emi12320-sup-0001-si
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Table S1. Bacterial strains and plasmids used in this study Strain Description Reference F- mcrA Δ (mrr-hsdRMS-mcrBC) Φ80dlacZ ΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG (Grant et al., 1990) Escherichia coli DH10B Mycobacterium smegmatis mc2155 Electrocompetent wild-type strain of M. smegmatis (Snapper et al., 1990) ∆hyd1 mc2155 with markerless deletion of MSMEG_2262 This work ∆hyd2 mc2155 with markerless deletion of MSMEG_2719 (Berney and Cook, 2010) ∆hyd3 mc2155 with markerless deletion of MSMEG_3931 This work ∆hyd12 mc2155 with markerless deletions of MSMEG_2262, MSMEG_2719 (aka Hyd3only) This work ∆hyd13 mc2155 with markerless deletions of MSMEG_2262, MSMEG_3931 (aka Hyd2only) This work ∆hyd23 mc2155 with markerless deletions of MSMEG_2719, MSMEG_3931 (aka Hyd1only) This work ∆hyd123 mc2155 with markerless deletions of MSMEG_2262, MSMEG_2719, MSMEG_3931 This work ∆dosR mc2155 with marked deletion of MSMEG_3944, HygR (O'Toole et al., 2003) Plasmids pJEM15 Escherichia coli-mycobacterial shuttle vector for in trans transcription fusions to lacZ; Kmr (Timm et al., 1994) pJEMhyd1-lacZ pJEM15 fused to promoter region of the Hyd1 operon This work pJEMhyd2-lacZ pJEM15 fused to promoter region of the Hyd2 operon This work pJEMhyd3-lacZ pJEM15 fused to promoter region of the Hyd3 operon This work pJEMhyd3T-lacZ pJEM15 fused to truncated promoter region of the Hyd3 operon (lacking DosR motifs) This work pX33 pPR23 (Pelicic et al., 1997) carrying a constitutive xylE marker; Gmr+ (Gebhard et al., 2006) pXhyd1KO pX33 harboring the MSMEG_2262 deletion This work construct. pXhyd2KO pX33 harboring the MSMEG_2719 deletion (Berney and Cook, construct. 2010) pXhyd3KO pX33 harboring the MSMEG_3931 deletion This work construct. 2 Table S2. Primers used in this study Name Sequence (5’-3’) Enzyme RT-PCR 2261-2262FW CGGGGATGTGGAAGACGCT 2261-2262RV ACGGATGCCACAACAGGTC 2262-2263FW CACGGTGTTCAAGACCCAGA 2262-2263RV GCCTGGCAGATGTTGCTTAT 2263-2264FW CGAAGATCAAGACTATCAGG 2263-2264RV TCAGGCGCAGGTAGCCGT 2264-2266FW TTAGGGAGACCTTACTTTCTGG 2264-2266RV GTCACTGGATGAACGGCCC 2266-2267FW GGGGCCGTTCATCCAGTG 2266-2267RV GCGTCGGGATCGAGGACGA 2267-2268FW ACCTCGACGAGATCGCCAAA 2267-2268RV TCGATCGAGATCACCGTGGG 2268-2269FW CACACCCACGGTGATCTCGA 2268-2269RV GCGACCGGCCGCACAGAGA 2269-2270FW ATTTCGCGTCCAACGGGGAG 2269-2270RV CGAGGGTTTCCCACTCGTC 2717-2718FW CCTCCAGTTCGCGCCTGT 2717-2718RV TGGTCCACGCGGGCCTTT 2718-2719FW CTCGTCGTCGGACTGCGTGT 3 2718-2719RV GACTTCGACGAGAACTACAG 2719-2720FW AGATGCGACTGGTGATGAAGT 2719-2720RV ATTCCCCGACAAGTTCATGC 2720-2721FW AGTTCACCGCGTTCGGCTCT 2720-2721RV CATCGAGAATGTCGGAAACCT 2721-2722FW CGAACTCGTGATGTTGAGC 2721-2722RV CTGTTCTGCTTCGACCTGAT 3926-3927FW TGTGTGAACCTTGTTCTCGTT 3926-3927RV CGGTCTGCCTTCTGCGACT 3927-3928FW GGAGTCGCAGAAGGCGAGA 3927-3928RV CCTTGGACGATGGGCGCTA 3928-3929FW GTCACGCCGCAGGCATCT 3928-3929RV CTGGACACACTCGCAGCC 3929-3930FW TGGTCACCAGGATCTTCGACT 3929-3930RV CGGCAGTGGAGTTCTCGC 3930-3931FW ACCGGCGATCACGGTCACTT 3930-3931RV GAACTGGGCATCCTGTTTCG 3931-3932FW CCTCTTCCCCTTCGTCGCT 3931-3932RV GATGCGATCAAGGCCGGTT 5’ RACE Hyd1RACE1 TGGTGTTGCCACTACAT Hyd1RACE2 GTCGGTCAACGAATCCA Hyd1RACE3 CGGACAACGTTGCCAGCGAGT Hyd2RACE1 GAACTCGTGATGTTGAGC 4 Hyd2RACE2 GGGCACAGCAGGATGA Hyd2RACE3 GCGGATGTCGAGATCGAGCCG Hyd3RACE1 ATGAAGGAATTGTTTCCAG Hyd3RACE2 GATCAGCGTGGCCACCAG Hyd3RACE3 GATCGTGAGGATGCCTTTGTCGT Hyd3RACEAlt1 TTGTTTCCAGGACTGCG Hyd3RACEAlt2 GTGGCCACCAGTCGG Hyd3RACEAlt3 TTTGTCGTAACCGGCCTTGATCG lacZ Fusions Hyd1LacZFW AAAAGTACTCGACCAGACGCGCGGCCT ScaI Hyd1LacZRV CGCGGATCCATTCTGACCGGCACTGTGAC BamHI Hyd2LacZFW AAATTTAGTACTGCCGTAGATCTCGATGACGC ScaI Hyd2LacZRV AAATTTGGATCCGGTGATCAGGTCGAAGCAGAA BamHI Hyd3LacZFW AAATTTAGTACTCGGACATGATGTCCACTCTCG ScaI Hyd3LacZRV AAATTTGGATCCTCGATCCGGATGATGTGGTCA BamHI Hyd3TLacZFW AAAAGTACTGACTTCGGTCCCTACCCGTC ScaI 2262KOLeftFW AAATTTACTAGTCCTGATACGCGAGCTAAGGAA SpeI 2262KOLeftRV GCCCGGCACGTTGGGCTCGTCTGCGTT 2262KORightFW CCAACGTGCCGGGCACGGTGTTCAA 2262KORightRV AAATTTACTAGTGAGCCAGTTGTCGTTCCAAT SpeI 2719KOLeftFW AAATTTACTAGTGCACACCGTCACGCATCAG SpeI 2719KOLeftRV CTTCGAGGAGGAGAACGATCGGGAGCA Deletions 5 2719KORightFW TCTCCTCCTCGAAGTCGATCTTGGTGTA 2719KORightRV AAATTTACTAGTTATTGGTGCGGGTTCGGTAA SpeI 3931KOLeftFW AAATTTACTAGTCGCTCATCTACAACTCCT SpeI 3931KOLeftRV CGACAACCACTGGATCACCCACAAGCTC 3931KORightFW TCCAGTGGTTGTCGGCGCAGGTAGGG 3931KORightRV AAATTTACTAGTGGAACGGCATTCCTCCTTGGT SpeI 6 Table S3. Annotation of the hydrogenase operons and adjacent maturation genes. Locus Gene Name Proposed Function hyd1 Operon MSMEG_2261 Hypothetical MSMEG_2262 hupS Hyd1 Small Subunit MSMEG_2263 hupL Hyd1 Large Subunit MSMEG_2264 hupD1 Endopeptidase of Hyd1 Large Subunit Precursor MSMEG_2266 Hypothetical MSMEG_2267 Complex Assembly (Contains TTP Repeats) MSMEG_2268 Putative [2Fe-2S] Cluster Protein MSMEG_2269 Conserved Hypothetical (Contains NHL Repeats) MSMEG_2270 Hypothetical hyd2 Operon MSMEG_2718 Putative [2Fe-2S] Cluster Protein MSMEG_2719 hhyL Hyd2 Large Subunit MSMEG_2720 hhyS Hyd2 Small Subunit MSMEG_2721 hypB Ni2+ Binding / Storage MSMEG_2722 hybA Ni2+ Insertion into Large Subunit hyd3 Operon MSMEG_3926 ctm1 Ca2+-Exporter (Type IIA P-type ATPase) MSMEG_3927 hupD2 Endopeptidase of Hyd3 Large Subunit Precursor MSMEG_3928 hyhL Hyd3 α Subunit MSMEG_3929 hyhS Hyd3 δ Subunit MSMEG_3930 hyhG Hyd3 γ Subunit MSMEG_3931 hyhB Hyd3 β Subunit MSMEG_3932 hspX Chaperone / Heat Shock Protein (Membrane-Bound) 7 Hyd1-Associated Maturation Genes MSMEG_2271 hypB1 Ni2+ Binding / Storage MSMEG_2272 hypA1 Ni2+ Insertion into Large Subunit MSMEG_2273 hypF1 Biosynthesis of CN- Ligands MSMEG_2274 hypC1 Transfer of Fe-(CN-)2-(CO) Moiety to Large Subunit MSMEG_2275 hypD1 Transfer of Fe-(CN-)2-(CO) Moiety to Large Subunit MSMEG_2276 hypE1 Biosynthesis of CN- Ligands Hyd2-Associated Maturation Genes MSMEG_2702 hypD2 Transfer of Fe-(CN-)2-(CO) Moiety to Large Subunit MSMEG_2703 hypC2 Transfer of Fe-(CN-)2-(CO) Moiety to Large Subunit MSMEG_2704 Transposase Interruption MSMEG_2705 hypE2 Biosynthesis of CN- Ligands MSMEG_2706 gmhA Phosphoheptose Isomerase MSMEG_2707 Hypothetical MSMEG_2708 Hypothetical MSMEG_2710 Hypothetical MSMEG_2711 hypF2 Biosynthesis of CN- Ligands MSMEG_2712 hypC3 Transfer of Fe-(CN-)2-(CO) Moiety to Large Subunit MSMEG_2713 hupD3 Endopeptidase of Hyd2 Large Subunit Precursor MSMEG_2714 Hypothetical (Membrane-Bound) MSMEG_2715 Conserved Hypothetical MSMEG_2716 Conserved Hypothetical MSMEG_2717 Conserved Hypothetical 8 Figure S1. Multiple sequence alignment of the amino acid sequences of hydrogenase large subunits. The mycobacterial large subunits were aligned with model examples of each group/subgroup of [NiFe]-hydrogenase using ClustalW. The cysteine residues that ligate the [NiFe] centre are underlined and are absent in the Ehr proteins. The Nterminal L1 and C-terminal L2 motifs surrounding the cysteine residues are highlighted in grey. Residues removed by endopeptidases during maturation are colored in red. Nterminal residues have been omitted from the Group 4 (1 – 152), Ehr (1 – 137), and EhrTB sequences (1 – 117). Group 1 (Desulfovibrio gigas) Group 2a (Anabaena variabilis) Group 2b (Ralstonia eutropha) Group 5 (Streptomyces avermilitis) Group 3a (Methanothermobacter marburgensis) Group 3b (Pyrococcus furiosus) Group 3c (Methanothermobacter marburgensis) Group 3d (Ralstonia eutropha) Group 4 (Escherichia coli) Ehr (Geobacter sulfurreducens) ----------------------SEMQG ------------------------MTI --------------------------M ---------MTTIIPEPSHKSESDG-L -------------------------MS ----------------------MRNLY --------------------------M --------------------------S MDYRQRPAPTTDAETYEFINELGDKKN ----------------MDYYQVAGDEV 05 03 01 17 02 05 01 01 179 148 Hyd1 (Mycobacterium smegmatis) Hyd2 (Mycobacterium smegmatis) Hyd3 (Mycobacterium smegmatis) EhrTB (Mycobacterium tuberculosis) ------------------------MTE ---------MTTTAPKPSDTEREPGQL ----------------------MNPEV MRTDAGPAPEFTDTGAFPFLAVEGPGV 03 18 05 142 NKIVVDPITRIEG--HLRIEVEVEGGKIKNAWSMSTLFRGLEMILKGRD-PRDAQHFTQR KTLDISPVGRVEGDLDVR--VEIEDGQVVNAWTHAELFRGFEIILRGKD-PQAGLIVTPR ERLVVGPFNRVEGDLEVN--LEVASGRVCSARVNATMYRGLEQILLHRH-PLDALVYAPR VEMAWDPITRIVGSLGIYTKIDFKQKEVVECHSTSSIFRGYSIFMKGKD-PRDAHFITSR ERIVISPTSRQEGHAELVMEVDDEGIVTKGRYFSITPVRGLEKMVTGKA-PETAPVMVQR IPITVDHIARVEGKGGVEIIVGDEG-VKEVKLNIIEGPRFFEAITIGKK-LEEALAIYPR VKLTMEPVTRIEGHAKITVHLDDAG-NVEDTRLHVMEFRGFEKFLQGRP-IEEAPRIVPR RKLVIDPVTRIEGHGKVVVHLDDDN-KVVDAKLHVVEFRGFEKFVQGHP-FWEAPMFLQR NVVPIGPLHVTSDEPGHFRLFVDGENIIDADYRLFYVHRGMEKLAETRMGYNEVTFLSDR HEVAVGPVHAGIIEPGHFRFQCHGEEVFHLEISLGYQHRGIEAGLAGGP-HPRTLQVMET 62 60 58 76 61 63 59 59 239 207 LDLFVSPLGRVEGDLDVR--VTINDGVVTSAWTEAAMFRGFEIILRGKD-PQAGLIVCPR VEMSWDPITRIVGSLGIYTKIDFENREVVECHSTSSIFRGYSLFMKGKD-PRDAHFITSR RTLSVGALARVEGEGALHVTLRDGA-VVGTQLNIYEPPRFFEAFLRGRA-HTEPPDLTAR YEIPVGPVHAGLIEPGHFRFSVAGETIVRLKARLWFVHRGIEKLFHGRP-ATAAVDLAER 60 77 63 201 ACGVCTYVHALASVRAVDNCVGVKIPEN------ATLMRNLTMGAQYMHDHLVHFYHLHA ICGICGASHLTSASWALDTAWNTTVPR------NAILARNLGQIVETIQSIPRYFYGLFA VCGICSVSQSVAASRALADLAGVTVPA------NGMLAMNLMLATENLADHLTHFYLFFM ICGICGDNHATCSCYTQNMAYGVQPPH------IGEWIVNLGEAAEYMFDHNIFQENLVG 116 114 112 130 Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group 1 2a 2b 5 9 Group Group Group Group Group Ehr 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 ICGVCPIPHTLASVEAIDDSLDIEVPK------AGRLLRELTLAAHHVNSHAIHHFLIAP ICSFCSAAHKLTALEAAEKAIGFTPRE------EIQALREVLYIGDMIESHALHLYLLVL ICGICDVQHHLAAAKAVDACFGFEPDD---VLPAAYKMREIMNWGSYMHSHGLHFYFLAA ICGICFVSHHLCGAKALDDMVGVGLKSGIHVTPTAEKMRRLGHYAQMLQSHTTAYFYLIV VCGICGFAHSTAYTTSVENAMGIQVPER------AQMIRAILLEVERLHSHLLNLGLACH AAGDTTIGHGQAYCMIMESLGGIKAPP------RAEVLRGIALELERLANHTGDLGAIAG 115 117 116 119 293 261 ICGICGGSHLYKSAYALDTAWRTHMPP------NATLIRNICQACETLQSIPRYFYALFA ICGICGDNHATCSCYAQNMAYGVQPPH------VAEWIVNLGEAAEYMFDHNIFQENLVG VCGICPVAYQVSACNAIEDACGVTVDP------EIVQLRRLLYCGEWIHSHALHIFLLHL ISGDTSAAHALAHSLAIEDALGIELPH------EVHRLRALIVELERLYNHAADLGALAN 114 131 117 255 LDWVNVANALN----------------------ADPAKAARLANDLSPRKTTTESLKAVQ IDLTNKKYRSS---------------------RFYDEAVRRFAAFTGKSYELGVTISSKP PDFTREIYAGR---------------------PWHTDATARFSPTHGKHHRLAIAARQRW VDFCEKMVSETNPGVLAQAEKTEAPHAGEHGYKTIADIMRSLNPFTGEFYREALQVSRWT --------------------------------------DFVPENLMADAINSVSEIRKNA PDYLGYSSP------------------------------LKMVNEYKKELEIALKLKNLG PDFIAGKDR-------------------------KTRNVFQIIKDAPDVALQAIELRKNA PEMLFGMDAPP-----------------------AQRNVLGLIEANPDLVKRVVMLRKWG ------------------------------------------FTGFDSGFMQFFRVRETS ------------------------------------------DVGYLPTSSFCGRIRGDF 154 153 151 190 137 147 151 156 311 279 IDLTNKNYAKS---------------------KLYDEAVRRFAPYVGTSYQPGVVLSAKP VDFCEKMVSETNPSVLAKAENTEAPHAGMHGYRTIADIMRALNPFTGEFYREALQVSRWT PDFLGHPDG------------------------------ISLAREHPELVERGLSLKKTG ------------------------------------------DVGYSLANAHAQRIRENL 153 191 147 273 AKVKALVESGQLGIFTNAYFLGGHPAYVLPAEVDLIATAHYLEALRVQVKAARAMAIFGA VEIYALFGGQWP-----------HSSYMVPGGVMCAPTLTDITRAWAILEYF-RTNWLEP FTLMGTLGGKWP-----------HTESVQPGGSSRAIDAAERVRLLGRVREF-R-CFLEQ REMFCLMEGRHV-----------HPSTLYPGGVGTVATIQLMTDYTTRLMRY-VEFMKKV QYVVDMVAGEGI-----------HPSDVRIGGMADNITELARKRLYAR-------LKQLK SWMMDVLGSRAI-----------HQENAILGGFGKLPSKETLEEMKA-------KLRESL LEIVRATGGRPI-----------HPTSSTPGGISTELDDETQKDLLQ-------KAQRNV QEVIKAVFGKKM-----------HGINSVPGGVNNNLSIAERDRFLNGEEGL-LSVDQVI MKMAEILTGARK-----------TYGLNLIGGIRRDLLKDDMIQTR-----------QLA LNMTAVLCGSRF-----------GRGLLVPGGTIFDLTPGLR-----------DDLLKRL 214 201 198 238 179 189 193 204 349 317 VEVYAIFGGQWP-----------HSSFMVPGGVMSAPTLSDVTRAIAILEHW-NDNWLEK REMFCLMEGRHV-----------HPSTLYPGGVGTVATIQLMTDYMTRLMRY-VEFMKKV NRIMEQIGGRAI-----------HPVNVRLGGFYSAPKPGDLKSLAA-------LLRKSL LRRNAAVTGHRL-----------LRGAIRAGGVALRALPDT----------------DEL 201 239 189 306 KNPHTQFTVVGGCTNYDSLRPERIAEFRKLYKEVREFIEQVYITDLLAVAGFYKNWAGIG VWLGCSLERYEEIQTYDDFMDWLEADIKHRESDLGFYWRMGLDIGLDRYGAGVGKYVSWG TLYAAPLEDVVALDSEVALWRWHAQAP--QAGDLRCFLTIAQDAALDQMGPGPGTYLSYG VPMHDDLFDFFYEAMPGYEKVGLRRTLLGCWGSFQDPA-VCNFSYKDMEKWGRAMFVTPG PKVNEHVELMIGLIEDKGLPEGLGVHNQPTLASHQIYGDRTKFDLDRFTEIMPES----SLAEYTFELFAKLEQYR------EVEGEITHLAVKPRGDVYGIYGDYIKASDGE-----ELAEATLELAVPIFEENIDLVNSLGNIETYHTGLVKNG-VWDVYDGIVRIKDKEGNLFRDYAQDGLRLFYDFHQKHRAQVDSFADVPALSMCLVGDDDNVDYYHGRLRIIDDDKHIVRQQMRREVQELVDVLLSTPNMEQRTVGIGRLDPEIARDFSNVGPMVRASG----------AEARRDLTNAIELLWSSPSVMGRLEGTGTLTREQALELGLVGPAARACG----------- 274 261 256 297 234 237 251 263 298 366 10 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr Hyd1 Hyd2 Hyd3 EhrTB 1 2a 2b 5 3a 3b 3c 3d 4 QWLGCSVDRWLENKTWNDVLAWVDENESQYNSDCGFFIRYCLDVGLDKYGQGVGNYLATG VPMHDDLFDFFYEALPGYEQVGLRRTLLGCWGSFQDPE-HCNFSYRDMEAWGRKMFVTPG DDALYTVREVADLDCP-------DVVFDHEFLALG-TADAYPIESGAIVRSAGP-----AALAVDLAEVATLTLANSVVYDRFAGTAVLHPDDASALGCLGYVARASG----------- 261 298 235 355 KTSNFLTCGEFPTDEYDLNSRYTPQGVIWGNDLSKVDDFNPDLIEEHVKYSWYEGADAHH YLPHEDKYQKPTIEG-RNAAMIMKSGVYDSFSDTHTLMDHTFARENTTHSWYDEGNADVH AYPQP------------EGGFCFAQGVWRSAQGRLDALDLAAISEDATSAWLVDQGGARH VVVDGKLVTTSLVDINLGIRILLGSSYYDDWTDQEMFVKNDPLGNPVDRRHPWNQHTNPH -----------------------------WYDDPEIAKRACSTIPLYDGRNVEVGPRARM ---------------------EFPSEDYKEHINEFVVEHSFAKHSHYKGKPFMVGAISRV ---------------------EFKPADYADTIAEHVKPYSWLKFPYIKDLGYPDGVYRVS ---------------------EFDYHDYLDHFSEAVEEWSYMKFPYLKELGREQGSVRVG ----------------------------HARDTRADHPFVGYGLLPMEVHSEQGCDVISR ----------------------------IKRDVRLDYPFGIYRMTHLPVATVFHGDVCAR 334 320 304 357 265 276 290 302 430 398 TYFEPSLYENPTIEG-RNAALIGRSGVFADG--RYFEFDQANVTEDVTHSFY-EGNRPLH VVVDGKLVTTSLVDINLGIRILLGHSYYDDWEDQEMFVKTDPLGNPVDRRHPWNQHTNPK ---------------------SFPLADFTTHVGEAQVPHSTALHAALDDGRYLTGPLARY ----------------------------LRSDARVEHPTIVLPITEIGAP---DGDVLAR 317 358 274 384 PYKG--------------VTKPKWTEFHGEDRYSWMKAPRYKGEAFEVGPLASVLVAYAK PFDR--------TTKPTQKNTKDFKNAYSWSTAVLHQDFGR----LEVGPLARQLVAGGQ PANG--------LTAPAP----DKVGAYTWNKAPRLAGAVL-----ETGAIARQLAG--PQKRDMEDKYSWVMSPRWFDGTDHLALDTGGGPLARLWSTALAGLVDIGYVQSTGHSVKI VEFQG---------------------------------------------FKERGVVAQH VNNK-------------------------------------------DLLYGRAKDLYES PLSR-------------------------------------------LNVADKMPDAAPK PLGR-------------------------------------------MNVTKSLP--TPL LKVR-----------------------------------------------------INE TMIR-----------------------------------------------------WLE 380 368 344 417 280 293 307 317 437 405 PFEG--------ETIPVNPEDGRRQGKYSWAKSPRYAVPGLGNVPLETGPLARRMAASAP PQKRDFDENYSWVMSPRWFDGKDHLALDTGGGPLARLWATALAGLVDIGYVRSTGNSVQI SLNS-------------------------------------------AHLPPIAREAATS YTVR-----------------------------------------------------RDE 369 418 291 391 KHEPTVKAVDLVLKTLGVGPEALFSTLGRTAARGIQCLTAAQEVEVWLDKLEANVKAGKD HGESWQHYDGFILD--AFQKMGGASIHLRQLAR----VHEIVKLYRQAERCLREFVLN-D -------AQPLVRD--AVARCG-ATVYTRVLAR----LVELARVVPLMEDWLQSLEIG-A NLPKTALKGPVEFE--WKIPQYGSNTIERDRARTYFQAYAAACALHFAEKALAEIRAGHT VARALEMKTALSRAIEILDELDTSAPVRADFDE--------------------------HKELLKGTNPFANN--LAQALELVYFIERAIDL----IDEVLIKWPVKERDKVEVR---AQDYFKEFQDKFG----YAQQTLLYHWARLIEV----LACAECAADALEG-DLSGE---AQEALERFHAYTKG--RTNNMTLHTNWARAIEI----LHAAEVVKELLHDPDLQKDQL-VYTALNMIDYGLDN--LPGGPLMVEGFTYIPHR--------------------------AQKSMDFIEEQLRQ--LPGGGHRVATAPCAGG---------------------------- 440 421 389 475 313 343 354 369 468 435 DAETHQDDDPLFAD--IYNAIG-PSVMVRQLAR----MHEGPKYYKWVRQWLDDLELK-E NLPKTALKGPVEFE--WKIPAKGSNTIERNRARTYFQAYAAACALHFAEKALTEIRAGRT AGLGAQCRNPFRSI--VVRAVEVVFAIEEALRI----IAEYQE--PPRPFVEVPAR---FAASAALAQHIVES--HTGPIEYAATLHPVGAP--------------------------- 421 476 339 422 11 Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr 1 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Ehr Hyd1 Hyd2 Hyd3 EhrTB 1 2a 2b 5 3a 3b 3c 3d 4 DLYTDWQYPTESQGVGFVNAPRGMLSHWIVQR-GGKIENFQHVVPSTWNLGPRCAERKLS PWYIKPKE-KDGRGWGATEASRGSLCHWIDIE-GGKIKNYQVIAATTWNVGPRDSEGVRG PYWASAHLPDQGAGVGLTEAARGSLGHWVSVR-DGRIDNYQIVAPTSWNFSPRDIAGQPG KTWEKFEVPDEGIGCGFTEAVRGVLSHHMVIR-DGKIANYHPYPPTPWNANPRDSFGTPG -------RGTGKLGIGAIEAPRGLDVHMAKVE-NGKIQFYSALVPTTWNIPTMGPATEG----------DGFGVSTTEAPRGILVYALKVE-NGRVAYADIITPTAFNLAMMEEH----KFPDSLERQAGDGVGIVEAPRGTLTHHYTCDENGLITKANIVVATIQNNPAMEMG----VLTPPPNAWTGEGVGVVEAPRGTLLHHYRADERGNITFANLVVATTQNHQVMNRT---------------FALGFAEAPRGDDIHWSMTGDNQKLYRWRCRAATYANWPTLRYMLR------------RLAVALTEGWRGEICHVAVTDDAGRFSRYKIVDPSFHNWQGLALALR-- 499 479 448 534 364 388 409 424 514 482 SFYTKPVEYAEGKGFGSTEAARGALSDWIVIE-DSKIKNYQVVTPTAWNIGPRDASEVLG KTWEKFEVPDEGIGCGFTEAVRGVLSHHMVIR-DGKIANYHPYPPTPWNANPRDSYGTPG ----------AGVGHGVSEAPRGLLYHRYEIGHDGLVRTATLIPPTAQNQAAIEHE--------------SSGIGIVEGWRGTIVHRVEIDVDGRITRAKVVDPSWFNWPALPVAMA-- 480 535 385 354 AVEQALIGTPIAD-----PKRPVEILRTVHSYDPCIACGVHVIDPESNQVHKFRIL---PIEEALIGTPIED-----SRDPVEVGHVARSFDSCLVCTVHAHDAKTGEELARFRTA--AVEKALEGAPVLQG----ETTPVAVQHIVRSFDPCMVCTVH------------------PYEDAVQGQPIFEENDREHFKGIDIMRTVRSFDPCLPCGVHMYLGEGKTLEKLHSPTQSV -------------------FHHEYGPHVIRAYDPCLSCATHVMVVDDEDKSVIKNEMVKI --VRMMAEKHYN---DDPERLKLLAEMVVRAYDPCISCSVHVVKL----------------IQKVAQDYIKPGVEVDDKIFNLMEMVIRAYDPCLSCATHTIDSQMRLATLEVYDSEGD --VRSVAEDYLGGHGEITEGMMNAIEVGIRAYDPCLSCATHALGQMPLVVSVFDAAGRLI ------------------GNTVSDAPLIIGSLDPCYSCTDRMTVVDVRKKKSKVVPYKEL ------------------GQQISDFPLCNKSFNLSYCGFDL------------------- 550 531 485 594 405 428 467 482 556 505 PIEQALVGSPIVD-----AEDPVELGHVARSFDSCLVCTVHAYDGKTGKELNRFVINGMV PYEDAVQGQPIFEENDREHFKGIDIMRTVRSFDPCLPCGVHMYLGGGKTLDLLHTPTQSA --LAVLVAANLD---RDDAELTQLCERAIRNHDPCISCATHFLTLTLDRG---------------------------DTIVPDFPLANKSFNQSYAGNDL------------------- 535 595 430 492 ------------------------------------TGE---------------------------------LVKRI-------DERAR-------ERYSIERKNSPLK ------------- 550 531 485 597 405 428 472 487 569 505 ------------TGD---------------------------------- 535 598 430 492 12 Figure S2. Multiple sequence alignment of the amino acid sequences of hydrogenase small subunits. The mycobacterial small subunit sequences were aligned with model examples of each group/subgroup of [NiFe]-hydrogenase using ClustalW. The cysteine residues proposed to ligate the iron-sulfur clusters are highlighted in yellow for the proximal cluster, blue for the medial cluster, and green for the distal cluster. Annotation was based on the three-dimensional structures of the Group 1 and Group 3a [NiFe]hydrogenases, as well as the predicted binding sites for other enzymes (Volbeda et al., 1995; Mills et al., 2013; Raleiras et al., 2013). Hydrogenases generally bind a [4Fe-4S] proximal cluster. The oxygen-tolerant Group 1 [NiFe]-hydrogenases instead encode a unique [4Fe-3S] cluster via six cysteine residues, though these residues are not conserved in oxygen-tolerant non-Group 1 enzymes (Fritsch et al., 2011). Alignments suggest that a cysteine residue in this cluster may be substituted for an asparagine in Group 2a enzymes and an aspartate in Group 5 residues. The medial cluster is generally a [3Fe-4S] species, but it is a [4Fe-4S] species in Group 3a enzymes. This alignment suggests Group 3b, Group 3c, and Group 5 hydrogenases may also bind a [4Fe-4S] medial cluster via a well-conserved additional cysteine residue. The distal cluster is also generally a [4Fe-4S] species. A solvent-exposed histidine substitutes for a cysteine residue in Group 1 enzymes, and likely Group 2b and Group 5 hydrogenases too. By contrast, alignment suggests a glutamine substitutes in Group 2a hydrogenases. The small subunits of the Group 3b, Group 4, and Ehr proteins are truncated and hence probably only ligate a proximal cluster. Group Group Group Group Group Group Group Group Group Group 1 O2-Sensitive (Desulfovibrio gigas) 1 O2-Tolerant (Ralstonia eutropha) 2a (Anabaena variabilis) 2b (Ralstonia eutropha) 5 (Streptomyces avermilitis) 3a (Methanothermobacter marburgensis) 3b (Pyrococcus furiosus) 3c (Methanothermobacter marburgensis) 3d (Ralstonia eutropha) 4 (Escherichia coli) Hyd1 (Mycobacterium smegmatis) Hyd2 (Mycobacterium smegmatis) Hyd3 (Mycobacterium smegmatis) EhrTB (Mycobacterium tuberculosis) ------------------MKFCTAVAVAMGM MVETFYEVMRRQGISRRSFLKYCSLTATSLG --------------------------------------------------------MNAPV --------------------MTAATPDTVGA --SLIARIKRFLGLEAEAKREEPEKEKSEPV -----------------------------------------------------------------------------MRAPHKDEIASHEL ----------MSNLLGPRDANGIPVPMTVDE 13 31 00 05 11 54 00 00 14 21 -----------------------------------------------------------------------------------------------MGWVAKIFRVGRVVEPAAPLPAAIAE 00 00 00 26 13 Group Group Group Group Group Group Group Group Group Group -GPAFAPKVAEALTAKKRPSVVYLH-NAECTGCSESLLRTVDPYVDELILDVIS-----LGPSFLPQIAHAMETKPRTPVLWLH-GLECTCCSESFIRSAHPLAKDVVLSMIS----------------------MTNVLWLQ-GGACSGNTMSFLNAEEPTVCDLIADFGI-----K -------CTGLASAKPGVLNVLWIQ-SGGCGGCSMSLLCADTTDFTGMLKSAGI-----H -A----DAGGAPADETPTIHILWINAGLSCDGDSVALTAAMQPSIEEIVLGVLPGLPKIA GASKEEVEKVAEENAKPRIGYIHL---SGCTGDAMSLTENYDILAELLTNMVDI------------------MGKVRIGFYAL---TSCYGCQLQLAMM-DELLQLIPNAEIV-----------------MAEKIKIGTMWL---GGCSGCHLSIADFHEKLLDVMEHADFE-----PATPMDPALAANREGKIKVATIGL---CGCWGCTLSFLDMDERLLPLLEKVTLL-----SIASMKASLLKKIKRSAYVYRVDC---GGCNGCEIEIFATLSPLFD------AE------ 65 84 37 52 66 105 37 39 65 66 Hyd1 Hyd2 Hyd3 EhrTB -----------------MASVLWFQ-GGACSGNTMSFLNADEPNVVDLIVDFGL-----D -M----PTEAAVKAEQALIHVLWINAGLSCDGDSVALTAATQPSIEEIALGALPGLPKIA -------------MSVPSLAVWKF---ASCDGCQLTLLDCEDELLTLAGEVRIA-----PPA--------GVRGSLQIRHVDA---GSCNGCEVEISGAFGPVYD------AE------ 37 55 38 63 Group1I Group1T Group2a Group2b Group5 Group3a Group3b Group3c Group3d Group4 -----MDYHETLMAGAGHAVEEALH------EAIKGDFVCVIEGGIPM---GDGGYWGKV -----LDYDDTLMAAAGHQAEAILEEIM---TKYKGNYILAVEGNPPL---NQDGMSCII V-----LWHPSLGLELGDNVQTLLWDCI---LGKIPLDILVFEGTVVNAP-NGTGEWNRF M-----LWHPSLSLESGVEQLQILEDCL---QGRVALHALCVEGAMLRGP-HGTGRFHLL VHWPLIDFECGPVGGS----DTFIEWFFKGERGEIDPFVLVVEGSIPNESIKPEGYWCGF ---------VYGQT-------LV------DLWEMPEMDLALVEGSVCLQD------------------CWFMI-------DR------DSIEDEKVDIAFIEGSVSTEE------------------FSPVL-----MDTK------YDEIPE-LDVVVIEGGIVNDE------------------RSSLT--------D------IKRIPERCAIGFVEGGVSSEE------------------RFGIK-------VV------PS--PRHADILLFTGAVTRAM---------- 111 133 88 103 122 133 65 68 92 92 Hyd1 Hyd2 Hyd3 EhrTB L-----LWHPSLGLELGNNAQKVFWDCA---KGERPLDIFVFEGTVIEAP-NGTGQMDMF VHWPLIDFECGPTGGA----DDFLAWFFRAERGELDPFVLVVEGSIPNEEIKNEGYWCGF ---------TFLzE-------AS------SAFTGGPYDISLVEGSITTPA------------------RFGAR-------LV------AS--PQHADALLVTGVVTHNM---------- 88 111 66 89 GR--RNMY-------DICAEVAPKAKAVIAIGTCATYGGVQAAKPNPTGT---------GG--RPFI-------EQLKYVAKDAKAIISWGSCASWGCVQAAKPNPTQA---------AD--RPMK-------DWLNDLAQAASFVVAVGDCATWGGIPAMEPNPSES---------AGTGVPMI-------EWVSRLAAVADYTLAVGTCAAYGGITAGGGNPTDA---------GD--NPETGQPITTSEWIDRLAPKALAVVAIGTCATYGGIHAMAGNPTGA-------------EHSL-------HELKELREKAKLVCAFGSCAATGCFTRYSRGGQQA--------Q----E--V-------ELVKKIRENAKIVVAVGACAVQGGVQSWSEKP-LEELWKKVYGDA ----N--R-------EFAEELREKAKFVISYGTCAVYGGIPGLRNLWDKDEVIEEAYINS ----N--I-------ETLEHFRENCDILISVGACAVWGGVPAMRNVFELKDCLAEAYVNS ----RSPA-------LRAWQSAPDPKICISYGACGNSGGIFHDL---------------- 152 174 129 146 170 173 111 115 139 125 Hyd1 Hyd2 Hyd3 EhrTB AG--RPMK-------DWVTDLAGAAQIVVAIGDCACFGGIPAMEPNPSGS---------GN--DPATGQPITTSEWLDRLTPKATAVVAVGTCATYGGIHAMAGNPTGA-------------D--E-------RRIREIREQSKILVTIGACATAGGIQALRNMSDIDEYLSVVYAQP ----AGPL-------RKTLEATPRPRVVIACGDCALNRGVFADA---------------- 129 159 113 122 Group 1I Group 1T --------VGVN-------EALG-----KLGVKAINIAGCPPNPMNFVGTVVHLL-T----------TPVH-------KVIT-----D--KPIIKVPGCPPIAEVMTGVITYMLTF--- 188 209 Group Group Group Group Group Group Group Group Group Group 1I 1T 2a 2b 5 3a 3b 3c 3d 4 1I 1T 2a 2b 5 3a 3b 3c 3d 4 14 Group Group Group Group Group Group Group Group 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Group 1I 1T 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group Group Group 1I 1T 2a 2b 5 3a 3b 3c 3d 4 Hyd1 Hyd2 Hyd3 EhrTB Group Group Group Group Group Group Group Group 1I 1T 2a 2b 5 3a 3b 3c --------QGLQFLKRKEGGFLGKDFRAKSGLPVINIPGCPSHPDWITQILVAI---ATG --------CGLQYEGDQPGGLLGLNYRSRAGLPVINVAGCPTHPGWVTDALALL---SAR --------MGVP-------DYLGWDWKSQAGIPIVCVPGCPIQPDNFSETLTYLLYQAAG ------------------PSHESFVPIADLIDVDLALPGCPPSPEIIAKTVVALLN---KVK---------------FQPKKAEPVSKYIKVDYNIYGCPPEKKDFLYALGTFLI---ITTP-NEEGVIP-SEDVPHLEGRVKPLGEVIDVDFEVPGCPPRSDVAAEVVMALLK---ATAVPGAKAVVPFHPDIPRITTKVYPCHEVVKMDYFIPGCPPDGDAIFKVLDDLVN-----------------------YCVWGGTDKIVPVDVYIPGCPPTPAATLYGFAMALG---- 178 195 215 211 152 169 195 161 --------TGLQFHKREKGGFLGPDFRSKMGLPVINVPGCPAHPDWITQILVAL---ATG --------MGVP-------DYLGWDWKSKAGIPIVCVPGCPIHPDNLAETLTYLLYMATD GYI---------------ETLATSTPPAAHVRVDYQLQGCPIDRGQLLDTLAALLI-----------------------YGVVGAVGEVVPVDVEIAGCPPTPAAIMAALRSVTGKL-- 178 204 154 159 -KGMPELDKQGRPVMFFGETVHDNCPRLKHFEAGE---FATSFGSPEAKKGYCLYELG--DRIPELDRQGRPKMFYSQRIHDKCYRRPHFDAGQ---FVEEWDDESARKGFCLYKMG-RIGDIALDELNRPQTFFNTFTQTGCTRNVHFAYKA---TTAEFG--Q--RKGCLFYDLGLLTASDLDTLGRPRFYADQLVHHGCTRNEYYEFKA---SAEK---PS--DLGCMMENMGAAPMIPLDDKLRPTWLFGATVHEGCDRAGYYEQGQ---FALTYDSPK-----CLVKLG-----------NDMDYLQPML-----DLAGYTEACGCDLQT-----KVVNQGLCIGCGT-----------GSWPEDIDYPVCLECRLNGHP--------------------CILLEKGEP ----------GEEIELPSTNLCEVCPREKPPEGLAMDFIKRQFEVGKPEDDLCLIPQGLI ----------GRPFDLPSSINRYD--------------------------------------------------LEQKIHARGPGELDEQPAEILHGDMVQPLRVKVDREARR------ 242 263 230 246 265 249 182 219 209 191 RAGDITLDDLHRPETFFKTFTQTGCTRVQFFEYKQ---STLSFGEGT-RTGCLFYEFG-QAPMIPLDDALRPKWLFGQSVHEGCDRAGYYEQGD---FATEYGSPK-----CIVKLG-----------GRKPRLPAKTVCAECKLRGIT--------------------CVLVAESIP ------------------------------------------------------------ 232 254 184 159 CKGPDTYNNCPKQLFNQ-VNWPVQAGHP-------CIACSEPNFWD----------LYSCKGPTTYNACSTTRWNEGTSFPIQSGHG-------CIGCSEDGFWD----------KGSCRGPMTHSSCNRILWNR-VSSKTRAGMP-------CLGCTEPEFPFFD------LKPGTCKGTQAHADCNTRLWNG-EGSCTRGGYA-------CISCTEPGFEE----------PGHCWGPVVK--CNVPKRGWMNGIGG-----CPNVGGICIACTMPGFPD----------KFMCAMA-----CQTRALDMTNGRPELNSDRCIK----CGICYVQCPRSWWPEEQ-IKKELGCLGPVTRAGCNAR---------------CPGFGVACIGCRGAIGYDVAWFDSLAKVFKECMGPATVSICGA---------------ECPSIAIPCRGCYGPTARVEDQGAKMISAIASD -----------------------------------------------------------LAGYRYGRQIADDYLTQLGQGEEQVARWLEAENDPRLNEIVSHLNHVVEEARIR------ 283 305 275 287 307 298 226 265 209 255 CRGPMTHSPCNRILWNR-QSSKTRAGMP-------CLGCTEPEFPHFD------LAPGTCWGPVVK--CNVPKRGWINGIGG-----CPNVGGICIGCTMPGFPD----------KFMCLGPVTHAGCGA---------------LCPSCHRGCYGCFGPAATPN--SAALIPLLRR------------------------------------------------------------ 277 296 226 159 PFYSA------------------------------------------------------FYDRLTGISQFGVEANADKIGGTASVVVGAAVTAHAA--ASAIKRASKK-NETSGSEH-VFKTQTIMG-VPKELPPG-----------VSNKNYAVLTMVAKDTAPKWAEEDFFTV--PFHQTPKVAGIPIGLPTDMPKAWFVALASLSKSATPKRVKLNATADHPLIAPAIRKTRLK PFMDEPPGAKV-----SSSASGAYGAVVRKLRTLTARTVDKEPKWRHTGDRITTGYRPPW L----------------------------------------------------------YKVE---KGMTKEE-IIERM------------KMFNGHDERVEKMVEKIFSGGEQ----ED-----KTVDPEE-VAEQLDD--------IVGTFYTFTLPAALIPMKIKKEGK------ 288 360 320 347 362 299 261 308 15 Group 3d Group 4 ----------------------------------------------------------------------------------------------------------------------- 209 255 Hyd1 Hyd2 Hyd3 HydTB VFKTQKVSGMIPKEVPEG-----------TDHLTYMGLAAAARIAAPQWSKEDMFVV--PFMDEPPGGKV-----STAASGLYGSAIRSLRHITGRTVDKEPRWRHRGTKLESGATRTW -------DGMTDGE-VDRVF------------STFNVTRFDAERNDR------------------------------------------------------------------------ 323 347 253 159 16 Figure S3: Neighbor-joining phylogenetic tree classifying the putative [NiFe]hydrogenases of Mycobacterium smegmatis and Mycobacterium tuberculosis based on alignments of their small subunit sequences. The branches are labeled with the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Poisson correction method and alignment gaps were only eliminated in pairwise sequence comparisons. The alignment of the small subunits is generally consistent with the alignment of the large subunits (Figure 1), thus validating the classifications. 17 Figure S4. Neighbor-joining phylogenetic tree analyzing the distribution of [NiFe]hydrogenases across the genus Mycobacterium based on alignments of their large subunit sequences. The branches are labeled with the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Poisson correction method and alignment gaps were only eliminated in pairwise sequence comparisons. This alignment demonstrates that [NiFe]-hydrogenases are widespread in the environmental species of mycobacteria. However, the obligate pathogens in the genus only contain the Ehr proteins. NuoB was selected as an outgroup. 18 Figure S5. Neighbor-joining phylogenetic tree analyzing the distribution of [NiFe]hydrogenases across the order Actinomycetales based on alignments of their large subunit sequences. The branches are labeled with the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Poisson correction method and alignment gaps were only eliminated in pairwise sequence comparisons. Group 5 and Group 3b [NiFe]-hydrogenases are common among the actinomycetes, whereas the other groups are more sparsely distributed. 19 Figure S6. Promoter regions of the hydrogenase operons. The entire promoter regions shown were cloned to produce promoter-lacZ fusions. The transcriptional starts, as determined by 5’ RACE for the hyd1 and hyd2 promoters, are boxed. The -10 region, -35 region, and predicted ribosomal binding sites are shown in bold. The three DosR motifs (corresponding to the consensus TSGGGACTWWAGTCCCSA) in the hyd3 promoter are highlighted in grey. Promoter Region of hyd1 Operon (MSMEG_2261-2270): -650 CGACCAGACGCGCGGCCTCGCGGACGTCCTCGGTGTAGAAGATCTCGTGATCGGCCAGCG CGGGCGGGACGCCGCGGCTGCGCACCTTTCGCCGCACGGGATCGCTGGTGCGGCGGATGT CGATGAACCCGAGCCGGGTCAGCCGTGACATCCGGGAACCCCCAGCAACGCCCGGCCTGA TACGCGAGCTAAGGAAAGCCTAACTTTGGTCAGCGATAACGGATTTCGGTCCCGCACCGC AACCCTCCTGATCGTCAGACGCGCATGAGAGCACCTATCTTTGCGCAATCCGGACAGGAA TTGCGCGAAAGCGATAGGCATCCGCGCATAGGCGAGGATATGTTTGCCGCGTCGCCGAAA CTCCGAGCGGGAGGATGGAGCGCACCTTTCGCCGCACGGGATCGCTGGTGCGGCGGATGT CGATGAACCCGAGCCGGGTCAGCCGTGACATCCGGGAACCCCCAGCAACGCCCGGCCTGA TACGCGAGCTAAGGAAAGCCTAACTTTGGTCAGCGATAACGGATTTCGGTCCCGCACCGC 20 AACCCTCCTGATCGTCAGACGCGCATGAGAGCACCTATCTTTGCGCAATCCGGACAGGAA TTGCGCGAAAGCGATAGGCATCCGCGCATAGGCGAGGATATGTTTGCCGCGTCGCCGAAA -35 -10 +1 CTCCGAGCGGGAGGATGGAATGGCCAGCAACGGTCACAGTGCCGGTCAGAAT RBS M A S N G H S A G Q N +62 Promoter Region of hyd2 Operon (MSMEG_2722-2718): -405 GCCGTAGATCTCGATGACGCGGCCCCGCGGCAGGCCGCCGATGCCGAGCGCCACATCCAG CGAGATGGAGCCGGTGGGGATCACCGAGATCGGCTGGCGCACCTCTTCGCCGAGGCGCAT CACCGAGCCTTTGCCGAAATTCTTGTCGATCTGGGCCATCGCCAGTTCGAGGGCCTTTTC GCGATCTGGGGCCTGCTGCGCCATGGTGGTGCCTCTCCGAGTAGTCGTGTCTGACCGGTG TTCCGATCGGTTGGCCGTGACGTTAGAGCAGACCACCGACAAGTCCGGTCGAACTCTTCA CCACAGTAGACGAACACCTGTTCGATTCAAGTGGACACGCCGAGCTGTCCGGACCCGCAT CGCGGTTTTGCGCCGGACGGCACACGACAGGCCTACCGTGGGAGCATCCCGGGTCCGATC -35 -10 +1 GGACAGCGATGCACGAGATGGCGATAACCCAGAGCGTCGTCGATGCGGTGTGTGAGCACG RBS M H E M A I T Q S V V D A V C E H CGGCGGGACGGCGCGTGCACAGCGTGCGCCTCGAGGTGGGCGCGTTGTGTGCGGTGGTCC A A G R R V H S V R L E V G A L C A V V 21 CCGACTCGATGCTGTTCTGCTTCGACCTGATCACC P D S M L F C F D L I T +170 Promoter Region of hyd3 Operon (MSMEG_3932-3926): CGGACATGATGTCCACTCTCGCCCGGGCCCGGTGCGGCCGATAGCGTCCGAGGACCGTTG ACGGAAGGCCTTAGGTCACCTGCGGGACCGTCAAAGTGCCCTTTCGCGCGAATTGTGCAG DosR Motif ATCCGCCGCGGATTCTGCTGCGGCCTGGAACGGCATTCCTCCTTGGTCGTTCCCCAGTTC CGGTAGTAGCGCCGCGCGGCATAAAGCCCGGCGACTCCGACGACGAGTTTCGCTACTGCG GTGGACATATCCCGAAATCTCGTCACTTCGGGGACAGAAAACCAGGGACGAAAGTTCGTG DosR Motif GTGACGGGACTTCGGTCCCTACCCGTCACCCGTGCCAGGTGGTCGGATCACTGACGTGGA DosR Motif AACGATTCCTACGAGTGAGGAGGACCGATGACCAAACTTCCTGAACGATCACGAGCACGC RBS M T K L P E R S R A R TCGCTCTTTCCGGAGATGTCCGACTTCTTCGCGGGTCTGCCGTCGTGGGCCTCGATCCGC S L F P E M S D F F A G L P S W A S I R CCGGTTTTCGGTGACCACATCATCCGGATCGA P V F G D H I I R I 22 Figure S7. Construction of unmarked deletion mutants in genes MSMEG_2262, MSMEG_2719 and MSMEG_3931 of M. smegmatis mc2155 and combinations thereof: double mutants: 22622719, 22623931, 27193931 and triple mutant: 226227193931. A. Schematic diagram of the vector pX33 with left and right flank of the target gene. B. Two-step approach for deletion of MSMEG_2719. The knockout construct consisted of two fragments flanking MSMEG_2719 on the left (LF) and right (RF) in pX33 (pX2719). Integration of the vector (thick black line) into the chromosome (thin black line) via the left flank (Int LF) or right flank (Int RF) and subsequent deletion of MSMEG_2719 (2719) are shown. Restriction sites of SmaI (S) and fragment sizes as detected in Southern hybridization are indicated. Southern hybridization analysis of the integration event: 1. Crossover: SmaI-digests of genomic DNA of wild-type mc2155 and a candidate colony (WT pX2719) were probed with radiolabeled left flank PCR product of the deletion construct. 2. Crossover: Southern hybridization analysis of 2719 and wild-type mc2155. Molecular masses are indicated in kb. M, marker. WT, wild-type. C. Two-step approach for deletion of MSMEG_2262 in WT strain and 2719 mutant using pX2262. Restriction sites of EcoRI (E) and fragment sizes as detected in Southern hybridization are indicated. D. Two-step approach for deletion of MSMEG_3931 in WT strain, single knockout strains 2719 and 2262 and double knockout strain 22622719 using pX3931. Restriction sites of EcoRI (E) and fragment sizes as detected in Southern hybridization are indicated. Drawings not to scale. 23 24 Figure S8. Trace output from hydrogen measurement of a wild-type M. smegmatis culture (OD600 = 2.53). No headspace was present in the measurement chamber. Arrows indicate time points where the electrode was temporarily removed and culture spiked with the indicated gases that were saturated in PBS. Hydrogen concentration (mmol L-1) 50 40 30 20 H2 + O2 O2 10 0 1.6 -10 1.7 1.8 1.9 Time since start (h) 25 Figure S9. Hydrogenase activity in wild-type whole cells supplemented with either 1 mM KNO3 or 1 mM fumarate (using deionized H2O as the vehicle), O2-saturated PBS was used where indicated. Oxidation of hydrogen was measured in cultures grown in HdB medium to early stationary phase in rubber stoppered serum vials with gradual depletion of oxygen. Rate of change in hydrogen concentration is given in nmoles hydrogen per minute per mg protein. Error bars represent standard deviation from three biological samples. 26 Figure S10. Hydrogenase activity in whole cells measured with hydrogen electrode. Oxidation of hydrogen was measured in cultures grown in carbon limited HdB medium to (A) stationary phase, or (B) early stationary phase in rubber stoppered serum vials with gradual depletion of oxygen. Unsaturated PBS was used instead of H 2-saturated PBS. Wild-type was compared to single mutants ∆hyd1, ∆hyd2, ∆hyd3, double mutants ∆hyd2∆hyd3 (Hyd1 only), ∆hyd1∆hyd3 (Hyd2 only), ∆hyd1∆hyd2 (Hyd3 only) and triple hydrogenase mutant ∆hyd1∆hyd2∆hyd3 (∆hyd123). Rate of change in hydrogen concentration is given in nmoles hydrogen per minute per mg protein. Error bars represent standard deviation from three biological samples. 27 28 References Berney, M. and Cook, G. M. (2010) Unique flexibility in energy metabolism allows mycobacteria to combat starvation and hypoxia. PLoS One 5: e8614. Fritsch, J., Scheerer, P., Frielingsdorf, S., Kroschinsky, S., Friedrich, B., Lenz, O. and Spahn, C. M. T. 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