Figure S1 Shewanella denitrificans OS217-reb1 Plesiocystis pacifica SIR-1-reb8 Polymorphum gilvum SL003B-26A1-reb1 Xanthomonas axonopodis pv. citri str. 306-reb6 Plesiocystis pacifica SIR-1-reb6 Azorhizobium caulinodans-reb3 Rhodospirillum centenum-reb1 Azorhizobium caulinodans-reb1 Plesiocystis pacifica SIR-1-reb3 Rhodospirillum centenum-reb2 Rhodospirillum centenum-reb5 Rhodospirillum centenum-reb4 Burkholderia ambifaria AMMD-reb2 Burkholderia ambifaria AMMD-reb1 Burkholderia ambifaria AMMD-reb3 Rhodospirillum centenum-reb3 Burkholderia lata-383 Plesiocystis pacifica SIR-1-reb5 Azorhizobium caulinodans-reb2 Plesiocystis pacifica SIR-1-reb4 Azorhizobium caulinodans-reb4 Plesiocystis pacifica SIR-1-reb7 Ruegeria pomeroyi DSS3-reb2 Pseudomonas fluorescens NEP1-reb2 Chromobacterium violaceum ATCC 12472-reb5 Plesiocystis pacifica SIR-1-reb2 Chromobacterium violaceum ATCC Polymorphum 12472-reb6 gilvum SL003B-26A1-reb2 Ruegeria pomeroyi DSS3-reb1 Pseudomonas fluorescens A506-reb3 Pseudomonas antarctica BS2772-reb3 Bukholderia Bukholderia sp. CCGE1003-reb1 sp. CCGE1003-reb2 Pseudomonas palleroniana Pseudomonas BS3265-reb3 protegens pf5-reb3 Pseudomonas aeruginosa PA14-PA14_27630 Pseudomonas aeruginosa PA14-PA14_27640 Pseudomonas synxantha BS33R-reb3 Pseudomonas aeruginosa AR0440-reb1 Pseudomonas libanensis BS2975-reb3 Pseudomonas mucidolens LMG2223-reb3 Pseudomonas aeruginosa LESB58-reb2 Pseudomonas aeruginosa AR0440-reb2 Pseudomonas chlororaphis B25-reb3 Pseudomonas aeruginosa LESB58-reb1 Pseudomonas aeruginosa LESB58-reb3 Chromobacterium violaceum ATCC 12472-reb3 Pseudomonas aeruginosa PA14-PA14_27685 Chromobacterium violaceum ATCC 12472-reb2 Polymorphum gilvum SL003B-26A1-reb3 Chromobacterium violaceum ATCC 12472-reb4 Ruegeria pomeroyi DSS3-reb3 Chromobacterium violaceum ATCC 12472-reb1 Burkholderia ambifaria AMMD-reb4 Pseudomonas fluorescens NEP1-reb1 Pseudomonas chlororaphis B25-reb1 Caedibacter taeniospiralis-rebD Pseudomonas protegens CHA0-reb1 Plesiocystis pacifica SIR-1-reb1 Pseudomonas protegens pf5-reb2 Pseudomonas synxantha BS33R-reb1 Pseudomonas fluorescens A506-reb1 Xanthomonas axonopodis pv. citri str. 306-reb3 Pseudomonas libanensis BS2975-reb1 Stenotrophomonas maltophilia R551-3-rebB Pseudomonas palleroniana BS3265-reb1 Caedibacter taeniospiralis-rebC Pseudomonas antarctica BS2772-reb1 Xanthomonas axonopodis pv. citri str. 306-reb4 Pseudomonas mucidolens LMG2223-reb1 Azospirillum brasilense Sp245-reb1 Pseudomonas protegens pf5-reb1 Pseudomonas protegens CHA0-reb2 Azospirillum brasilense Sp245-reb2 Burkholderia glumae BGR1-reb3 Pseudomonas mucidolens LMG2223-reb2 Pseudomonas chlororaphis B25-reb2 Burkholderia gladioli BSR3-reb1 Pseudomonas synxantha BS33R-reb2 Pseudomonas fluorescens A506-reb2 Burkholderia gladioli BSR3-reb2 Pseudomonas libanensis BS2975-reb2 Pseudomonas palleroniana BS3265-reb2 Burkholderia glumae BGR1-reb5 Pseudomonas antarctica BS2772-reb2 Saccharophagus Xanthomonas degradans campestris 2-40-reb pv. campestris str. ATCC Burkholderia glumae BGR1-reb4 Xanthomonas axonopodis pv. citri str. 306-reb1 Xanthomonas oryzae pv. oryzae PXO99A-reb Burkholderia pseudomallei 305-reb Burkholderia mallei SAVP1-reb1 Marinomonas mediterranea MMB1-reb2 Burkholderia glumae BGR1-reb6 Marinomonas mediterranea MMB1-reb1 Aliivibrio fischeri ES114-reb4 Stenotrophomonas maltophilia R551-3-reb1 Aliivibrio fischeri ES114-reb5 Shewanella denitrificans OS217-reb2 Aliivibrio fischeri ES114-reb1 Caedibacter taeniospiralis-rebB Aliivibrio fischeri ES114-reb2 Aliivibrio fischeri ES114-reb3 Aliivibrio fischeri ES114-reb6 Caedibacter taeniospiralis-rebA Burkholderia gladioli BSR3-reb3 Maricaulis maris MCS10-reb Xanthomonas axonopodis pv. citri str. 306-reb2 Stenotrophomonas maltophilia R551-3-rebA Burkholderia glumae BGR1-reb2 Azospirillum brasilense Sp245-reb3 Marinomonas mediterranea MMB1-reb4 Marinomonas mediterranea MMB1-reb3 Shewanella denitrificans OS217-reb4 Burkholderia gladioli BSR3-reb5 Shewanella denitrificans OS217-reb5 Azospirillum brasilense Sp245-reb4 Shewanella denitrificans OS217-reb3 Burkholderia gladioli BSR3-reb4 Stenotrophomonas maltophilia R551-3-reb2 Xanthomonas axonopodis pv. citri str. 306-reb5 0.4 Burkholderia glumae BGR1-reb1 Supplementary Figure 1. Phylogenetic tree for Reb proteins from 38 bacteria (comprised of pseudomonads represented in Figure 1a and complete genomes in Figure 6 of Raymann et al.1. C. taeniospiralis RebA, RebB, and RebC are highlighted in yellow. The cluster containing C. taeniospiralis RebD and its Pseudomonas orthologs is shown in blue. We have assigned the designation “RebP” to Reb orthologs that cluster with P. aeruginosa PA14_27630 and PA14_27640 (shown in purple). Green circles at the end of each branch indicate the presence of FecI2 homologues in the respective strains, while red circles indicate its absence. Pseudomonas psychrotolerans Pseudomonas citronellolis Figure S2 Pseudomonas salegens Pseudomonas xinjiangensis Pseudomonas resinovorans Pseudomonas sabulinigri Pseudomonas guangdongensis Pseudomonas furukawaii Pseudomonas aeruginosa PA14 PA14 aeruginosa Pseudomonas Pseudomonas denitrificans denitrificans Pseudomonas Pseudomonas oryzae Pseudomonas pohangensis Pseudomonas sihuiensis Pseudomonas stutzeri Pseudomonas mendocina Pseudomonas extremorientalis Pseudomonas pseudoalcaligenes Pseudomonas alcaliphila Pseudomonas xanthomarina Pseudomonas simiae Pseudomonas knackmussii Pseudomonas trivialis Pseudomonas Pseudomonas extremaustralis Pseudomonas veronii poae Pseudomonas alkylphenolia Pseudomonas cerasi Pseudomonas antarctica (1/2) Pseudomonas asplenii Pseudomonas orientalis Pseudomonas Pseudomonas fuscovaginae entomophila Pseudomonas rhodesiae Pseudomonas monteilii Pseudomonas plecoglossicida Pseudomonas lurida Pseudomonas mosselii Pseudomonas palleroniana (1/1) Pseudomonas fulva Pseudomonas tolaasii Pseudomonas parafulva Pseudomonas cremoricolorata Pseudomonas cedrina Pseudomonas oryzihabitans Pseudomonas umsongensis Pseudomonas azotoformans Pseudomonas Pseudomonas alcaligenes agarici Pseudomonas granadensis Pseudomonas synxantha (3/8) Pseudomonas putida Pseudomonas rhizosphaerae Pseudomonas libanensis (2/2) Pseudomonas Pseudomonas reinekei moraviensis Pseudomonas brenneri Pseudomonas Pseudomonas fluorescens koreensis (7/19) Pseudomonas mandelii Pseudomonas mucidolens (2/2) Pseudomonas amygdali Pseudomonas Pseudomonas prosekii lini Pseudomonas yamanorum Pseudomonas frederiksbergensis Pseudomonas syringae Pseudomonas savastanoi Pseudomonas protegens (7/8) Pseudomonas arsenicoxydans Pseudomonas avellanae Pseudomonas cichorii Pseudomonas fragi Pseudomonas corrugata Pseudomonas psychrophila Pseudomonas taetrolens Pseudomonas brassicacearum Pseudomonas viridiflava Pseudomonas thivervalensis Pseudomonas chlororaphis (47/48) Pseudomonas mediterranea 0.02 Supplementary Figure 2. Phylogenetic tree showing all Pseudomonas species with complete genomes available in the Pseudomonas Genome DB. Indicated in parentheses are the number of full genomes with rebP1 homologues out of the total number of full genome sequences available for a species. Species with partial genome sequences are marked by red asterisks. Figure S3 a WT ΔrebP1P2 ΔrebP1P2::rebP1P2 b MCS-gfp PrebP1-gfp WT WT ΔrebZΔfecI2 ΔrebZ ΔfecI2 c d PrebP1-mScarlet and PPA1/04/03-gfp in WT PrebP1-mScarlet in ΔrebZΔfecI2::rebZ fecI2 Supplementary Figure 3. a, SEM images of SDS-insoluble fractions prepared from the indicated strains. Scale bar = 500 nm. b, Fluorescence microscopy images of top-down views of three-day-old PA14 colony biofilms expressing PrebP1-gfp. Dotted lines demarcate the edge of the biofilm. Scale bar = 25 mm. c, Representative image of thin section prepared from a three-day-old biofilm containing the constructs PrebP1-mScarlet and PPA1/04/03-gfp. Scale bar = 10 μm. d, Left panels: Bright-field and fluorescence microscopy images of top-down views of three-day-old colony biofilms containing PrebP1- mScarlet in the ∆rebZ∆fecI2 complemented strain background. Dotted lines demarcate the edge of the biofilm. Scale bar = 25 mm. Right panel: Representative image of a thin section prepared from a three-day-old biofilm formed by the complemented strain expressing PrebP1-mScarlet. Dotted lines denote the air-biofilm interface (top) and the biofilm-agar interface (bottom). Scale bar = 10 μm. Supplementary Table 1: Strains Strain Number Description Source Appears in figure Pseudomonas aeruginosa UCBPP-PA14 Clinical isolate UCBPP-PA14. (2) 2c-f PA14 ΔpelB-G LD2720 PA14 with deletion in PA14_24490-24560. (3) 1b-d, Table S1 PA14 ΔpelB-G LD2936 PA14 with deletion in PA14_24490-24560 This study S4a ΔPA14_27630-27640 and PA14_27630-27640. PA14 ΔpelB-G LD3026 PA14 ΔpelB-G ΔPA14_27630-27640 with This study S4a ΔPA14_27630-27640:: wild-type PA14_27630-27640 PA14_27630-27640 complemented back into the site of deletion. PA14 LD3224 PA14 with PrebP1-mScarlet inserted at the This study 2a-b attB::PrebP1-mScarlet attB site using pLD3210. PA14 LD3319 PA14 ΔPA14_27690-27700 with This study 1e-f ΔPA14_27690-27700 PrebP1P2-mScarlet inserted at the attB attB::PrebP1-mScarlet site using pLD3210. PA14 ΔPA14_27690 LD3148 PA14 ΔPA14_27690 with PrebP1-gfp This study S4b attB::PrebP1-gfp inserted at the attB site using pLD2757. PA14 ΔPA14_27700 LD3149 PA14 ΔPA14_27700 with PrebP1-gfp This study S4b attB::PrebP1-gfp inserted at the attB site using pLD2757 and an extra copy of rebP1 N-terminally tagged with gfp under the control of a weak RBS. PA14 attB::PrebP1- LD3657 PA14 with PrebP1-mScarlet inserted at the This study S4c mScarlet attB site