A Kurthia_sp._538-KA26 Firmicutes 100 Bacillus_thuringiensis Geobacter_sulfurreducens Deltaproteobacteria Crocosphaera_subtropica Cyanobacteria 100 Crocosphaera_subtropica_ATCC_51142 60 Crocosphaera_watsonii_WH_8501 Vulgatibacter_incomptus 97 Zymomonas_mobilis - “Cand. Deianiaeaceae” [D] 100 100 Gluconacetobacter_diazotrophicus Komagataeibacter_hansenii_ATCC_23769 - [A] 100 Achromobacter_piechaudii_ATCC_43553 - Midichloriaceae [M] 100 Bordetella_avium 100 87 Bordetella_parapertussis - [R] 74 Bordetella_pertussis_Tohama_I Other 39 Bordetella_bronchiseptica Acinetobacter_soli - Holosporaceae [H] 33 Acinetobacter_albensis 100 53 Acinetobacter_baumannii 63 Acinetobacter_junii 100 100 Neisseria_iguanae 100 Bacteroidetes 99 Neisseria_canis Neisseria_wadsworthii Planctomycetes 100 100 Neisseria_polysaccharea Neisseria_lactamica PVC: Chlamydiae 68 Neisseria_gonorrhoeae 62 Neisseria_meningitidis_MC58 98 Rickettsiales_bacterium_Ac37b [R] Ehrlichia_ruminantium_str._Gardel [A] 100 100 Ehrlichia_chaffeensis_str._Arkansas [A] 67 Ehrlichia_canis [A] Rickettsiales 100 Anaplasma_phagocytophilum [A] 100 GROUP 2 100 Anaplasma_marginale_str._St._Maries [A] 49 Anaplasma_centrale_str._Israel [A] Mariniblastus_fucicola 77 Vibrio_cholerae_B33 Escherichia_coli_str._K-12_substr._MG1655 74 100 Candidatus_Hamiltonella_defensa__Bemisia_tabaci_ 100 85 Wigglesworthia_glossinidia 33 100 Xylella_fastidiosa 72 Pseudomonas_aeruginosa_PAO1 Methylococcus_capsulatus_str._Bath 29 Rickettsiales_endosymbiont_of_Stachyamoeba_lipophora [R] Rickettsiales_endosymbiont_of_Trichoplax_sp._H2 [M] 60 100 Candidatus_Aquarickettsia_rohweri [M] 100 Legionella_longbeachae_D-4968 3 GROUP 100

100 Legionella_pneumophila_str._Corby Rickettsiales Legionella_pneumophila Magnetospirillum_magneticum Nitrococcus_mobilis_Nb-231 14 Parachlamydia_acanthamoebae_str._Halls_coccus Chlamydia_pneumoniae_CWL029 39 Chlamydia_avium_10DC88 25 100 100 98 Chlamydia_psittaci_02DC21 Fig. S1 Chlamydiales_bacterium_38-26 Criblamydia_sequanensis_CRIB-18 99 100 95 Candidatus_Rubidus_massiliensis 9 Chlamydia_sp._32-24 100 Candidatus_Paracaedibacter_symbiosus [H] 44 Candidatus_Odyssella_thessalonicensis [H] Candidatus_Deianiraea_vastatrix [D] Candidatus_Midichloria_mitochondrii_IricVA [M] 39 Occidentia_massiliensis [R] 91 100 Neorickettsia_helminthoeca_str._Oregon [A] 100 Neorickettsia_sp._179522 [A] BOOM Neorickettsia_sennetsu [A] GROUP 1 100 98 Neorickettsia_risticii_str._Illinois [A] 100 Neorickettsia_risticii [A] 100 Rickettsia_endosymbiont_of_Ixodes_scapularis [R] 100 Rickettsia_buchneri [R] 41 Lawsonia_intracellularis_N343 99 100 Lawsonia_intracellularis_PHE/MN1-00 49 Candidatus_Fokinia_solitaria [M] see Fig. 2C Rickettsiales_endosymbiont_of_Peranema_trichophorum [M] 92 Acinetobacter_sp. (likely contaminant; other Acinetobacter group above) Holospora_obtusa_F1 [H] 75 Holospora_curviuscula [H] 92 100 100 99 Holospora_undulata_HU1 [H] Holospora_elegans_E1 [H] Legionella_endosymbiont_of_Polyplax_serrata 100 Cardinium_endosymbiont_of_Sogatella_furcifera 100 Cardinium_endosymbiont_of_Encarsia_pergandiella 100 Cardinium_endosymbiont_cBtQ1_of_Bemisia_tabaci 100 Wolbachia_endosymbiont_of_Ctenocephalides_felis_T [A] 69 Wolbachia_endosymbiont_of_Nilaparvata_lugens [A] 91 Wolbachia_endosymbiont_of_Laodelphax_striatellus [A] Wolbachia_endosymbiont_wNfla [A] 100 100 Wolbachia_endosymbiont_wNleu [A] 96 Wolbachia_endosymbiont_of_Armadillidium_vulgare_str._wVulC [A] Wolbachia_sp._KTCN [A] 71 100 Wolbachia_sp._SYDL [A] 100 Wolbachia_sp._TIH [A] 73 Wolbachia_endosymbiont_of_Cimex_lectularius [A] 0.1 sub./site 37 Wolbachia_sp._TUA [A] B 27 Betaproteobacteria_bacterium_RIFCSPLOWO2_12_FULL_63_13_OGA51326.1 Fig. S1 Chromatiales_bacterium__ex_Bugula_neritina_AB1__OED35259.1 Dongia_mobilis_WP_133614772.1 Oceanibacterium_hippocampi_SLN12270.1 100 Oceanibacterium_hippocampi_WP_085881512.1 27 Thiotrichales_bacterium_MBS36085.1 Candidatus_Finniella_inopinata_WP_130153669.1 [H] 16 Candidatus_Paracaedibacter_symbiosus_WP_084756013.1 [H] 100 91 Candidatus_Paracaedibacter_acanthamoebae_AIK96411.1 [H] 32 100 Candidatus_Paracaedibacter_acanthamoebae_WP_075261567.1 [H] Gammaproteobacteria_bacterium_RBG_16_37_9_OGT09541.1 100 Coxiellaceae_bacterium_HBC71581.1 85 99 Candidatus_Nucleicultrix_amoebiphila_WP_157111114.1 [H] 100 Caedimonadaceae_bacterium_KAB2833547.1 [H] 98 Caedimonas_varicaedens_WP_062139989.1 [H] Caedibacter_sp._37-49_OJX11967.1 [H] 82 98 Candidatus_Paracaedimonas_acanthamoebae_AIL13301.1 [H] 50 Caedibacter_sp._38-128_OJX05467.1 [H]

Clade comprised 57 Candidatus_Hepatobacter_penaei_WP_082192079.1 [H] of intracellular 97 Holospora_undulata_WP_006304416.1 [H] bacterial species 100 Holospora_undulata_HU1_ETZ05432.1 [H] Rickettsiales_bacterium_Ac37b_WP_038601953.1 [R] 22 100 Cardinium_endosymbiont_of_Culicoides_punctatus_WP_133281577.1 Cardinium_endosymbiont_of_Culicoides_punctatus_TDG95756.1 73 83 Candidatus_Amoebophilus_asiaticus_WP_012473281.1 100 Candidatus_Amoebophilus_sp._36-38_OJW68886.1

22 Rickettsiales_endosymbiont_of_Stachyamoeba_lipophora_WP_125215760.1 [R] Verrucomicrobia_bacterium_CG1_02_43_26_OIO60485.1 11 82 Verrucomicrobia_bacterium_GWC2_42_7_OHE71373.1 Candidatus_Odyssella_thessalonicensis_WP_010302065.1 [H] 24 Candidatus_Paracaedibacter_symbiosus_WP_075553232.1 [H] 75 79 Candidatus_Odyssella_thessalonicensis_WP_033444796.1 [H] 99 Candidatus_Odyssella_thessalonicensis_WP_010303140.1 [H] Rickettsiales 100 Candidatus_Aquarickettsia_rohweri_WP_126044659.1 [M] - Anaplasmataceae [A] 14 Rickettsiales_endosymbiont_of_Trichoplax_sp._H2_WP_154510730.1 [M] - Midichloriaceae [M] Rickettsiales_endosymbiont_of_Trichoplax_sp._H2_MSO13531.1 [M] - Rickettsiaceae [R] 100 Other Alphaproteobacteria 100 Rickettsiales_endosymbiont_of_Trichoplax_sp._H2_WP_154510822.1 [M] - Holosporaceae [H] Candidatus_Aquarickettsia_rohweri_WP_126045186.1 [M] 73 100 Rickettsiales_endosymbiont_of_Trichoplax_sp._H2_WP_154512549.1 [M] Betaproteobacteria 17 Gammaproteobacteria Rickettsiales_endosymbiont_of_Peranema_trichophorum_WP_130121325.1 [M] Bacteroidetes endosymbiont_of_Acanthamoeba_sp._UWC8_WP_052646582.1 [H] 97 100 PVC: Verrucomicrobia Candidatus_Jidaibacter_acanthamoeba_KIE05270.1 [M] 100 Candidatus_Jidaibacter_acanthamoeba_WP_053332606.1 [M] 27 81 Wolbachia_pipientis_WP_070065361.1 [A] 97 wCfeT [A] Other Wolbachiae (31 sequences) [A] 100 Candidatus_Phycorickettsia_trachydisci_WP_106874536.1 [R] 62 98 Occidentia_massiliensis_WP_019231221.1 [R]

100 Orientia_chuto_WP_045796892.1 [R] 59 100 (11 sequences) [R] 99 spp. (40 sequences) [R] 0.1 sub./site 84 wCfeT proteins Top Blastp hit Protein Size Accession Size Annotation Taxon Max, tot. Cov. E value %ID

WP_168464803 309 APR98706 311 Patatin-like phospholipase wFol 514, 514 100% 0.0 78.14% WP_168464804 72 AZU37351 74 ParD-like family antidote wBta 107, 107 97% 7e-29 72.86% WP_168464805 96 OAM00647 92 RelE/ParE family toxin wDacA 154, 154 95% 5e-47 80.43% ------44 ------WP_168464806 262 APR98707 257 PHA03095 (Ank repeats) wFol 321, 321 98% 1e-107 60.31% WP_168464807 82 APR99034 86 XRE family transcriptional regulator wFol 111, 111 97% 5e-30 68.75%

WP_168464808 648 APR98618 651 DNA ligase (NAD(+)) LigA wFol 1007, 1007 99% 0.0 73.92% WP_168464809 79 APR97852 96 XRE family transcriptional regulator wFol 139, 139 98% 4e-41 89.74% WP_168464414 545 WP_143688845 493 Group II intron RevTranscriptase/maturase wStr 992, 992 89% 0.0 99.59% WP_168464810 310 APR98606 312 Recombination-promoting nuclease/put. tnp wFol 506, 506 100% 3e-179 79.35% WP_168464811 301 APR98615 302 Hypothetical protein (put. rhoptry protein) wFol 421, 421 100% 9e-146 74.17% WP_168464812 226 APR98945 217 DNA repair protein RadC wFol 367, 367 96% 7e-127 84.86%

wCfeT e a b 6 c z g f e d σ 4 h v j

WOVitA1 a b 6 σ 4 c d e f g z h v j i

TNP EAM RadC Patatin Other ortholog Tail σ σ factor Head ATPase Base Recombinase

Fig. S2 BioB BioF BioH BioC BioD BioA

Phylogenetic Group (see Class I: Completely conserved gene order Figure S1A)

Neorickettsia spp. 1 “BOOM”

Wolbachiae 1 “BOOM”

Peranema trichophorum endosym. 1 “BOOM”

Rickettsia buchneri (2 copies) 1 “BOOM”

Class IIA: Partially conserved gene order (rogue BioA)

Trichoplax sp. H2 endosymbiont 293 GROUP 3

”Cand. Aquarickettsia rohweri GROUP 3

“Cand. mitochondrii 900 1 “BOOM”

Stachyamoeba lipophora endosym. GROUP 3

Class IIB: Partially conserved gene order (rogue BioB)

“Cand. Fokinia solitaria” 1 “BOOM”

“Cand. Occidentia massiliensis” 3 1 “BOOM”

Class III: No conservation of gene order

“Cand. Deianiraea vastatrix” 856 41 1 “BOOM”

Anaplasma marginale 106 130 217 193 A. phagocytophilum 218 298 26 154 152 GROUP 2 A. centrale 279 186 223 108

Ehrlichia canis 234 68 155 197 E. ruminatum 209 62 153 215 GROUP 2 E. chaffeensis 257 203 116 66

Rickettsiales bacterium str. Ac37b 556 247 329 GROUP 2

“Cand. Deianiaeaceae” Midichloriaceae Anaplasmataceae Rickettsiaceae Fig. S3 A chaffeensis ------MSDHYK--LVLSNPQGLHITYRQLLGN----KASIIFFGGFNSNMQGTKATALY 48 U Anaplasma centrale MLRQSCVGAVVGEEKRLELGNSSYISYMQTTVQS---PVSVVFFGGFMSDMHGTKAQHLF 57 Rickettsiales bacterium Ac37b ------MNIAFN-VQKLLLSNNNYIAYSKINSKTQNELPGIIFFSGFNSNMQGTKARNLT 53 helminthoeca ------MQTKYL------DTSHGRIAYQTFDNNP---EVGVLFMTGLASDMSGRKSERLR 45 Rickettsia buchneri ------MHKLYN------KTQDKFIVYDNYRIINTN-IPSVIFLHGLMSSMKSTKAIYLI 47 wCle ------MDYCK----LFGESGKYIAYRKLQGR----RTSIVFFGGFASNMNGTKATAIY 45 wPip_Pel ------MNYCK----LLDGSNGHIAYRKLQGK----KASIVFFSGFASNMDGTKATAVY 45 Neoehrlichia lotoris ------MTDYSPSFLVLDHNNNHKIAYRQLKKNSN--LPSILLLGGFGSNMYGEKATALY 52 ------MHKLYN------KTQDKFIVYDNYRIINTN-IPSVIFLHGLMSSMQSTKAIYLI 47 Orientia tsutsugamushi Boryong ------MHKIYT------LDDGRFIAYRQHKSQKNS-LINIIFLHGMMSNMSGKKSSYLY 47 . * * .:::: *: *.* . *: : Ser DYCKSHNLGLILFDYLGHGQSDGQFTDYNISDWYKNCIEIITQLTPTNRPKIIIGSSMGA 108 U Anaplasma centrale EYCKSHGVHCTVFDYFGHGSSSGEFQECTISDWYASCVSVVESL--TSAPLVIVGSSMGG 115 Rickettsiales bacterium Ac37b EYCQNNNYNFIKFDYLGHGLSSGIFHECTIGIWLENCLSIIDNL--TTDKHIFIGSSMGG 111 Neorickettsia helminthoeca SFCENNQVAFTRFDYFGHGRSEGSFLHGNISKWTENALEVLERV--TTGKQILVGSSMSG 103 Rickettsia buchneri DYCKKNNYNFIVFDNFGHGNASGQFEDQTISDWLEGVSLILDKL--IDKEAILVGSSMGG 105 wCle KFCQENDVALVLFDYFGHGHSSGDFTDYTISDWQKNCTRVINEL--TSSKQIIIGSSMGG 103 wPip_Pel KFCQENDIALVLFDYFGHGNSSGDFADYTISDWQKNCAKVISEL--TSNKQIIIGSSMGG 103 Neoehrlichia lotoris NYCNKHNLNLTVFDYLGHGHSSGNFTDYTIGDWYKNCISVIESL--TNGPQIIIGSSMGG 110 Rickettsia typhi DYCKKNNYNFIVFDNFGHGNAYGQFEDQTISDWLEGVALILDKL--IETEAILIGSSMGG 105 Orientia tsutsugamushi Boryong QLCQEEDLNFLAFDNYGHGNSSGRFIDQTIESWFDATRAIMYHTS-NNFKNIIVGSSLGG 106 . *:. ** *** : * * .* * :: :::***:..

Ehrlichia chaffeensis WLMLLVAISHQDKVSHLISLAGAPDFTESLIFQKLNTQQKDELYKYGQITL--SQNSNNM 166 U Anaplasma centrale WLMLLTALSHGRRVRGLVGMAPAPDFTESL---DLSESQRAEMMRTGKTIK--NTDNC-- 168 Rickettsiales bacterium Ac37b WLALLASILRPEKVAGIICIAAAPDFTENLIWNTLSLEEKNKLQTQGIIKL--SSNYCEG 169 Neorickettsia helminthoeca WMMFKIAEKHPEKVKGLVGIAAAPDFTED-FLEGLTHETKQALEKNGYFTF--TRNRDE- 159 Rickettsia buchneri WLALLAALRFPDKIKGLVCVAPAPDFTEN-IWQNISLNDQNKMQKEGILEV--SGKNCEH 162 wCle WLMLLTALQIPERIAALIGVSSAPDFTEDLIFKQLSGKQKEELDSKGVVDF--TSGRC-- 159 wPip_Pel WLMLLTALQFPEKIAALIGISSAPDFTEDLIFKQLSGKQKEELGSKGVIDF--TSEHC-- 159 Neoehrlichia lotoris WLMLLIAQSYPHKVISLLGLAPAPDFTENLIFNKLTQEQKDCLHTNNQIIF--TFNKYED 168 Rickettsia typhi WLALLAALRFPDKIKCLICVAPALDFTEN-IWQNISLNDQNKMKKEGIIEV--SSENCQH 162 Orientia tsutsugamushi Boryong WLAMLAAIKNEIEISGVVALAPAIDFTETLIWNKLTEKNKNSMIHTGYIELGGTGNTCNN 166 *: : : : :: :: * **** :. . . : . . Asp Ehrlichia chaffeensis YSYVITRNLIEDGRKHLLLNQESINITCPITLIHGMNDDTVPYQTSITVAEKIKSDNVNL 226 U Anaplasma centrale -SYVITKKLIDDGKAHLLMNKREIAVECPMVLIHGMDDTVVPYQVSLEIAGKVKSGDVRV 227 Rickettsiales bacterium Ac37b -EYEISLKLIEEAREHLILNK-PLDIKCPIYLLHGMADKDVPYNFSLDLVNSISSQDITV 227 Neorickettsia helminthoeca -KLVITKTLLDDGKKNLILTQ-RIKVPCPVVLLHGLADDIVSYRKSIELAELIESSPVEV 217 Rickettsia buchneri -KYPISYKLIEDAKKHLLLTKKQIDINTPLHIIHGMLDEDVPYNVSVKLLEKITSKQIVM 221 wCle -AYKITKNLIEDGRKKLLLNKETIDINSPVRLLHSINDKDVPYQTSLNLAERVKSTDVEV 218 wPip_Pel -AYKITKNLIEDGRKNLLLNREAIDINCPVRLLHSINDKDVPYQTSLNLAEKIKSTDVEV 218 Neoehrlichia lotoris RSYDITDNLIKDGRKHLLLNNDNININCPVILIHSMSDLVVPYSTSIHVAEKITSTNVNL 228 Rickettsia typhi -KYPISYKLIEDAKKHLLLRKQQIEINIPVHIIHGMLDKNVPYNVSVKLLEKITSKQIVM 221 Orientia tsutsugamushi Boryong -KYHISYNLICNARKYLLLNKPTINIQCPIAIIHGMQDQEVPYQGSIDLINKVQTHYSTL 225 *: .*: :.. *:: . : : *: ::*.: * *.* *: : : : : His Ehrlichia chaffeensis HLIKSANHNLSDDTSLNIIFKYIKEAVEQSIQVK 260 U Anaplasma centrale HLSKSGTHRLTDEHSLGLMLESVKGLMRPSVPV- 260 Rickettsiales bacterium Ac37b KLVKDAGHGMSTKVNLYLLYNTINELITKITNK- 260 Neorickettsia helminthoeca RLIKGADHSMSDPTSITVLTDTVRALI------244 U = characterized BioU Rickettsia buchneri KLIKDGHHNLSREEDLKVMTNSLEEVISLSNIK- 254 wCle HLTKSAEHNMSDSHSLKILFQAIREFLPTV---- 248 = taxa with BioH wPip_Pel HLIKSAEHNMSDNHSLKILFKTIREFLPGEIYN- 251 = no biotin synthesis Neoehrlichia lotoris HLIKSGNHYLRDEHSLNVTFSAIQSLLAQC---- 258 Rickettsia typhi KLIKDGNHNLSRKEDLKVIANSLEEMISNIK--- 252 Orientia tsutsugamushi Boryong KLLKYADHFLSDSVSLSHISYAIKEIINARLV-- 257 .* * . * : .: : :

Fig. S4 B Fig. S4 Otsu Orientia tsutsugamushi Boryong (Otsu) 100.00 Nhel Neorickettsia helminthoeca (Nhel) 31.56 100.00 Acen U Anaplasma centrale (Acen) 34.01 37.08 100.00 Rtyp Rickettsia typhi (Rtyp) 40.48 31.97 35.92 100.00 Rbuc Rickettsia buchneri (Rbuc) 41.50 34.43 38.87 88.49 100.00 R37b Cle Rickettsiales bacterium Ac37b (R37b) 41.11 37.30 36.90 46.22 45.85 100.00 w Pip_Pel wCle 38.21 40.25 44.90 37.14 36.73 44.13 100.00 w wPip_Pel 37.10 43.15 43.15 39.43 37.50 46.80 84.27 100.00 Nlot Neoehrlichia lotoris (Nlot) 40.24 38.93 45.82 37.60 37.60 41.57 52.02 52.82 100.00 Echa U Ehrlichia chaffeensis (Echa) 41.67 37.45 45.02 41.37 39.44 41.96 52.82 54.98 60.24 100.00

C WP_026986745 Fodinicurvata fenggangensis U = characterized BioU 100 WP_051511448 Skermanella stibiiresistens = taxa with BioH = no biotin synthesis AHX11391 Neorickettsia helminthoeca

AIL65244 Rickettsiales bacterium Ac37b

81 CAM79807 Orientia tsutsugamushi Boryong 100 88 KDO03679 Rickettsia buchneri

100 AAU03813 Rickettsia typhi

52 ECH0326 (characterized BioU) Ehrlichia chaffensis U 68 ACZ49562 Anaplasma centrale U 37 KJV69137 Neoehrlichia lotoris

56 XP_029675926 Formica exsecta 48 arthropod (ant) mitochondria Rickettsiales XP_011860388 Vollenhovia emeryi - Anaplasmataceae 92 CAQ55098 wPip_Pel - Rickettsiaceae 55 Other Alphaproteobacteria 0.2 sub./site BAO99902 wCle

D NCBI Taxon Blastp results (%ID) taxid BioV BioG BtsA BioJ BioZ * EstN1 BioK BioU

768 Anaplasma NSS NSS NSS NSS 33 ~27; (2) NSS Present

943 Ehrlichia NSS NSS NSS NSS 33 ~27; (2) NSS Present

2021221 Rickettsiales endosymbiont of NSS NSS NSS NSS 33 NSS NSS NSS Trichoplax sp. H2

2602574 Ca. Aquarickettsia rohweri NSS NSS NSS NSS 33 NSS NSS NSS

1528098 Rickettsiales bacterium Ac37b NSS NSS NSS NSS 35 31; AIL65750 NSS Present

752179 Occidentia massiliensis NSS NSS NSS NSS 35 NSS NSS NSS

2163644 Ca. Deianiraea vastatrix NSS NSS 37; (1) NSS 30 NSS NSS NSS

(1) QED23496 (5-methyltetrahydropteroyltriglutamate--homocysteine S-methyltransferase); only 12% query coverage * % similarity to FabH; BioZ is similar to FabH but was able to complement E. coli ΔbioH mutants (PMID: 11320134). (2) similarity to a cohort of MhpC-like Abhydrolase 5 proteins (unrelated to BioU; E. chaffeensis Arkansas protein is ECH_0221).