both ASCT and SCS had slowest growth, suggesting competitive inhibition. Strains with ΔsucC backgrounds had fastest growth, but at the cost of achieving lower final densities. Strains were grown in M9 minimal media supplemented with 0.3% w/v sodium acetate and 75 mg/ml carbenicillin for plasmid maintenance. OD 600 readings were taken hourly. Lines represent replicates (n = 3 for each strain). NATURE MICROBIOLOGY DOI: /nmicrobiol

3 Supplementary Figure 2. Rectangular tree representation of asct phylogeny in Fig. 1c. Values at nodes indicate bootstrap support from maximum-likelihood analysis (1,000 replicates). Bar, substitutions per site asct only asct and succd incomplete TCA cycle Bacteroides salanitronis DSM copy 1 Bacteroides coprocola DSM copy 1 Prevotella dentalis DSM 3688 Bacteroides salanitronis DSM copy 2 Bacteroides coprocola DSM copy 2 Bacteroides dorei CL03T12C01 Bacteroides vulgatus ATCC 8482 Bacteroidetes Bacteroides fragilis NCTC 9343 Bacteroides thetaiotaomicron 7330 Bacteroides pyogenes DSM Bacteroides helcogenes P Parabacteroides distasonis ATCC 8503 Parabacteroides johnsonii DSM Porphyromonas gingivalis 381 Achromobacter denitrificans USDA-ARS-USMARC Bordetella hinzii F582 Castellaniella defragrans 65Phen Herbaspirillum hiltneri N3 Agrobacterium tumefaciens Ach5 Brucella suis ATCC Brucella melitensis ATCC Pseudomonas fluorescens NCIMB Pseudomonas aeruginosa Cu1510 Acinetobacter johnsonii XBB1 Acinetobacter baumannii ATCC Massilia sp. WG5 Pseudogulbenkiania ferrooxidans 2002 Alpha-, Beta-, and Laribacter hongkongensis HLHK9 Gammaproteobacteria Vitreoscilla stercoraria DSM 513 Bradyrhizobium sp. BTAi1 Azoarcus sp. BH72 Limnohabitans planktonicus II-D5 Comamonas testosteroni CNB-2 copy 2 Burkholderia stagnalis ABCPW-33 Burkholderia cepacia LO6 Burkholderia territorii MSMB1838 Burkholderia cepacia ATCC Burkholderia pseudomallei ST1381 Sphingobium japonicum BiD32 copy 1 Sphingobium japonicum BiD32 copy 2 Blastochloris viridis ATCC Gluconacetobacter diazotrophicus PAl 5 Komagataeibacter hansenii ATCC Acetobacter tropicalis LMG 1663 copy 1 Commensalibacter intestini A911 Acetobacter cerevisiae LMG 1625 Acetobacter tropicalis LMG 1663 copy 2 Alphaproteobacteria Acetobacter papayae JCM Acetobacter okinawensis JCM Acetobacter aceti 1023 Acetobacter aceti NBRC Acetobacter nitrogenifigens DSM Propionibacterium propionicum F0230a Propionibacterium freudenreichii DSM Propionibacterium acnes ATCC Corynebacterium ulcerans FRC58 Actinobacteria Corynebacterium glutamicum ATCC Corynebacterium diphtheriae NCTC Corynebacterium argentoratense DSM Rhodococcus equi 103S Stenoxybacter acetivorans DSM Delftia acidovorans SPH-1 Comamonas testosteroni CNB-2 copy 1 Snodgrassella alvi wkb12 Snodgrassella alvi wkb2 Conchiformibius kuhniae DSM Conchiformibius steedae DSM 2580 Kingella denitrificans ATCC Betaproteobacteria Simonsiella muelleri ATCC Moraxella catarrhalis Gammaproteobacteria Kingella kingae KKKWG1 Alysiella crassa DSM 2578 Bergeriella denitrificans NBRC Kingella oralis ATCC Neisseria weaveri ATCC Neisseria elongata subsp. glycolytica ATCC Neisseria bacilliformis ATCC BAA-1200 Clostridium kluyveri DSM 555 NATURE MICROBIOLOGY DOI: /nmicrobiol

4 Supplementary Figure 3. Prevalence of asct in the Neisseriaceae. The maximum likelihood algorithm was used to construct a whole genome phylogeny, based on 363 orthologous genes (108,434 amino acid residues). Branches are colored based on presence of asct and succd in tip taxa, and extrapolated using parsimony to interior branches. Circles indicate nodes with % bootstrap support (1,000 replicates). Bar, substitutions per site Leeia oryzae DSM Amantichitinum ursilacus IGB-41 Chitiniphilus shinanonensis DSM Andreprevotia chitinilytica DSM asct only Deefgea rivuli DSM Chitinibacter sp. ZOR0017 asct and succd Chitinibacter tainanensis DSM succd only Chitinilyticum aquatile DSM Chitinilyticum litopenaei DSM Aquaspirillum serpens DSM 68 Microvirgula aerodenitrificans DSM Laribacter hongkongensis HLHK9 Gulbenkiania indica DSM 1790 Gulbenkiania mobilis MB1 Pseudogulbenkiania sp. NH8B Pseudogulbenkiania ferrooxidans 2002 Vogesella sp. EB Chromobacterium haemolyticum T124 Chromobacterium subtsugae PRAA4-1 Chromobacterium piscinae ND17 Chromobacterium violaceum ATCC Chromobacterium vaccinii MWU205 Vitreoscilla stercoraria DSM 513 Vitreoscilla sp. SN6 Stenoxybacter acetivorans DSM Snodgrassella alvi wkb12 Snodgrassella alvi wkb2 Neisseria shayeganii 871 Eikenella corrodens ATCC Eikenella sp. NML Conchiformibius kuhniae DSM Conchiformibius steedae DSM 2580 Alysiella crassa DSM 2578 Simonsiella muelleri ATCC Kingella kingae KKKWG1 70 Kingella denitrificans ATCC Kingella oralis ATCC Neisseria weaveri ATCC Neisseria wadsworthii 9715 Neisseria bacilliformis ATCC BAA-1200 Neisseria elongata subsp. glycolytica ATCC Bergeriella denitrificans NBRC Neisseria sp. oral taxon 014 str. F0314 Neisseria mucosa ATCC 256 Morococcus cerebrosus CIP Neisseria macacae ATCC Neisseria sicca ATCC Neisseria flavescens SK114 Neisseria subflava NJ9703 Neisseria cinerea ATCC Neisseria polysaccharea ATCC Neisseria lactamica Neisseria gonorrhoeae FA 1090 Neisseria meningitidis MC58 family Neisseriaceae Burkholderia cepacia ATCC Azoarcus sp. BH72 Nitrosospira multiformis ATCC other Betaproteobacteria 0.2 NATURE MICROBIOLOGY DOI: /nmicrobiol

5 Supplementary Table 1. Genes, plasmids, and strains used in this study. SUPPLEMENTARY INFORMATION Description Reference Gene SALWKB2_RS03080 asct from Snodgrassella alvi wkb2 NZ_CP AD928_RS13430 asct from Acetobacter cerevisiae LMG 1625 NZ_LHZA00000 DIP_RS20725 asct from Corynebacterium diphtheriae NCTC NC_ DR90_RS06235 asct from Moraxella catarrhalis ATCC NZ_CP F911_00479 asct from Acinetobacter baumannii ATCC NZ_APRG00000 BF9343_RS00125 asct from Bacteroides fragilis NCTC 9343 NC_ Plasmid pbad-ebfp2 (control) pbad backbone, EBFP2 (blue fluorescent protein) insert [15] pwk1 pbad backbone, SALWKB2_RS03080 insert This study pwk24 pbad backbone, DIP_RS20725 insert This study pwk25 pbad backbone, AD928_RS13430 insert This study pwk29 pbad backbone, DR90_RS06235 insert This study pwk34 pbad backbone, F911_00479 insert This study pwk39 pbad backbone, BF9343_RS00125 insert This study pwk31 (control) pbad backbone, F911_00479 insert (reversed) This study Strain E. coli CGSC 7636 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ -, rph- 1, Δ(rhaD-rhaB)568, hsdr514 E. coli CGSC 8788 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), ΔsucC777::kan, λ -, rph-1, Δ(rhaD-rhaB)568, hsdr514 E. coli CGSC F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), ΔsucD778::kan, λ -, rph-1, Δ(rhaD-rhaB)568, hsdr514 [16] [16] [16] NATURE MICROBIOLOGY DOI: /nmicrobiol

6 Supplementary Table 2. Growth curve data for Fig. 1b, means and standard deviations. Strain identities as follows: 7636, E. coli CGSC 7636; wke9, pbad-ebfp2 in CGSC 8788; wke11, pbad- EBFP2 in CGSC 11810; wke5, pwk1 in CGSC 8788; wke3, pwk1 in CGSC 11810; wke17, pwk24 in CGSC8788; wke15, pwk24 in CGSC 11810; wke21, pwk25 in CGSC 8788; wke23, pwk25 in CGSC 11810; wke27, pwk29 in CGSC 8788; wke29, pwk29 in CGSC 11810; wke33, pwk34 in CGSC 8788; wke34, pwk34 in CGSC 11810; wke35, pwk39 in CGSC 8788; wke36, pwk39 in CGSC OD600 means Hour 7636 wke9 wke11 wke5 wke3 wke17* wke15* wke21 wke23 wke27 wke29 wke33 wke34 wke35 wke Standard deviations Hour 7636 wke9 wke11 wke5 wke3 wke17* wke15* wke21 wke23 wke27 wke29 wke33 wke34 wke35 wke * n = 3 for each strain, except wke17 and wke15, where n = 2. NATURE MICROBIOLOGY DOI: /nmicrobiol

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