Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes

(1) Sample sites.We previously analyzed total DNA extracted from deep-sea sediments obtained from the IODP survey (Leg 201) of the Peruvian trench and continental margin for the presence of Chloroflexi 16S rRNA and rdh genes (16) using PCR and nanoliter-quantitative PCR (qPCR) (13). On the basis of these screening results and the results of previous metagenomic studies of this region (1), we identified samples from continental margin sites 1227 and 1229 and trench site 1230 as promising for future investigation for Chloroflexi by single-cell genomics. The surface waters of the Peruvian continental margin are part of an upwelling system that is biologically active and high in total organic carbon, about 2 to 3% by weight (17, 18). Samples were taken from three sites, 1227, 1229, and 1230, at sediment depths 0.3, 1, and 7.3 m below sea floor (mbsf), respectively, corresponding to estimated sediment ages of 1.4, 16, and 64 kiloyears (kyr) (S. D’Hondt, personal communication) (Table 1) (19, 20). All sample sites were located within the sulfate reduction zone. Areal carbon oxidation rates were estimated to be between 2 and 5 µmol cm⁻² year⁻¹ (21, 22). Site 1227 is located in a small, fault-bounded sediment pond within the Trujillo Basin at a water depth of 427 m. Based on maximum alkalinity and dissolved organic carbon (DOC) concentration measurements, site 1227 is considered to be highly biologically active (17). Site 1229 is located on the Peru continental shelf at a water depth of 150.5 m. Previous clone libraries from these sediments revealed that Chloroflexi were among the most predominant bacterial groups (5, 23). A more detailed metagenomic study revealed that Chloroflexi contributed 12% to 16% to the total gene pool, independent of depth (1). Site 1230 is located at 5,086 m deep on the lower slope of the Peru Trench at the subduction zone between the continental crust and accretionary complex (19).

(2) Single-cell genome recovery, assembly statistics, and phylogeny.To develop more refined metabolic hypotheses for the lifestyle of deep-sea sediment Chloroflexi, we recovered single cells from drilling cores of sites 1227, 1229, and 1230; sorted them into 384-well plates by fluorescence-activated cell sorting; lysed the cells; and amplified the genomes via multiple displacement amplification (MDA) (13, 24). Out of 1,260 wells sorted, three wells containing Chloroflexi were identified by PCR screening with broad eubacterial primers targeting the 16S rRNA gene: well 653N14 from site 1227, wells 657A03 and 657K04 from site 1229, and well 662N06 from site 1230. The amplified genomic DNA from these wells was then sequenced. After assembly, we noticed that well 657K04 contained two 16S rRNA genes from two distinct Chloroflexi subphyla (25). Each of the 16S rRNA gene sequences was identical to the sequence of either single-cell genome 657A03 or 662N06. Tetranucleotide frequency analyses (TNA) and GC content were sufficient to distinguish the two genomes in silico (see Fig. S2 in the supplemental material). Reads mapping to each of these two genomes in well 657K04 were coassembled with the reads from corresponding well 657A03 or 662N06, depending on 16S rRNA gene sequence identity and TNA. The combined assembly of 657A03 and 657K04 is referred to as DscP3 (deep-sea single cell, population 3) (Table S1). Since the 16S rRNA gene sequence of 662N06 is identical to DscP2 (13), the combined assembly of 662N06 and 657K04 is referred to as DscP2-2. As the genomic content from well 653N14 was from a single cell, the assembly of the genomic data was named Dsc4 (deep-sea single cell 4).

Classification of the 16S rRNA gene sequences revealed that the single-cell-derived genomes belong to cells of Chloroflexi subphyla I and II (Fig. 1) (26). DscP3 and Dsc4 are unclassified Chloroflexi of subphylum I, most closely related to single cells An-B04, An-B16, An-B22, and An-J10 from the Okinawa Trough (15). DscP2-2 is a member of the class-level clade Dehalococcoidetes of Chloroflexi subphylum II with a 16S rRNA gene sequence identical to the previously reported DscP2; these populations are most closely related to Okinawa Trough single cell De-I04 and terrestrial RBG_16-57-8, RBG_13_46_14, and RBG_16_51_9 obtained from Rifle, CO, aquifer sediments (13, 15, 27) (Fig. 1).

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FIG 1 

Rooted maximum likelihood tree of 16S rRNA gene. Evolutionary distance tree of the bacterial phylum Chloroflexi derived from comparative analysis of 16S rRNA gene sequences showing the phylogenetic relationship between members of the phylum with Escherichia coli as the outgroup. The sequences were aligned with the method described in reference 89 and masked using Gblocks (96), and the phylogenetic tree was constructed using the maximum likelihood method using PhyML (97) and the TN93 substitution model. Nodes highly supported by bootstrap resampling (100 replicates) are represented by black (≥90%), gray (≥70%), or white (≥50%) circles, respectively. Names in orange are genomes presented in this study, and those in blue are other marine Chloroflexi.

Genome completeness estimates using tRNAs and marker genes from CheckM (see Table S1, reference 28) predicted the size of the genomes of the subphylum I members DscP3 and Dsc4 to be between 2 and 2.7 Mb (conservative completeness estimates of 64% and 49%, respectively), significantly smaller than the 3.2- to 3.5-Mb genome size estimated for closely related An-B04, An-B16, An-B22, and An-J10 (15), though the discrepancy is likely due to the incompleteness of these genomes. On the other hand, for DscP2-2 we estimate the genome size to be around 1 to 1.3 Mb (36% completeness), similar to the 1.38-Mb genome size previously predicted for population DscP2 (13). Below, we report our metabolic reconstruction from single-cell genomic data for DscP3 and Dsc4 first, followed by DscP2-2.

(3) Predicted Metabolism of DscP3 and Dsc4.We analyzed the genomes of DscP3 and Dsc4 with respect to catabolic pathways. Central metabolic pathways are predicted to include complete pathways for glycolysis/gluconeogenesis and the Wood-Ljungdahl pathway (WLP, see below). The annotation of multiple sugar transporters suggests external carbohydrates as substrates for the glycolytic pathway, and possibly the consumption of the released reducing equivalents by the WLP. The presence of Rnf (rhodobacter nitrogen fixation) and Hdr-IFO (heterodisulfide-reductase-associated ion-translocating Fd:NADH oxidoreductase) provides potential means of chemiosmotic energy conservation. No evidence for respiration of inorganic electron acceptors was found. Because of the presence of these genes, we hypothesized that DscP3 and Dsc4 have the genomic potential for a homoacetogenic lifestyle.

(3.1) (i) Wood-Ljungdahl pathway.Homoacetogenic bacteria are anaerobic, facultative autotrophs that are able to grow by oxidation of organic or inorganic substrates coupled to the reduction of carbon dioxide to produce acetate via the Wood-Ljungdahl pathway (WLP). Although most known homoacetogenic bacteria are found within the genera Clostridium and Acetobacterium, this metabolism is present in 23 different bacterial genera (29). Despite their phylogenetic diversity, the hallmark of a homoacetogenic metabolism is the use of the WLP for CO₂ reduction to acetate coupled to a membrane-bound enzyme complex for energy conservation (29–31). The ubiquity of the homoacetogenic bacteria in diverse environments can be attributed to the metabolic flexibility of these microorganisms. Electron donors for the WLP include a large variety of organic and inorganic substrates including formate, hexoses, pentoses, cellulose, alcohols, hydrogen (H₂), and CO (30). Homoacetate fermentation, more commonly referred to as homoacetogenesis, is a form of acetogenic metabolism that uses the Embden-Meyerhof-Parnas (glycolytic) pathway for the oxidation of glucose to 2 molecules of acetate and 2 molecules of CO₂ which are subsequently reduced to an additional molecule of acetate (31).

The WLP consists of two branches—the methyl-branch and the carbonyl-branch. In the forward (reductive) direction the methyl group of acetate is formed in the methyl-branch through the three-step reduction of CO₂ and the carbonyl-branch generates the carbonyl group of acetate via the one-step reduction of a second molecule of CO₂. In the methyl-branch, one molecule of CO₂ is reduced to formate, catalyzed by a formate dehydrogenase. Formyl-tetrahydrofolate synthetase (Fhs) covalently binds the formyl group to tetrahydrofolate (THF) coupled to hydrolysis of ATP to produce formyl-THF. The bifunctional enzyme, methenyl-THF cyclohydrolase/methylene-THF dehydrogenase (FolD) abstracts a water molecule from formyl-THF to yield methenyl-THF, which is then reduced to methylene-THF. The final reduction step is catalyzed by methylene-THF reductase (MetF), which produces methyl-THF. The methyl group is then transferred by a methyltransferase and a corrinoid iron-sulfur protein to CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) which also catalyzes the one-step reduction of CO₂ to CO in the carbonyl-branch. The CODH/ACS catalyzes the final carbonylation of methyl-THF by CO to form acetyl-CoA.

The WLP is utilized by a wide variety of anaerobic microorganisms for both C1 metabolism and energy conservation by coupling folate-mediated C1 metabolism to either CO₂ reduction (forward) or acetate oxidation (reverse). In fact, the WLP is considered to be one of the most ancient pathways for biomass and ATP production (30, 31, 100). In the forward direction, homoacetogenic bacteria utilize the WLP for acetate production and energy conservation. In some sulfate-reducing bacteria, the WLP operates in reverse by coupling the exergonic reduction of sulfate to sulfide to the oxidation of acetate (35). The incomplete WLP found in Dehalococcoides spp. has been implicated in the unconventional strategy of generating methyl-tetrahydrofolate for methionine biosynthesis (101).

As we found in the genomes of DscP3 and Dsc4 genes encoding a complete WLP, we compared in more detail the genes encoding the predicted pathway enzymes and associated operon architectures in Chloroflexi DscP3 and closely-related Dsc4 with those of two well-studied homoacetogenic bacteria, the Gram-positive Moorella thermoacetica and Acetobacterium woodii (31, 32). While both of these homoacetogenic bacteria carry the genes of the catabolic WLP for acetate formation from two CO₂ with H₂ as electron donor, they differ in key genes and mechanisms for energy conservation (31).

Energy conservation in M. thermoacetica is based on H₂-cycling and a H₂-dependent NADP⁺ reduction, where both H₂-cycling and NADPH oxidation are associated with CO₂ reduction in the WLP. In the net energy-conserving module of M. thermoacetica, NADPH is bifurcated into reduced ferredoxin and NADH; the former being used for CO₂ reduction to CO. NADH is further bifurcated by the hexaheteromeric HdrABC-MvhD-MetVF complex (Moth_1191-1196) to reduce methylene-THF and an unknown, low redox potential electron carrier, which is speculated to reduce H⁺ to H₂ via a membrane-associated, H⁺-pumping hydrogenase (Ech) (103). Thus, a HdrABC-MvhD-MetVF methylene-THF reductase, a NADPH-bifurcating Nfn, and an Ech are the defining enzymes for an idiosyncratic catabolism of M. thermoacetica.

In contrast, Acetobacterium woodii contains a linear, H₂-dependent energy metabolism, where a soluble tetrameric electron-bifurcating hydrogenase (HydABCD) catalyzes the oxidation of hydrogen to the generation of NADH and a reduced ferredoxin (31). Energy conservation is mediated by a Na⁺-translocating Rnf complex, coupling the oxidation of reduced ferredoxin to the reduction of NAD⁺. The reducing equivalents of NADH are subsequently funneled into the methyl-branch of the WLP. Homoacetogenic metabolism from glucose in A. woodii involves the production of 2 acetate, 2 NADH, and 2 reduced ferredoxin. Both NADH and 1 reduced ferredoxin are utilized in the WLP. The Rnf complex transfers 0.5 ferredoxin to generate 0.5 NADH, which, combined with the remaining 0.5 ferredoxin, generates hydrogen via HydABCD in the electron-confurcating direction (102). Thus, key enzymes of an A. woodii homoacetogenic catabolism are an NADH-dependent methylene-THF reductase, an electron-bifurcating hydrogenease, an Rnf, and the absence of Nfn (30, 37, 106, 107).

The DscP3 genome contains a hdrABC-mvhD-metVF gene cluster in an operon similar to that of M. thermoacetica (Fig. 2) (103). Unlike M. thermoacetica however, the hdrC gene was present in a translational fusion with hdrB as found also in some sulfate-reducing bacteria (104) and methanogens without cytochromes such as Methanothermobacter marburgensis (36, 105). A BLAST comparison of this DscP3 operon to genomes of other sequenced Chloroflexi and Chloroflexi-containing metagenomes found similar gene clusters in contigs from shotgun metagenomes of the White Oak River Basin (33) as well as the SAG An-B22, from the Okinawa Trough (15) indicating conservation of the herein-identified hdrABD-mvhD-metVF gene cluster among marine Chloroflexi. Uniquely, DscP3 has two non-identical sets of metV and metF genes sharing only 42.8% (bit score, 257) and 40.9% (bit score, 71.6) amino acid sequence similarity, respectively. The first methylene-THF reductase, MetF_1, has 93% amino acid similarity to the MetF of An-B22 and, in a comparison to M. thermoacetica and A. woodii, has an amino acid sequence identity closer to M. thermoacetica (54.5%; bit score, 367) than to A. woodii (38.3%; bit score, 236). The second copy, MetF_2, is closest in sequence identity to the MetF of A. woodii (56.8%; bit score, 329) relative to M. thermoacetica (42.23%; bit score, 244). In the genomes of homoacetogens, metV and metF are arranged in sequence; however, genes encoding a putative two component regulatory system, phoBR, flank metF_1 in DscP3. No other known Chloroflexi clade contains the metV gene required for MetF function in homoacetogens, making the Moorella-type HdrABC-MvhD-MetVF complex of DscP3 unique to marine members of Chloroflexi subphylum I.

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FIG 2 

Gene order and domain composition between the methylene tetrahydrofolate reductase and associated genes from Moorella thermoacetica and marine Chloroflexi. Several sequenced Anaerolineae populations and single cells from marine sediments, including DscP3, share similarities in operon structure with Moorella thermoacetica. An-B22 is a single-cell genome from the Okinawa Trough (15), SM23_63 and SM23_83 are from White Oak River metagenomes (33), and Anaerolineae bacterium 4484-236 is from a Guayamas Basin metagenome (NCBI accession no. MZGP00000000). Dashed double lines indicate the end of a contig. Unlabeled genes are nonconserved hypothetical proteins. Genes colored gray are upstream of the hdrABC-metVF-mvhD operon in M. thermoacetica. Fer4 (red) is the 4Fe-4S-binding domain (PF00037); Pterin_bind (orange) is the pterin-binding protein family (PF00809); CCG (yellow) is the cysteine-rich domain (PF02754); Pyr_redox_2 (green) is the pyridine nucleotide-disulfide oxidoreductase family, which contains a small NAD(P)H-binding domain within a larger FAD domain (PF07992); FlpD (blue) is the methyl-viologen reducing hydrogenase delta subunit family containing 4 conserved cysteine residues (PF02662); MTHFR_C (indigo) family is the C-terminal domain of MetF containing the FAD-binding site (PF12225); MTHFR (violet) is the MetF family, including the FAD-binding domain and TIM barrel (PF02219).

The bifunctional methenyl-THF cyclohydrolase/methylene-THF dehydrogenase (folD) of DscP3 has 58% (bit score, 329) amino acid identity to that gene of M. thermoacetica (Moth_1516) and 54% (bit score, 252) identity to the folD of Methanosarcina barkeri (Mbar_82315). Experimental evidence showed that FolD in these two microorganisms is specific for NADPH as electron donor rather than NADH as in A. woodii (AWO_RS04705) or Clostridium ljungdahlii (AWO_RS04705) (31, 38, 39, 108). Because of the higher sequence similarity between NADPH-specific FolD of M. barkeri and M. thermoacetica to those of DscP3 and Dsc4, we hypothesize that folD in the herein-identified deep-sea Chloroflexi are likewise specific for NADPH.

(3.2) (ii) Proton-translocating membrane-associated complexes in DscP3 and Dsc4.The genomes of the subphylum I Chloroflexi DscP3 and Dsc4 contain genes encoding an Rnf complex, a heterodisulfide-reductase-associated ion-translocating Fd:NADH oxidoreductase (Hdr-IFO), a partial NADH-ubiquinone oxidoreductase (Nuo), and an F-type ATP synthase.

(3.2.1) Rhodobacter nitrogen fixation complex.The Rhodobacter nitrogen fixation (Rnf) complex couples the exergonic reduction of NAD⁺ with reduced ferredoxin (Fd) to proton or sodium translocation and can function anabolically in the energy-consuming, Fd-reducing direction, such as in Rhodobacter capsulatus, Pseudomonas, Azotobacter, and Escherichia species, or catabolically in the energy-conserving, NAD⁺-reducing direction, as in Acetobacterium woodii, Clostridium kluyveri, and Methanosarcina acetivorans (40–42). The flavoprotein maturation enzyme, ApbE, is frequently found directly adjacent to the rnf operon, and in Rhodobacteria and sulfate reducers, it is often annotated as rnfF (43, 44). Based on operon organization, Rnf can be classified into three distinct types (Fig. S5) (42). The rnfABCDGE type is found in Rhodobacter, Pseudomonas, Azotobacter, or Escherichia and seems to be primarily of anabolic function to generate reduced Fd from NADH for biosyntheses (109, 110, 111). The rnf(B)CDGEA organization is found in species of Chlorobium, Bacteroides, and Prosthecochloris (42). The third configuration, rnfCDGEAB, is found in Acetobacterium woodii, Methanosarcina acetivorans, members of the Clostridia genera, and some Desulfovibrio species, among others (48, 107, 112, 113, 114). In Clostridium kluyveri, M. acetivorans, and A. woodii, Rnf (rnfCDGEAB) is the key complex in the energy conservation pathway during homoacetogenesis (31). DscP3 and Dsc4 both harbor genes for a complete RnfCDGEAB directly preceded by ApbE (Fig. S5).

(3.2.2) Heterodisulfide-reductase-associated ion-translocating Fd:NADH oxidoreductase.In DscP3 and Dsc4 (as well as in An-B16), genes are present encoding a putative heterodisulfide-reductase-associated ion-translocating Fd:NADH oxidoreductase (Hdr-IFO) postulated to be involved in reverse electron transport in Syntrophobacter fumaroxidans (45). This gene cassette encodes HdrABC subunits and two putative oxidoreductase domain-containing proteins indicative of Fd:NAD(H) oxidoreductase. The genes encoding Hdr-IFO are conserved among bacteria capable of syntrophic metabolism: M. thermoacetica DSM 512 (Mothe_RS03705 to -RS03675), Clostridium halophilum (SAMN02194393_03034 to -03037), Desulfovirgula thermocuniculi (G4G4_RS0108870 to -RS0108895), Thermincola potens (TherJR_1949 to -1955), Syntrophoaceticus schinkii strain Sp3 (SSCH_160001 to -160008), Syntrophomonas wolfei (SWOL_RS02005 to -RS02050), Syntrophorhabdus aromaticivorans strain UI (SynarDRAFT_0715 to -0709), and Desulfovibrio vulgaris (DVU2404 to -2399) (Fig. S6). In a study of Desulfovibrio vulgaris strain Hildenborough, it was revealed that this gene cassette, but not the Rnf operon, was expressed when grown in a lactate-degrading syntrophic coculture with a hydrogenotrophic methanogen (46–49). A previously published survey of publicly available genomes of bacteria capable of syntrophic metabolism indicated that, with the exception of Desulfovibrio, the genomes of syntrophic bacteria contain either Rnf or Hdr-IFO (45). In syntrophic bacteria, Rnf and Hdr-IFO are thought to be involved in reverse electron transport, using NADH and an ion motive force to reduce Fd (45).

(3.2.3) NADH-ubiquinone oxidoreductase-like complex.A functional NADH-ubiquinone oxidoreductase (Nuo) operon generally contains 14 subunits in three modules, NADH-binding (N), proton-translocating (P), and quinone-reducing (Q) modules, which are hypothesized to have emerged through the combination of separate functional modules (50, 51). Like the genome of DEHC11 from Aarhus Bay, the genomes of DscP3 and Dsc4 lack the genes for the Nuo N module (nuoE to -G), leaving only 11 subunits in the operon (52). The 11-subunit Nuo complex bears a resemblance to Ech, except that it lacks the [NiFe]-binding motifs in subunits nuoB and nuoD that are required for hydrogenase function (51). The N module, however, is frequently absent in Eubacteria and Archaea, and the 11-subunit Nuo may couple with another complex encoded elsewhere in the genomes of these microorganisms (51). The partial genome most closely related to DscP3 and Dsc4, An-B22, does contain both an 11-subunit Nuo and the nuoEFG genes for a complete NADH module in a separate operon, indicating that a Nuo complex may be part of the core metabolism of deep-sea Chloroflexi.

(3.3) (iii) Genomic signatures for electron donors for the Wood-Ljungdahl pathway.In homoacetogenic metabolism, reducing equivalents for the WLP are generated by oxidizing glucose to two molecules of acetate and two molecules of CO₂ via glycolysis, pyruvate:ferredoxin oxidoreductase (PFOR), phosphotransacetylase, and acetate kinase (31). In our single-cell assemblies, DscP3 and Dsc4, we found all required genes for glycolysis and gluconeogenesis except those for carbohydrate activation. The presence of PFOR, phosphotransacetylase, and acetate kinase provides a link for acetate formation from pyruvate. Putative α-glucoside ABC transporters identified as carbohydrate-binding module family 50, chitin or peptidoglycan specific, were identified in the genomes of both DscP3 and Dsc4, indicating a possible input for the glycolytic pathway. Genomes of closely related Chloroflexi, An-B10 and An-B22, contain genes for xylose, hexose, and multiple-sugar ABC transporters (15).

The genome of DscP3 contains an l-lactate dehydrogenase (LDH), and both DscP3 and Dsc4 contain l-lactate permease. The pathway for acetogenic fermentation of lactate has recently been described in A. woodii (115). In A. woodii, the LDH forms a complex with EtfAB and uses flavin-based electron confurcation to drive the highly endergonic NAD⁺-dependent oxidation of lactate with the exergonic reduction of NAD⁺ from reduced ferredoxin. The oxidation of pyruvate proceeds as in heterotrophic fermentation, yielding 1 acetate, 1 CO₂, and 1 ATP molecule per pyruvate. The stoichiometry of acetogenic fermentation of lactate by A. woodii is the production of 2 acetate, 2 CO₂, 2 ATP, and 4 NADH molecules per molecule of lactate (115). The WLP serves to balance the reducing equivalents produced. Unlike heterotrophic fermentation, this involves the input of energy to generate reduced ferredoxin and H₂ from 2 NADH. The electron-bifurcating hydrogenase can produce H₂ from 0.5 molecule of reduced ferredoxin and 0.5 molecule of NADH. The remaining 1.5 molecules of NADH are used by Rnf to produce the necessary 1.5 molecules of reduced ferredoxin coupled with the transport of a proton or sodium ion with the chemiosmotic gradient. As mentioned above, DscP3 and Dsc4 contain lactate dehydrogenase, EtfAB, and Rnf. To provide the reducing power for acetogenic lactate fermentation, an electron-bifurcating hydrogenase or formate dehydrogenase (described below) is needed.

In order to identify other potential electron donors for the WLP, we examined the genomes of DscP3, Dsc4, and related marine Chloroflexi for the presence of hydrogenases and formate dehydrogenases. Although the genomes of DscP3 and Dsc4 are incomplete, we found no genomic evidence for the presence of hydrogenases or hydrogenase expression/maturation genes. Other closely related, yet also incomplete, sequenced Chloroflexi single-cell genomes, An-B16, An-J10, and An-L06, do contain hydrogenase expression/maturation genes hypABCDEF while An-B22 does not (15). This makes it difficult to determine whether hydrogenase genes in DscP3 and Dsc4 are either absent or just not amplified as a result of the bias of the MDA reaction. However, both the DscP3 and Dsc4 genomes do encode multiple putative formate dehydrogenases.

(3.3.1) Formate dehydrogenases.Three distinct types of formate dehydrogenases (FDHs) were found to be encoded by the genomes of DscP3 and Dsc4: an NAD(P)-dependent FDH (NAD-FdhAB), a formate:quinol oxidoreductase (FdnGHI), and a heterodisulfide reductase-associated FDH (HdrBCA-MvhD-FdhBA).

(3.3.1.1) NAD-dependent formate dehydrogenase.A putative heterodimeric NAD(P)-dependent FDH (FdhAB) complex was identified in the genome of DscP3. Previously, the genome of SAG An-B22 was also hypothesized to contain genes for an NAD-dependent FDH (15). The α subunits of the NAD-FdhAB from DscP3 and An-B22 share 78% amino acid identity, but the sequence of An-B22 contains a selenocysteine residue in the active site (15). The cytoplasmic, selenocysteine-containing, NADP-dependent FdhAB of M. thermoacetica (MOTH_2312 to -2314) is involved in the generation of formate in the first step of the CO₂ reduction of the methyl branch of the Wood-Ljungdahl pathway (10). The α subunits of FdhAB in DscP3 and the NADP-dependent FdhAB of M. thermoacetica, respectively, share 43% (bit score, 961) amino acid identity; however, the β subunits are dissimilar. FdhB of M. thermoacetica contains a 4Fe-4S cluster-binding domain, a ferredoxin-binding domain, and an NADP-binding domain. On the other hand, the β subunit of FdhAB from DscP3 contains a thioredoxin-like 2Fe-2S ferredoxin, an NAD(P)-binding domain, an flavin mononucleotide (FMN)-binding domain, a soluble ligand-binding domain, and a 4Fe-4S cluster-binding domain. It is unclear whether or not the predicted NAD(P)-binding site is specific to NAD⁺ or NADP⁺. The best BLASTP hit for the β subunit of the FdhAB from DscP3 is to the NADH-ubiquinone oxidoreductase subunit F (NuoF) of Anaerolinea thermophila UN-1 with 53% (bit score, 519) amino acid identity.

The best BLASTP hit for the α subunit was to the FdhA of the thermophilic sulfate-reducing bacterium Desulfotomaculum thermosubterraneum (60%; bit score, 1057). The NAD-dependent FDH of D. thermosubterraneum (BUA65_RS16050 to -RS16065) and other Desulfotomaculum species is hypothesized to be electron confurcating, driving the unfavorable oxidation of NADH generated during propionate degradation to the exergonic oxidation of ferredoxin to produce formate (54). A similar NAD-dependent FDH complex (HylCBA-FdhA) in Clostridium acidurici (Curi_c29380 to -c29410) functions in the reverse (bifurcating) direction during uric acid fermentation (55). The bifurcating/confurcating HylCBA-FdhA complex in C. acidurici (Curi_c29380 to -c29410) and that in D. thermosubterraneum (BUA65_RS16050 to -RS16065) each consist of four subunits: HylC, HylB, HylA, and the catalytic Fdh α subunit, FdhA. HylC contains a 2Fe-2S cluster-binding domain; HylB contains a 2Fe-2S iron-sulfur-binding domain, an NAD-binding domain, a soluble ligand-binding domain, an FMN-binding domain, and two 4Fe-4S cluster-binding domains; and HylA contains one 2Fe-2S and three 4Fe-4S cluster-binding domains. FdhA contains one 2Fe-2S and one 4Fe-4S cluster-binding domain, a selenocysteine, and a molybdopterin-binding domain. The genome of DscP3 contains genes for the two subunits FdhA and FdhB, which bear resemblance to HylA and the NAD-binding subunit, HylB, respectively, with a thioredoxin-like 2Fe-2S cluster-binding domain instead of two 4Fe-4S cluster-binding domains (Fig. 3). The similarities between the subunits of the heterotetrameric electron-bifurcating/confurcating HylCBA-FdhA from C. acidurici and Desulfotomaculum spp. indicate that the predicted heterodimeric NAD-dependent FDH of DscP3 might also be electron bifurcating/confurcating. If FdhAB is an electron bifurcating Fdh, we hypothesize that formate could be oxidized to CO₂ during acetate oxidation via the WLP, and as an electron confurcating Fdh, CO₂ could be reduced to formate as the first intermediate of the methyl branch of the WLP. This bifurcating/conforcating formate dehydrogenase may also provide a link between lactate fermentation and the WLP, similar to the electron-bifurcating hydrogenase in A. woodii (115).

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FIG 3 

NAD-dependent formate dehydrogenase. (A) Subunit similarity between the NAD-dependent formate dehydrogenase from DscP3 and the electron-bifurcating/confurcating formate dehydrogenases of Clostridium acidurici and Desulfotomaculum thermosubterraneum (54, 55). (B) Predicted electron-bifurcating formate dehydrogenase from D. thermosubterraneum. (C) Predicted NAD-dependent formate dehydrogenase from single-cell population DscP3.

(3.3.1.2) Formate:quinol oxidoreductase.The formate:quinol oxidoreductase complex, often referred to as formate dehydrogenase-N (FdnGHI), is a three-subunit, membrane-integral protein found in the genomes of both DscP3 and Dsc4. FdnI is the catalytic subunit, encompassing a putative molybdopterin-binding domain containing a TAT-signal peptide sequence; FdnH contains two 4Fe-4S cluster domains; and FdnG is predicted to be membrane bound and to contain two cytochrome b domains (56). The FdnI subunit of DscP3 is an incomplete sequence, being cut off by the end of a contig, but Dsc4 contains a second FdnI subunit directly downstream of FdnGHI, sharing 48% amino acid similarity with the first. The first FdnI sequence, FdnI_1, displays highest similarity to the α subunit of the membrane-bound FDH-like oxidoreductase complex in Dehalogenimonas lykanthroporepellens (Dehly_0443) and Dehalococcoides ethenogenes 195 (DET0187) at 56% (bit score, 1,104) and 49% (bit score, 752) amino acid identity, respectively (Fig. S3). The second FdnI, FdnI_2, is predicted to be selenocysteine containing and has 68% (bit score, 1,067) amino acid similarity to Bacillus sp. strain NC2-31.

The FdnH and FdnG subunits of the FdnGHI complex in Dsc4 are most similar to the iron-sulfur subunit of the polysulfide reductase from Calderihabitans maritimus (KKC1_02500) (58%; bit score, 320) and the cytochrome b-containing integral membrane subunit (SAMN06269301_1443) of an FDH from Geobacter sp. strain DSM 9736 (41%; bit score, 248), respectively (57). The FdnH and FdnG subunits from DscP3 are most similar to those in Bacillus massilosenegalensis with 46% (bit score, 160) amino acid identity for the iron-sulfur β subunit and 39% (bit score, 244) for the cytochrome b subunit. In Bacillus, FdnGHI is hypothesized to mediate the effects of acidity through the oxidation of formate, generating NAD(P)H which transfers electrons to the electron transport chain through cytochrome b (58). Two membrane-associated FDHs in Escherichia coli, FdnGHI and FdoGHI, were found to exhibit substrate promiscuity and to have hydrogen-oxidizing activity (56). Similar to the membrane-associated FDH in Bacillus spp. and E. coli, the FdnGHI of DscP3 and Dsc4 may likely be a formate- and/or hydrogen-oxidizing enzyme.

Interestingly, in terrestrial Dehalococcoides spp. the operon encoding the FDH-like complex consists of two genes: one encoding a formate dehydrogenase catalytic subunit homologue with similarities to FdnI and the second encoding a membrane-bound subunit homologous to FdnH but lacking the cytochrome b domains. The operon is missing an iron-sulfur cluster-containing FdnH homologue and is instead hypothesized to associate with the Hup hydrogenase complex (DET0110 to -0112) as a part of quinone-independent organohalogen respiration via reductive dehalogenases (11, 12, 53, 59). The FdnI homologue of Dehalococcoides spp. contains a serine residue instead of a cysteine or selenocysteine at a key position in the active site, which is hypothesized to be the cause for the lack of formate dehydrogenase activity (53). Notably, the FDH-like protein of Dehalococcoidetes and the associated 4Fe-4S cluster-containing subunit of the Hup operon are closest in sequence similarity to the FdnI (48%; bit score, 262) and FdnH (35%; bit score, 191) subunits, respectively, from Dsc4. The FDH-like complexes of Dehalogenimonas and Dehalococcoides, while membrane bound, have no cytochrome b subunit. Dehalogenimonas, Dehalococcoides, and other subphylum II Chloroflexi do not synthesize quinones. Menaquinones, however, have been recovered from cell extracts of facultative anaerobic members of Chloroflexi subphylum I, Ardenticatenia and Caldilinea (60, 61). Like these other members of subphylum I, we hypothesize that DscP3 and Dsc4 are capable of quinone-dependent electron transfer as supported by the presence of the genes menA and ubiE/menG, which catalyze the synthesis of menaquinone from 1,4-dihydroxy-2-naphthoate.

(3.3.1.3) Heterodisulfide reductase-associated formate dehydrogenase.A selenocysteine-containing FDH was found downstream of the mvhD, hdrA, and hdrBC genes that encode a potential HdrBCA-MvhD-FdhBA similar to that of Desulfobacula toluolica Tol2 (TOL2_RS06275 to -RS06300), an aromatic-compound-degrading, sulfate-reducing bacterium from marine sediment (Fig. S4) (62). It has been observed in methanogenic metabolism that FDH can couple with complexes containing HdrA-like proteins for electron bifurcation (63). FdhA of this putative complex is selenocysteine containing and is, with 59% (bit score, 852) amino acid identity, most closely related to the α subunit of the four-subunit NAD-dependent FDH of the halophilic anaerobe Selenihalanaerobacter shriftii (B5D41_RS09265). The β subunit, FdhB, contains an iron-sulfur cluster-binding domain and has highest amino acid similarity at 40% (bit score, 206) to the FdhB of D. toluolica Tol2. Interestingly, the reductive dehalogenase RdhA sequence found in DEHC11 from Aarhus Bay was found to be most similar to a homologue found in D. toluolica Tol2 (14).

(3.4) Anaerobic benzoate oxidation.A complete anaerobic benzoate oxidation pathway was discovered in the genomes of DscP3 and Dsc4 (Fig. 4). This is the first record of a complete anaerobic benzoate degradation pathway found in a Chloroflexi genome. While it was previously reported that the subphylum II Chloroflexi member DEHJ10 from Aarhus Bay contains subunits of BCR (52), our finding is the first record of complete BCR genes in members of subphylum I.

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FIG 4 

Reconstructed energy metabolism of DscP3 and Dsc4. All pathways found to be supported by genomic evidence in DscP3 and/or Dsc4. Reduced electron carriers are shown in red, oxidized electron carriers in blue, and ATP in orange. Abbreviations: F6P, fructose-6-phosphate; PFK, phosphofructokinase; FBP, fructose-1,6-bisphosphatase; F1,6BP, fructose bisphosphate aldolase; TPI, triose phosphate isomerase; PGK, phosphoglycerate kinase; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PGM, phosphoglycerate mutase; THF, tetrahydrofolate; Fd, ferredoxin; ME, malic enzyme; GADP, glyceraldehyde-3-phosphate; GADPH, glyceraldehyde-3-phosphate dehydrogenase; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; PK, pyruvate kinase; LDH, lactate dehydrogenase; FumR, fumarate reducatse; SCoAS, succinyl-CoA synthase; OGDH, 2-oxoglutarate dehydrogenase; Fdn, formate dehydrogenase-N; PFOR, pyruvate-ferredoxin oxidoreductase; PTA, phosphotransacetylase; AK, acetate kinase; Rnf, Rhodobacter nitrogen fixation complex; FdhAB, formate dehydrogenase H; FTS, 10-formyltetrahydrofolate synthetase; FolD, bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methylene-tetrahydrofolate cyclohydrolase; MTHFR, 5,10-methylenetetrahydrofolate reductase; AcsE, methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase; CoFeSP, corrinoid iron-sulfur protein; ACS, acetyl-CoA synthase; CODH, carbon monoxide dehydrogenase; BCL, benzoate-CoA ligase; BCR, benzoyl-CoA reductase; Hdr-IFO, heterodisulfide reductase-associated ion-translocating ferredoxin oxidoreductase; Dch, cyclohexa-1,5-diene-1-carboxyl-CoA hydratase; Had, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase; Oah, 6-oxycylohex-1-ene-1-carbonyl-CoA hydrolase; HPDhd, 3-hydroxypimeloyl-CoA dehydrogenase; PimB, acyl-CoA acetyltransferase; GcdH, glutaryl-CoA dehydrogenase; EHt, enoyl-CoA hydratase; 3-HbcdH, 3-hydroxyacyl-CoA dehydrogenase; ETF, electron transport flavoprotein; TCA, tricarboxylic acid.

The first step in anaerobic benzoate degradation is benzoate-CoA ligase, which catalyzes the conversion of benzoate to benzoyl-CoA. The second step is the reductive dearomatization of benzoyl-CoA to cyclohex-1,5-diene-1-carbonyl-CoA with reduced ferredoxin by benzoyl-CoA reductase (BCR) (64). After we discovered BCR in the single-cell genomes of DscP3 and Dsc4, we reanalyzed SAG An-B16 from the Okinawa Trough and found that this SAG also contains these genes (15). Amino acid sequence and gene order comparisons revealed the BCRs of these marine Chloroflexi to be of class I (bzdNOPQ), the same class as previously found in DEHJ10 and most similar to the BCRs of the well-studied facultative Azoarcus spp. and Thauera aromatica (Fig. S7) (14, 65, 66). In contrast to the ATP-independent reversible class II BCRs (bamBCDEFGHI) found in Syntrophus aciditrophicus, Desulfococcus multivorans, Geobacter spp., and other obligate anaerobic benzoate degraders, class I BCRs are unlikely participants in benzoate fermentation, because the amount of energy available from benzoate oxidation is insufficient to support the energy requirement of the ATP-dependent class I BCR (64, 67–69). Rather, class II BCRs are found associated with anaerobic benzoate oxidation coupled with iron, sulfate, or proton reduction. The sole known exception is the hyperthermophilic archaeon Ferroglobus placidus, which utilizes a class I BCR despite its growth via iron reduction (70). It has been hypothesized that for the class II BCRs, Hdr-IFO allows for the generation of reduced ferredoxin to provide some of the necessary reducing power for benzoyl-CoA reduction (45).

In the anaerobic benzoate degradation pathway, cyclohex-1,5-diene-1-carbonyl-CoA is subsequently metabolized either to 2,3-didehydro-pimeloyl-CoA (using the Rhodospeudomonas palustris BadK-BadH-BadI pathway) or to 3-pimeloyl-CoA (using the Dch-Had-Oah pathway) (64). The genomes of DscP3, Dsc4, and An-B22 contain all of the genes required for the degradation of benzoyl-CoA to 3-hydroxypimeloyl-CoA, including those for a benzoate-CoA ligase (bzdA), the four subunits of Azoarcus-type BCR (bzdNOPQ), ferredoxin (bzdM), and the BCR ferredoxin-regenerating system (korAB and bzdV), as well as genes for the modified β-oxidation pathway (bzdWXY and dch-had-oah).

Further oxidation of 3-hydroxypimeloyl-CoA produces three molecules of acetyl-CoA and CO₂. Many microorganisms contain multiple acyl-CoA reductase and enoyl-CoA hydratase family proteins that might participate in the degradation of 3-hydroxypimeloyl-CoA (71). In Azoarcus sp. strain EbN1, multiple gene clusters have been identified encoding enzymes for β-oxidation (72). Therefore, any of the acyl-CoA reductase and enoyl-CoA hydratase genes within the genomes of DscP3 and Dsc4 could be responsible for the oxidation of 3-hydroxypimeloyl-CoA.

The genome of DscP3 contains genes for electron transfer flavoproteins (Etfβα) and an Etf-linked acyl-CoA dehydrogenase (ACAD) with an Etf dehydrogenase (fixABCX) system. ACADs catalyze the reversible oxidation of acyl-CoA to a 2,3-enoyl-CoA involved in β-oxidation (73). While members of an acyl-CoA dehydrogenase family share the same reaction mechanism and a high degree of sequence identity, they differ widely in their substrate-binding specificity. The nonbifurcating acyl-CoA dehydrogenase-Etf system has mostly been studied in mammals in which electrons are transferred from fatty acid-oxidizing ACADs to a membrane Etf-quinone oxidoreductase (Etf-QO) that transfers the electrons to the quinone pool (74). In members of Clostridium, Etfαβ is known to form a stable complex with a propionyl-CoA dehydrogenase (Pcd) or butyryl-CoA dehydrogenase (BcdA) (75, 76). The BcdA-Etfβα complexes of Firmicutes are bifurcating, coupling the NADH-dependent exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin (Fd). The latter is key for energy conservation by the formation of either NADH via Rnf or hydrogen via Ech (76, 77). An exception is the Pcd-Etfβα complex of Clostridium propionicum, in which a 15-amino-acid insertion into the Etf α subunit is proposed to keep the protein in a conformation which catalyzes the NADH-dependent reduction of acryloyl-CoA to propionyl-CoA but does not support the reduction of Fd (75). The fixABCX system, which has been described in several syntrophic and diazotrophic bacteria, contains an Etfβα homologue, FixAB, and is proposed to bifurcate electrons from NADH to Fd, FixX, and the Etf-quinone oxidoreductase homologue, FixC, with subsequent electron transfer to the quinone pool (78). While the substrate of ACAD in DscP3 is unknown, Etf has been observed to be the electron acceptor for glutaryl-CoA dehydrogenase (79). Thus, we hypothesize that the electrons from the oxidation of glutaryl-CoA to crotonyl-CoA in benzoate degradation are confurcated with a reduced ferredoxin to form NADH or funneled to the quinone pool via the FixABCX system.

(4) New insights into DscP2-2.Our previous analysis of the partial genome of DscP2, originating from two single cells from site 1230 (13), identified hydrogenase accessory proteins encoded by hypABCDEF, an archaeal-type H⁺ ATPase (subunits ABCDEFI), a [NiFe]-hydrogenase, and a partial F₄₂₀-reducing hydrogenase and genes encoding the following enzymes required for the WLP: formate dehydrogenase, formyltetrahydrofolate synthetase, CO-dehydrogenase/CO-methylating acetyl-CoA synthase complex (α and β subunits), methyltetrahydrofolate reductase, and methylene tetrahydrofolate methyltransferase. Besides a 16S rRNA gene identical to DscP2, the here-reported DscP2-2 genome also shares 275 coding sequences (CDS) (>97% nucleotide identity) with DscP2, including carbon monoxide oxidoreductase/acetyl-CoA synthase from the WLP and the archaeal-type H⁺ ATPase (13). Moreover, the here-reported DscP2-2 genome contains 321 unique coding sequences, including an Rnf complex and genes homologous to the bifurcating NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase (Nfn) (Fig. 5).

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FIG 5 

Metabolic pathways found in DscP2/DscP2-2. Genes identified in either DscP2 or DscP2-2 are shown in black, and those not found but hypothesized to be present are in gray. Proton-translocating complexes are in green, and electron-bifurcating complexes are in orange. Figure abbreviations are the same as in Fig. 4 with the addition of the following: ACoAS, acetyl-CoA synthase; IDH, isocitrate dehydrogenase; Aco, aconitase; Tkt, transketolase; E4P, erythrose-4-phosphate; G6P, glucose-6-phosphate; X5P, xylose-5-phosphate; HK, hexokinase; PGI, phosphoglucose isomerase; PC, pyruvate carboxylase; ArgA, N-acetylglutamate synthase; ArgB, acetylglutamate kinase; ArgC, N-acetylglutamylphosphate reductase; ArgD, N-acetylornithine aminotransferase; ArgE, acetylornithine deacetylase; OTC, ornithine transcarbamoylase; ArgG, argininosuccinate synthase; ArgH, argininosuccinate lyase; L2HGDH, l-2-hydroxyglutarate dehydrogenase; GtcA, glutaconate CoA-transferase.

As previously mentioned, partial genomes of DscP3 and Dsc4 contain genes encoding an Rnf complex. The genes for this Rnf complex in the subphylum I Chloroflexi are identical to the one found in DscP2-2. The presence of these genes in all three genomes indicates that they might have been shared via horizontal gene transfer (80). While this is not the first time that an Rnf complex has been found in a member of the phylum, it is the first reported observation of an Rnf complex in a member of Chloroflexi subphylum II (81).

The NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase (NfnBA) is a heterodimeric enzyme that catalyzes the reversible oxidation of NADH and ferredoxin with the reduction of NADP⁺ via flavin-based electron bifurcation (82). The [2Fe-2S]-containing NfnB subunit shows sequence similarities to bacterial dihydroorotate dehydrogenase (pyrK), and NfnA, which harbors two [4Fe-4S] clusters and flavin adenine dinucleotide (FAD), shows similarity to the small subunit of NADPH-dependent glutamate synthase (GltD) (83). Because of the similarities of the subunits to other functional genes and the inconsistency of nomenclature in genome annotations, their location on the genome is paramount to identifying their putative function. Homologues of gltD are found in all domains of life and are part of known or putative electron transfer systems (84). For example, NfnBA is often annotated as SudBA in the genomes of sulfate-reducing bacteria and archaea (85, 86), which catalyze the reduction of polysulfide to H₂S using NADPH as electron donor. In DscP2-2, the genomic context vicinal to nfnBA looks remarkably similar to a conserved region near the origin of replication of Dehalococcoides spp. (Fig. S8) (87). In fact, an examination of Dehalococcoides and Dehalogenimonas genomes reveals putative nfnBA genes conserved in all members of the clade. The putative NfnBAs with highest amino acid similarity to members of Chloroflexi subphylum II are the acetate-oxidizing, sulfate-reducing Deltaproteobacteria member Thermodesulforhabdus norvegica and the acetate-oxidizing syntrophic firmicute Syntrophaceticus schinkii.