Metabolic reconstruction of Db. autotrophicum HRM2 based on known growth substrates and metabolic capacities. Complete oxidation of organic substrates and CO₂ fixation under autotrophic growth conditions proceeds via the Wood–Ljungdahl pathway (blue box). The sulfate reduction is given in a dark red box, heterodisulfide reduction is given in a dark orange box and electron transfer from Qmo to sulfate reduction is given in an orange box. All selenium-dependent proteins are marked by dark grey backgrounds and an asterisk (*). The cytoplasmic membrane is given in light grey. Arrows indicate metabolic flows; dashed lines indicate assumed or putative electron flows. For reasons of simplicity, cytochromes, multihaem proteins and the following proteins with one or more paralogues are displayed only once in the figure: Acs, CysAW, EtfAB, FdhAB, GlpAB, HdrA, HdrL, MvhD, PorABC, RnfA–E, Sat, Suc, SulP, TmcBCA. Abbreviations are: CM, cytoplasmic membrane; OM, outer membrane; Acs/CODH, acetyl-CoA synthase/CO dehydrogenase; Acs, acetyl-CoA synthetase; AcsF, CODH maturation factor; Adh, aldehyde dehydrogenase; AMP/ADP/ATP, adenosine-monophosphate/-diphosphate/-triphosphate; AprAB, adenylylsulfate reductase; AtpA–I, ATP synthase F0/F1; Cit, citrate synthase; CO₂, carbon dioxide; Cdh1/2, carbon monoxide dehydrogenase; CdhA–E, bifunctional acetyl-CoA synthetase/carbon monoxide dehydrogenase; DctPQM, TRAP-type C4-dicarboxylate transporter; DsrABC, dissimilatory sulfite reductase complex; EtfAB_ox/EtfAB_red, electron transfer flavoprotein, oxidized and reduced form; FabGH, 3-oxoacyl-acyl carrier synthase/reductase; FadDBA, long-chain fatty acid-CoA ligase; FdhABCD, formate dehydrogenase; Fd_ox/Fd_red, ferredoxin, oxidized and reduced form; FdrABC, fumarate reductase; Fhs, formate-tetrahydrofolate ligase; Fdx, ferredoxin, 4Fe-4S cluster; Fhs, formate-tetrahydrofolate ligase; FolD, methylenetetrahydrofolate dehydrogenase; FumAC, fumarate hydratase; GlpAB, glycerol-3-phosphate dehydrogenase; HdrADFL, heterodisulfide reductase; HmeCDEP, Hdr-like menaquinol-oxidizing complex; HydA, [Fe]-only fusion hydrogenase; HyfBCDEFGI, hydrogenase Hyf homologue; HynAB, periplasmic [Ni/Fe] hydrogenase; HysAB, periplasmic [Ni/Fe/Se] hydrogenase; Idh, isocitrate dehydrogenase; LdhAB, lactate dehydrogenase; LldP, l-lactate permease; Mae, malic enzyme; MetF-ABC, methylenetetrahydrofolate reductase; MQ/MQ-H₂, menaquinone pool, oxidized and reduced form; MvhADG, methylviologen non-reducing hydrogenase; NAD⁺, NADH/H⁺, nicotinamide-adenine dinucleotide, oxidized and reduced form; NADP⁺, NADPH/H⁺, nicotinamide-adenine dinucleotide phosphate, oxidized and reduced form; NqrA–F, Na⁺-translocating NADH-quinone reductase; PccB, propionyl-CoA carboxylase; Pfl, pyruvate formate lyase; PorABC, pyruvate:ferredoxin oxidoreductase; PPi, pyrophosphate; QmoABC, quinone-interacting membrane-bound oxidoreductase complex; RnfA–E, electron transport complex protein; Sat, sulfate adenylyl transferase; Sbm, methylmalonyl-CoA mutase; SdhABC, succinate dehydrogenase/fumarate reductase; SseA, thiosulfate sulfur transferase; SucCD, succinyl-CoA synthetase; SulP, high-affinity H⁺/SO₄²⁻ symporter; TmcABC, acidic type II cytochrome complex; Tst, thiosulfate sulfur transferase; X/XH₂, unknown carrier of reducing equivalent, oxidized and reduced form.

Genomic context of the hdrA, hdrF, hdrD and hdrL genes of Db. autotrophicum HRM2. Deduced heterodisulfide reductase genes are marked in red. The HdrA/HdrL proteins are encoded at nine genetic loci, one of which contains a tandem associated hdrL/hdrA copy (VIII). Each hdrA or hdrL locus is associated with a methylviologen non-reducing hydrogenase subunit D (mvhD), beside the hdrA4 locus, which is followed by a deduced hdrD2 gene activity (VIII). The loci V, VI, VII and VIII contain genes for a formate dehydrogenase subunit B, which is followed directly by formate dehydrogenase subunit A genes in loci V and VI. In locus VIII hdrA4 gene is associated with genes for deduced orthologues of subunits HdrB and HdrC, giving an hdrACB-like operon as can be found in Desulfovibrio species. The HdrF1 and HdrF2 genes are associated with genes for anaerobic glycerol-3-phosphate dehydrogenase activity (GlpAB) which probably transfers electrons from dicarboxylic acid degradation and genes for dicarboxylic acids transporters (Dct). hdrF3 is clustered with electron transfer flavoprotein subunits EtfBA, which might transfer electrons from the β-oxidation of fatty acids. Abbreviations are: hdrA, heterodisulfide reductase, subunit HdrA; hdrL, predicted heterodisulfide reductase/glutamate synthase fusion protein, subunit HdrL; hdrF, heterodisulfide reductase subunit HdrF; hdrD, heterodisulfide reductase, subunit HdrD (HdrCB homologous protein); dsrA, dissimilatory sulfite reductase, α-subunit; dsrB, dissimilatory sulfite reductase, β-subunit; dsrC, dissimilatory sulfite reductase, γ-subunit; mvhD, methylviologen non-reducing hydrogenase, iron-sulfur subunit MvhD; mvhG, methylviologen non-reducing hydrogenase, iron-sulfur subunit MvhG; mvhA, methylviologen non-reducing hydrogenase, nickel-iron subunit MvhA; glpA, anaerobic glycerol-3-phosphate dehydrogenase large subunit; glpB, anaerobic glycerol-3-phosphate dehydrogenase small subunit; etfA, electron transfer flavoprotein α-subunit; etfB, electron transfer flavoprotein β-subunit; dctP1 TRAP-type C4-dicarboxylate transporter, periplasmic solute-binding component; dctM1, TRAP-type C4-dicarboxylate permease, large subunit; dctQ1, TRAP-type C4-dicarboxylate permease, small subunit; 1, HysB, periplasmic [Ni/Fe/Se] hydrogenase, large subunit; 2, HysD, periplasmic [Ni/Fe/Se] hydrogenase, small subunit; 3, HyaD2, hydrogenase expression/formation protein; 4, hypothetical protein; 5, response regulator; 6, histidine kinase; 7, ferredoxin; 8, Fe/S cluster protein; 9, Aor5, tungsten-containing aldehyde reductase; 10, CheY-like regulator; 11, putative regulatory protein.

Metabolic heat map of Db. autotrophicum HRM2 (white-orange-red scale on similarity; metabolic genes versus organisms). In the red column 206 metabolic genes of Db. autotrophicum HRM2 are grouped into seven functional categories: hydrogenase, membrane complexes, hdr genes, sulfate reduction, substrate utilization, C1-metabolic pathway and formate dehydrogenases. First and best bidirectional alignments with the proteins of 67 phylogenetically important, but taxonomically diverse prokaryotes are given by colour-coded boxes. Colours correspond to similarity as indicated on the top left panel. A white background indicates no similarities and no hits, dark-red boxes indicate high similarities and best hits. The grey ladders on the left and the right side indicate the 206 genes compared. Paralogous genes in Db. autotrophicum HRM2 were not indicated by name but by corresponding boxes, e.g. 17 grey boxes for acd1–17. The complete bidirectional blast data with 700 to date sequenced prokaryotes genomes and the 4947 genes of Db. autotrophicum HRM2 are given in Table S1. Order of organism abbreviations follows the grouping used in this figure: Archaea:M.mz, Methanosarcina mazei; R.ic, Rice cluster RC1; H.ma, Haloarcula marismortui ATCC 43049; M.th, Methanothermobacter thermoautotrophicus str. ΔH; M.st, Methanosphaera stadtmanae; T.ac, Thermoplasma acidophilum; A.fu, Archaeoglobus fulgidus; M.ja, Methanococcus jannaschii; M.ka, Methanopyrus kandleri; S.ac, Sulfolobus acidocaldarius DSM 639; H.bu, Hyperthermus butylicus; Chlamydiae: C.tr, Chlamydia trachomatis; Firmicutes: M.pn, Mycoplasma pneumoniae; D.et, Dehalococcoides ethenogenes 195; B.bu, Borrelia burgdorferi; D.ha, Desulfitobacterium hafniense Y51; Mo.th, Moorella thermoacetica ATCC 39073; S.au, Staphylococcus aureus MRSA252; O.ih, Oceanobacillus iheyensis; B.su, Bacillus subtilis 168; C.te, Clostridium tetani E88; C.ac, Clostridium acetobutylicum; γ-proteobacteria: H.in, Haemophilus influenzae; V.fi, Vibrio fischeri ES114; E.co, Escherichia coli K12; X.ca, Xanthomonas campestris; A.ci, Acinetobacter sp. ADP1; P.pu, Pseudomonas putida KT2440; β-proteobacteria: N.eu, Nitrosomonas europaea; A.ar.,‘Aromatoleum aromaticum’ EbN1; T.de, Thiobacillus denitrificans ATCC 25259; B.ma, Burkholderia mallei ATCC 23344; R.eu, Ralstonia eutropha JMP134; Deinococcus-Thermus: D.ra, Deinococcus radiodurans; Planctomycetes: R.ba, Rhodopirellula baltica; α-proteobacteria: C.cr, Caulobacter crescentus; Z.mo, Zymomonas mobilis ZM4; M.lo, Mesorhizobium loti; R.et, Rhizobium etli CFN 42; A.tu, Agrobacterium tumefaciens C58; S.me, Sinorhizobium meliloti; R.pa, Rhodopseudomonas palustris CGA009; N.wi, Nitrobacter winogradskyi Nb-255; B.ja, Bradyrhizobium japonicum; Cyanobacteria: P.ma, Prochlorococcus marinus CCMP1375; Syn, Synechocystis PCC6803; Nos, Nostoc sp.; α-proteobacteria: B.ba, Bdellovibrio bacteriovorus; Sy.a, Syntrophus aciditrophicus SB; D.ps, Desulfotalea psychrophila LSv54; D.au, Desulfobacterium autotrophicum HRM2; A.de, Anaeromyxobacter dehalogenans 2CP-C; P.ca, Pelobacter carbinolicus; G.me, Geobacter metallireducens GS-15; G.su, Geobacter sulfurreducens; D.vu, Desulfovibrio vulgaris Hildenborough; D.de, Desulfovibrio desulfuricans G20; diverse Gram-negative bacteria: B.fr, Bacteroides fragilis NCTC 9434; F.nu, Fusobacterium nucleatum; C.tp, Chlorobium tepidum TLS; S.ru, Salinibacter ruber DSM 13855; H.py, Helicobacter pylori J99; C.je, Campylobacter jejuni; P.ac, Propionibacterium acnes KPA171202; C.gl, Corynebacterium glutamicum ATCC 13032; M.tu, Mycobacterium tuberculosis H37Rv; T.ma, Thermotoga maritima; A.ae, Aquifex aeolicus.

Genomic organization of the Wood–Ljungdahl pathway. In Db. autotrophicum HRM2 the genes encoding the key enzymes from the Wood–Ljungdahl pathway are organized in four operon-like structures in a single chromosomal locus. The genes encoding a bifunctional acetyl-CoA synthase/CO dehydrogenase (ACS/CODH) form one colinear group. The genes of the methyl branch of the Wood–Ljungdahl pathway are organized in three distinct groups containing (i) methylene-tetrahydrofolate reductase (MetF1), (ii) methylene-tetrahydrofolate dehydrogenase (FolD) and (iii) C1-tetrahydrofolate synthetase (THF synthetase) (Fhs).

Pfam (domain scans analysis) and domain alignments of four types of heterodisulfide reductase subunits (Hdr) of Db. autotrophicum HRM2. Relevant Pfam domains are given in colour codes. All Hdr proteins depicted contain selenocysteine; the HdrA and the HdrL type of the proteins are colocated with methylviologen non-reducing hydrogenase subunit D (mvhD). The deduced HdrF protein contains a domain with several transmembrane regions indicating a possible membrane integration of the protein. In contrast, HdrD, HdrA and HdrL have domain structures typical for soluble proteins.