“Candidatus Galacturonibacter soehngenii” Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of “Candidatus Galacturonibacter soehngenii,” from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of “Ca. G. soehngenii” was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the “Ca. G. soehngenii” MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of “Ca. G. soehngenii” enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of “Ca. G. soehngenii.”

In a recent study on D-galacturonate-limited, anaerobic enrichment cultures, we identified the dominant bacterium as a species from a novel genus within the Lachnospiraceae, for which we proposed the name "Candidatus Galacturonibacter soehngenii." The Lachnospiraceae family is part of the phylum Firmicutes, which includes several genera that harbor acetogens (Drake et al., 2008;Ragsdale and Pierce, 2008;Schuchmann and Muller, 2013;Valk et al., 2018). Fermentation product stoichiometries of the enrichment cultures were consistent with an acetogenic dissimilation of galacturonate. The overall stoichiometry is shown in Equation (2) (Valk et al., 2018).
Metagenome analysis of the enrichment culture revealed homologs of most structural genes for WLP enzymes, but no homologs were found for genes encoding subunits of the canonical CODH/ACS complex (EC 2.3.1.169) (Valk et al., 2018).
The goal of the present study was to further investigate the presence of a possible alternative configuration of the WLP in "Ca. G. soehngenii." To analyze in vivo activity of the WLP, D-galacturonate-limited enrichment cultures were co-fed with 13 C-labeled bicarbonate, followed by analysis of 13 C in the methyl and carboxyl groups of acetate. To investigate whether canonical WLP genes might have been overlooked in the initial metagenomics analysis, a fully closed metagenome-assembled genome (MAG) sequence of "Ca. G. soehngenii" was constructed using long-read nanopore sequencing, and meta-transcriptome analysis was performed to analyze the expression levels of genes of interest. Additionally, CO dehydrogenase activity was analyzed in cell extracts.

Reactor Setup and Operation
Chemostat cultures were grown in 1.2 L laboratory bioreactors (Applikon, Delft, The Netherlands), which were stirred at 300 rpm and kept at 30 • C. Anaerobic conditions were maintained by flushing the headspace with nitrogen gas, at a flow rate of 120 mL min −1 . Culture pH was controlled at 8 ± 0.1 by automatic titration (ADI 1030 Biocontroller, Applikon, Delft, The Netherlands) of 1 M NaOH. The dilution rate was 0.09 ± 0.01 h −1 and the working volume of 0.5 L was kept constant by peristaltic effluent pumps (Masterflex, Cole-Parmer, Vernon Hills, IL, United States) coupled to electrical level sensors. Bioreactors were inoculated (10% v/v) with 50 mL samples of D-galacturonate-limited, anaerobic chemostat enrichment cultures (Valk et al., 2018), stored in 30% v/v glycerol at -20 • C. Cultures were run in continuous mode and after at least 6 days (18 generations) stable product composition and biomass concentration were established. System stability was assessed by online monitoring of CO 2 production and offline monitoring of fermentation products and optical density. When measurements varied by less than 10% over multiple volume changes, without a clear upward or downward trend, samples were taken during subsequent cycles.

Medium
The cultivation medium contained (g L −1 ):

Analysis of Substrate and Extracellular Metabolite Concentrations
To determine substrate and extracellular metabolite concentration, reactor sample supernatant was obtained by centrifugation of culture samples (Heraeus Pico Microfuge, Thermo Fisher Scientific, Waltham, MA, United States). Concentrations of D-galacturonate and extracellular metabolites were analyzed with an Agilent 1100 Affinity HPLC (Agilent Technologies, Amstelveen, The Netherlands) equipped with an Aminex HPX-87H ion-exchange column (BioRad, Hercules, CA, United States) operated at 60 • C with a mobile phase of 5 mM H 2 SO 4 and a flow rate of 0.6 mL min −1 . CO 2 and H 2 concentrations in the bioreactor exhaust gas were measured using a Prima BT Bench Top mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) after the gas was cooled by a condenser (4 • C).

Biomass Dry Weight
Twenty milliliter of culture broth samples were filtered over predried and pre-weighed membrane filters (0.2 µm Supor-200, Pall Corporation, New York, NY, United States), which were then washed with demineralized water, dried in a microwave oven (Robert Bosch GmbH, Gerlingen, Germany) for 20 min at 360 W and reweighed. Carbon and electron balances were constructed based on the number of carbon atoms and electrons per mole, while biomass composition was assumed to be CH 1.8 O 0.5 N 0.2 (Roels, 1983).

Quantitative Fluorescent in situ Hybridization (qFISH) Analysis
Fluorescent in situ hybridization was performed as described previously (Daims et al., 2005), using a hybridization buffer containing 35% (v/v) formamide. Probes were synthesized and 5 labeled with either 5(6)-carboxyfluorescein-Nhydroxysuccinimide ester (FLUOS) or with one of the sulfoindocyanine dyes (Cy3 and Cy5; Thermo Hybaid Interactiva, Ulm, Germany) ( Table 1). The general probe EUB338mix, labeled at both 3 and 5 ends with Cy5, was used to identify all eubacteria in the sample. Microscopic analysis was performed with a LSM510 Meta laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). The qFISH analysis was based on at least 29 fields of view at 6730 × magnification, using DAIME (version 2.1) software (DOME, Vienna, Austria; Daims et al., 2006). The bio-volume fractions of "Ca. G. soehngenii" and Enterobacteriaceae populations were calculated as the ratio of the area hybridizing with specific probes relative to the total area hybridizing with the universal EUBmix probe set (Amann et al., 1990;Daims et al., 1999).

Labeling Experiment 13 C-Labeled Sodium Bicarbonate Addition
A 1 M NaH 13 CO 3 solution was used to replace the regular 1 M NaOH solution as a pH titrant in steady-state D-galacturonatelimited enrichment cultures (pH 7.8 ± 0.1, D = 0.1 h −1 , T = 30 • C). Broth was collected on ice every 2 h for 8 consecutive hours and centrifuged (12,000 × g, Heraeus Pico Microfuge, Thermo Fisher Scientific, Waltham, MA, United States) before the supernatant was collected and stored at -20 • C until analysis by NMR. CO 2 , H 2 and 13 CO 2 concentrations in the exhaust gas were measured by MS (Prima BT Bench Top MS, Thermo Fisher Scientific, Waltham, MA, United States) after the gas had been cooled by a condenser (4 • C).
Illumina and Nanopore Sequencing, Metagenome Assembly, and Genome Binning DNA The metagenomic-assembled genome of "Candidatus Galacturonibacter soehngenii" described by Valk et al. (2018) was used as template for preparing the metagenome libraries. The DNA extraction, Illumina sequencing, metagenomic assembly and binning process is described

Genome Assembly
The assembling of the contigs from the "Candidatus Galacturonibacter soehngenii" genome bin into a single scaffold based on the long Nanopore reads was done using SSPACE-LongRead scaffolder v. 1.1 (Boetzer and Pirovano, 2014). GapFiller v. 1.11 (Boetzer and Pirovano, 2012) or by manual read mapping and extension in CLC Genomics Workbench v. 9.5.2 (Qiagen, Hilden, Germany) were used to close gaps in the draft genome with the previously assembled Illumina data. Finally, manual polishing of the complete genome was done to remove SNPs and ensure a high-quality assembly. The meta-genome has been submitted to the sequence read archive (SRA) 1 with accession number SRR10674409, under the BioProject ID PRJNA566068.

Trimming and Mapping of rRNA Reads
Raw RNA reads in FASTQ format were imported into CLC Genomics Workbench v. 9.5.5 and trimmed for quality, requiring a minimum phred score of 20 and a read length of 45. Reads from each sample were hereafter mapped to CDSs obtained from the MAG of "Ca. G. soehngenii" with a minimum similarity of 98% over 80% of the read length. Reads per kilobase of transcript per million mapped reads (RPKM) were calculated based on raw read-counts and the length of each CDS. The meta-transcriptome mapped to the genome of "Ca. G. soehngenii" are shown in Supplementary Data Sheet S2.

Plasmid and Strain Construction
Gene F7O84_RS11645 was codon optimized for expression in Escherichia coli with the GeneArt online tool and integrated behind the TEV recognition site of the pET151/D-TOPO expression vector by GeneArt (GeneArt GmbH, Regensburg, Germany). The resulting plasmid was transformed into a chemically competent E. coli strain BL21 according to manufacturer's instructions (NEBuilder HiFi DNA Assembly Master Mix chemical transformation protocol (E2621), New England Biolabs, Ipswich, MA, United States) and named pUD1074. The plasmid sequence of pUD1074 has been deposited at the NCBI GenBank 2 with the corresponding accession number MN498128.

Heterologous Expression of the Putative CO Dehydrogenase Candidate
All E. coli cultures were performed in 120 mL capped bottles with 50 mL of mineral medium (Diender et al., 2016). Prior to inoculation, the bottles were autoclaved at 120 • C after which the mineral media was supplemented with autoclaved (120 • C, 20 min); glucose 5 g L −1 , peptone (BD Bacto Difco, Thermo Fisher Scientific, Waltham, MA, United States) 1 g L −1 , yeast extract (BD Bacto Difco, Thermo Fisher Scientific, Waltham, MA, United States) 2 g L −1 and cysteine 1 g L −1 . Additionally, 0.05 g L −1 ampicillin was added and the gas phase was exchanged with air, with a final pressure of 170 kPa. All E. coli cultures used for measurements were inoculated with overnight grown precultures (1:50 v/v) and incubated at 37 • C and shaken (300 rpm) until oxygen was depleted (2-3 h). Subsequently 1 mL (250 g L −1 ) glucose, 1 mL reducing agent (0.4 M cysteine) and 1 mL IPTG (40 mM) were added. After 3 h (at 30 • C, unshaken) of incubation, the cells were harvested and processed anaerobically according to Diender et al. (2016). Enzymatic activity analysis was conducted using a modified method initially described by Diender et al. (2016). The essays were performed in an anaerobic environment using 100-300 µL of cell extract with both CO and hydroxylamine as substrate. To increase metal cofactor availability, 1:200 (v/v) metals solution was added to the assay buffer which contained in (g L −1 ); HCl 1.8, H 3 BO 3 0.0618, MnCl 2 0.06125, FeCl 2 0.9435, CoC l2 0.0645, NiCl 2 0.01286, ZnCl 2 0.0677, CuCl 2 0.01335.

Homology Protein BLAST Analysis
The sequence of the putative CODH (F7O84_RS11645) was blasted with the BLASTp (version 2.2.28+; Altschul et al., 1997) tool of the JGI-IMG/M database (Markowitz et al., 2012), with default parameter settings. Finished genomes from members of the Lachnospiraceae family in the public JGI-IMG/M database (Markowitz et al., 2012) were selected for analysis, Supplementary Table S4. The stains identified in the BLAST search, or closely related strains (Supplementary Table S5) were subsequently analyzed in KEGG (Kanehisa et al., 2014) for presences of the CODH/ACS complex with pathway map 1200.

RESULTS
Physiological Characterization of D-Galacturonate-Limited Enrichment Cultures Dominated by "Ca. G. soehngenii" Anaerobic, galacturonate-limited chemostat enrichment cultures were used to study the physiology of "Ca. G. soehngenii" cultures. In a previous study (Valk et al., 2018), the relative abundance of "Ca. G. soehngenii" in such cultures did not exceed 65%, based on metagenomic analysis, and formate and H 2 were detected in the liquid and gas phases, respectively. It was hypothesized that, in these experiments, a low in situ hydrogen partial pressure limited in vivo WLP activity, as it was expected that hydrogen was used as reductant for the production of acetate from formate or CO 2 . To investigate this possibility, head space flushing instead of sparging was applied, using N 2 gas. This caused an increase in the hydrogen partial pressure in the media broth (De Kok et al., 2013). Additionally, the dilution rate was decreased from 0.125 to 0.1 h −1 . Analysis of the abundance of "Ca. G. soehngenii" in the resulting enrichment cultures by quantitative fluorescence in situ hybridization (qFISH) indicated that 86.5 ± 2.6% of the bio-volume of qFISH-detectable cells consisted of "Ca. G. soehngenii." The major side population Enterobacteriaceae represented 13.8 ± 2.4% of the bio-volume. As these two subpopulations together accounted for 100.2 ± 5.0% of the bio-volume, it was assumed that any other, minor, subpopulations did not significantly influence the stoichiometry of catabolic fluxes.
Product yields and biomass-specific conversion rates of the D-galacturonate-limited anaerobic enrichment cultures dominated by "Ca. G. soehngenii" ( Table 2) showed acetate as dominant catabolic product (0.57 ± 0.03 Cmol (Cmol galacturonate −1 ). Carbon and electron recoveries were 94 and 92%, respectively, indicating that all major fermentation products were identified. As observed previously (Valk et al., 2018), this acetate yield on galacturonic acid was significantly higher than the combined yields of formate and hydrogen. This difference was interpreted as indicative for acetogenesis by one of the dominant organisms, of which only the "Ca. G. soehngenii" MAG was shown to harbor homologs for most WLP structural genes (Ragsdale and Pierce, 2008;Valk et al., 2018). Yields of hydrogen and formate on galacturonate (0.02 ± 0.01 mol Cmol galacturonate −1 ) and 0.02 ± 0.01 (Cmol galacturonate −1 ), respectively were significantly lower than found in a previous study on "Ca. G. soehngenii" (Valk et al., 2018). This observation is consistent with a higher in vivo contribution of the WLP as a result of a higher hydrogen partial pressure and/or lower specific growth rate in the present study.

Incorporation of 13 C-Labeled Bicarbonate Into Acetate Corroborates Acetogenic Fermentation
A simple model was constructed to predict formation of labeled acetate, using biomass-specific conversion rates measured in pseudo-steady state enrichment cultures as inputs (Supplementary Calculations S1, S2 and Supplementary Figure S1). Model simulations predicted that, after 8 h, approximately 15% of the acetate produced by the enrichment culture should be labeled. To investigate if CO 2 was indeed incorporated into acetate via acetogenic fermentation, 13 C-labeled bicarbonate was fed to a "Ca. G. soehngenii" enrichment chemostat culture. However, after 8 h, the fraction of 13 C in the methyl group of acetate increased to 2.0%. This increase represented only a small increase relative to the 1% natural abundance of 13 C ( Table 3; Rumble et al., 2017). In contrast, after 8 h of 13 C-bicarbonate feeding, the enrichment culture showed a 21.5% abundance of 13 C in the carbonyl-group of acetate (Table 3).
Significant Activity of CO Dehydrogenase in Cell Extracts of "Ca.

G. soehngenii" Enrichment Cultures
In the WLP, 13 C-labeled CO 2 incorporation into the carbonylgroup of acetate involves activity of CO dehydrogenase (COOS, EC 1.2.7.4). To investigate the presence of this key enzyme in "Ca. G. soehngenii, " an anaerobic enzyme activity assay was performed on cell extracts of enrichment cultures, using CO as electron donor and methyl viologen (MV) as electron acceptor (Diender et al., 2016). These assays revealed a CO dehydrogenase activity of 2.1 ± 0.6 µmol min −1 (mg protein) −1 . Reduction of MV in the absence of either CO or cell extract was below detection limit [<0.05 µmol min −1 (mg protein) −1 ].

Identification of Two Putative Novel CO Dehydrogenase Genes in a Newly
Obtained Single-Scaffold MAG of "Ca. G. soehngenii" Previous analysis of the "Ca. G. soehngenii" MAG (Valk et al., 2018) was based on an assembly made with shortread DNA sequencing data. To identify if putative CODH/ACS complex genes had been missed in this analysis due to incomplete assembly, long-read Oxford Nanopore sequencing (Deamer et al., 2016;Jain et al., 2016) was used to improve the previously assembled "Ca. G. soehngenii" MAG. The resulting genome assembly consisted of 8 contigs and was estimated to have a 98% completeness and contained no genetic contamination with sequences from other organisms according to checkM ( Table 4). As in the previous study, homologs were detected for most structural genes associated with the WLP ( Table 5), but none of the annotated genes in the predicted proteome showed homology with known CODH/ACS genes (Vallenet et al., 2006;Valk et al., 2018). A search in the newly assembled "Ca. G. soehngenii" MAG sequence for homologs of signature genes of the six other known pathways for inorganic carbon fixation did not point toward their involvement in carbon metabolism (Supplementary Table S2). CO dehydrogenases contain highly conserved amino-acid motifs (Pfam or protein-family domains) associated with their nickel-iron-sulfur clusters (Eggen et al., 1991(Eggen et al., , 1996Maupin-Furlow and Ferry, 1996;Jeoung and Dobbek, 2011;Techtmann et al., 2012;El-Gebali et al., 2018). The newly assembled "Ca. G. soehngenii" MAG sequence did not reveal hits for the Pfam domain of the CO dehydrogenase α-subunit of the CODH/ACS complex (PF18537) (Darnault et al., 2003).
However, two open reading frames F7O84_RS02405 and F7O84_RS11645, harbored the PF03063 Pfam domain, which is associated with the hybrid cluster protein (HCP) and the catalytic center of the Ni-CODH family (van den Berg et al., 2000;Wolfe et al., 2002). Although HCP has been associated with hydroxylamine reductase activity, its catalytic activity has Completeness and contamination were estimated with CheckM (Parks et al., 2015).
Frontiers in Microbiology | www.frontiersin.org not been experimentally confirmed and, moreover, sequence motifs in HCP showed high similarity with the functional domain of Ni-CODHs making it an interesting candidate genes for the CODH function of the WLP in "Ca. G. soehngenii" Wolfe et al., 2002;Aragão et al., 2003;Almeida et al., 2006). A closer inspection of the genetic context of both genes showed many flanking genes encoding hypothetical proteins in their close vicinity, but no genes previously associated with acetogenesis.

Homologs of Acetogenesis Genes Are Transcribed in D-Galacturonate-Limited "Ca. G. soehngenii" Enrichment Cultures
A meta-transcriptome analysis of the enrichment cultures showed significant transcript levels of most homologs of known WLP genes, which were approximately 10-fold lower than those of homologs of structural genes encoding Entner-Doudoroffpathway enzymes involved in galacturonate catabolism ( Table 6).
A notable exception was the extremely low transcript level of a putative formate dehydrogenase gene (F7O84_RS07405; EC 1.17.1.9). A candidate gene for pyruvate-formate lyase (PFL, EC 6.2.1.3) was highly transcribed (F7O84_03160 , Table 6). These observations suggested that formate generated by PFL, rather than CO 2 , was the major substrate for the methyl branch of the WLP in "Ca. G. soehngenii." Homologs of Rnf cluster (F7O84_03275-3295; EC 7.2.1.2) and hydrogenase (F7O84_0945-50, F7O84_04820; EC 1.12.7.2) genes, which were previously implicated in acetogenesis (Biegel and Müller, 2010;Müller, 2014, 2016), showed high transcript levels ( Table 6). Of the two candidate genes for CO dehydrogenase, F7O84_RS11645 showed the highest transcript level ( Table 6). As, under the experimental conditions, no hydroxylamine reductase activity was expected, this result reinforced the candidature of F7O84_RS11645 as possible CO dehydrogenase gene. In an attempt to directly investigate if F7O84_RS11645 encoded a functional CO dehydrogenase, its open reading frame was cloned into high-copy-number E. coli expression vector. However, enzyme assays with cell extracts of the resulting E. coli strain did not yield consistent evidence for either CO dehydrogenase or hydroxylamine dehydrogenase activity (Supplementary Table S3).

Identification of Proteins With a High Homology of the Putative CODH Within Other Members of the Lachnospiraceae Species
A protein BLAST search (Altschul et al., 1997) of the putative CODH (F7O84_RS11645) was done to investigate if presence of the putative CODH gene also coincided with an apparently incomplete WLP in other members of the Lachnospiraceae family. Indeed, 13 sequenced members of the Lachnospiraceae family showed predicted proteins with a high homology with the putative CODH (Supplementary Table S4). 9 of the 13 Lachnospiraceae members were present in the KEGG database (Kanehisa et al., 2014; Supplementary Table S5), and subsequently analyzed on the presence or absence of the CODH/ACS complex. All organisms contained only a partial WLP, with the ACS genes not identified. In seven of the members, respectively Lachnoclostridium saccharolyticum, Lachnoclostridium phytofermentans, Pseudobutyrivibrio xylanivorans, Butyrivibrio fibrisolvens, Pseudobutyrivibrio xylanivorans, and both Roseburia species the full CODH/ACS complex was not identified. Further study is required to elucidate the relevance of the putative CODH for acetogenic metabolism.

DISCUSSION
Incorporation of carbon from 13 C labeled bicarbonate into the carbonyl group of acetate supported our previous conclusion, based on product profiles, that acetogenesis occurs in anaerobic, galacturonate-limited enrichment culture of "Ca. G. soehngenii" (Valk et al., 2018). A much lower labeling of the methyl group of acetate indicated that, instead of carbon dioxide, the methyl branch of the WLP in the "Ca. G. soehngenii" enrichment cultures predominantly used formate as a substrate, generated in the anaerobic fermentation of galacturonate (Figure 1). This conclusion is consistent with the low transcript levels of the only putative formate dehydrogenase gene (F7O84_RS07405; EC 1.17.1.9; Table 6) identified in the "Ca. G. soehngenii" MAG, the high transcript level of a putative pyruvate-formate lyase gene (F7O84_RS03160, EC 6.2.1.3; Table 5) and the low net production rates of formate in the anaerobic enrichment cultures ( Table 2). In contrast, previous labeling studies on acetogens harboring the WLP showed marginal preferential labeling of the carboxyl moiety of acetate (Wood and Harris, 1952;O'Brien and Ljungdahl, 1972;Schulman et al., 1972), indicating the use of extracellular CO 2 as substrate for both the methyl-and carbonylgroups of acetate. While the observed labeling pattern was consistent with acetogenic metabolism of galacturonate via a WLP, this did not rule out involvement of another pathway for carbon fixation in acetate. Involvement of the hydroxypropionate bi-cycle, 3-hydroxypropionate/4-hydroxybutyrate cycle and dicarboxylate/hydroxybutyrate cycle were excluded since no homologs were found in the "Ca. G. soehngenii" MAG for the majority of genes associated with these three pathways (Supplementary Table S2). Key genes were also missing for the reductive pentose phosphate cycle (rPPP) and reductive citric acid cycle (rTCA) (Supplementary Table S2) and, moreover, neither of these pathways could explain preferential labeling of the carboxyl group of acetate (Alberts et al., 2002;Shimizu et al., 2015). No gene candidates were identified for the glycine cleavage (GCV) system (Supplementary Table S2 and Supplementary Figure S2) and 13 C-labeled bicarbonate fed into this pathway should result in equal labeling of the methyl and carbonyl groups of acetate (Figueroa et al., 2018;Supplementary Figure S2). Additionally, none of the routes would require the high CO dehydrogenase enzyme activity measured in cell extracts of the "Ca. G. soehngenii" enrichment culture. This analysis leaves the WLP as the only known carbon fixation pathway consistent with the observed stoichiometry of fermentation products, the labeling pattern of acetate and, with the notable exception of the CODH complex, genome and transcriptome analysis of "Ca. G. soehngenii." Homologs of structural genes encoding enzymes of an adapted Entner-Doudoroff pathway for galacturonate metabolism were highly expressed in the galacturonate-limited, anaerobic "Ca. G. soehngenii" enrichment cultures ( Table 6). Since conversion of one mole of galacturonate into two moles of pyruvate via this pathway is redox-cofactor neutral, redox equivalents for acetogenesis needed to be derived from pyruvate dissimilation (van Maris et al., 2006;Kuivanen et al., 2019). Pyruvate:ferredoxin oxidoreductase (F7O84_RS03200, FIGURE 1 | Graphical representation of the proposed pathway for acetogenic D-galacturonate catabolism in "Candidatus Galacturonibacter soehngenii." The conversions of known and annotated genes identified in the MAG and transcribed in the meta-transcriptomic analysis "Ca. G. soehngenii" are colored black, the proposed CO dehydrogenase candidate colored yellow and the unidentified acetyl-CoA synthase colored red. With pyruvate:ferredoxin oxidoreductase (pfor, EC 1.2.7.1), pyruvate formate lyase (pfl, EC 6.2.1.3), ferredoxin hydrogenase (hydABC, EC 1.12.7.1) and the Rnf-cluster (rnfABCDEG, EC 7.2.1.2) explicitly shown. EC 1.2.7.1) has been reported to couple fermentation and WLP in other anaerobes (Drake et al., 1981;Menon and Ragsdale, 1996b;Schuchmann and Müller, 2014). Strong, highly transcribed homologs of structural genes for PFOR and for a ferredoxin hydrogenase (EC 1.12.7.2) ( Table 6; F7O84_RS03200 and F7O84_0945-50, F7O84_04820 respectively) indicated that it also fulfils this role in "Ca. G. soehngenii." The significant CO dehydrogenase (CODH) (Weghoff and Müller, 2016) activities in cell extracts enrichment cultures, combined with the incorporation of 13 C from bicarbonate in acetate strongly suggested the presence of a functional CODH enzyme in "Ca. G. soehngenii." Two highly conserved classes of CODH enzymes have been described (King and Weber, 2007;Techtmann et al., 2012). Aerobic CODH enzymes (coxSML complex; EC 1.2.5.3) have a Mo-Cu-Se associated active site and only use CO as substrate (Schübel et al., 1995;Dobbek et al., 1999). Strictly anaerobic Ni-Fe-S associated CODH (cooS, EC 1.2.7.4) can use also CO 2 as substrate (Doukov et al., 2002;Techtmann et al., 2012). A close functional relationship between Ni-CO dehydrogenases and hydroxylamine reductases was shown when a single aminoacid substitution was shown to change a Ni-CO dehydrogenase into a hydroxylamine reductase . Since no strong homologs of canonical aerobic or anaerobic CODH genes were identified, the HCP homolog F7O84_RS11645 is therefore the best candidate for the observed CODH activity. Our inability to demonstrate stable CODH activity in cell extracts upon expression of F7O84_RS11645 in E. coli could have many causes, including improper folding, metal or cofactor requirements (Ensign et al., 1990;Kerby et al., 1997) or requirement of additional subunits or other proteins (Bonams and Luddent, 1987;Bonam et al., 1989;Ensign and Ludden, 1991;Aragão et al., 2008;Bar-Even et al., 2012a). The immediate genetic context of F7O84_RS11645 showed many ORFs encoding predicted conserved proteins with unknown function. Coexpression of fosmid libraries (Shizuya et al., 1992;Ho et al., 2018) of the "Ca. G. soehngenii" MAG together with the plasmid used in this study in an E. coli strain, may be helpful in resolving the genetic requirements for CODH activity in this organism.
It remains unclear how the CODH-dependent carbonyl branch and formate-dependent methyl branch of a WLP pathway in "Ca. G. soehngenii" organism are linked. The present study is not the first in which carbon fixation linked to the WLP was observed in the absence of a full complement of canonical WLP structural genes (Zhuang et al., 2014;Figueroa et al., 2018). However, no clear physiological nor phylogenetic connections were detected between "Ca. G. soehngenii" and the organisms studied previously, a strict dehalogenide-respiring Dehalococcoides mccartyi strain from the Chloroflexi phylum and the phosphite-oxidizing Deltaproteobacterium "Candidatus Phosphitivorax anaerolimi" Phox-21, respectively.
This study illustrates how quantitative analysis of metabolite formation by chemostat enrichment cultures, combined with 13 C-labeling, (meta-)genome assembly and annotation, metatranscriptome analysis and biochemical assays can raise new and surprising questions about intensively studied metabolic pathways. Based on our results, involvement of a novel inorganic carbon assimilation pathway, which produces a similar labeling and product profile as the WLP, cannot be fully excluded. However, despite the wide distribution of the CODH/ACS complex in Bacteria and Archaea (Schuchmann and Müller, 2016), the available evidence appears to point in the direction of an as yet unidentified link between the methyl and carbonyl branches of the WLP. Further research to resolve this issue may benefit from additional labeling studies with 13 C-bicarbonate, 13 C-formate or partially labeled D-galacturonate combined with metabolome analysis and in vitro enzyme activity studies of formate dehydrogenase. Such studies are complicated by our current inability to grow "Ca. G. soehngenii" in pure cultures (Valk et al., 2018). The organisms shown in the Supplementary Table S4 might be interesting alternative organisms to study in more detail, as they are available in pure culture. It would therefore be relevant to identify if any of these organisms exhibit a similar acetogenic metabolism, with an incomplete complement of WLP enzymes, to further explore this intriguing metabolic conundrum.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the European Nucleotide Archive (ENA) under the BioProject ID PRJNA566068, NCBI GenBank accession number MN498128.

AUTHOR CONTRIBUTIONS
ML, JTP, and LV designed the experiments, interpreted the results, and wrote the manuscript. LV did all cultivations and labeling study. LV and MD performed the enzyme activity assays and heterologous experiment. LV and JFP performed the qFISH analysis. GS made the model. MSD performed the experimental work for the metatranscriptomic and meta-genomic analysis. MSD, LV, and PN analyzed the data. All authors read and approved of the final manuscript.