Comparative Genomics of Syntrophic Branched-Chain Fatty Acid Degrading Bacteria

The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading “Syntrophomonas wolfei subsp. methylbutyratica” strain JCM 14075T (=4J5T), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1T, and non-BCFA-metabolizing S. wolfei subsp. wolfei strain GöttingenT. We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in β-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct β-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems (i.e., electron-bifurcating hydrogenase, formate dehydrogenase, and membrane-bound hydrogenase) and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H2 generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific β-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism.

The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading "Syntrophomonas wolfei subsp. methylbutyratica" strain JCM 14075 T (=4J5 T ), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1 T , and non-BCFA-metabolizing S. wolfei subsp. wolfei strain Göttingen T . We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in β-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct β-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems (i.e., electron-bifurcating hydrogenase, formate dehydrogenase, and membrane-bound hydrogenase) and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H 2 generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific β-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism.
Key words: syntroph, branched-chain fatty acid, genomics, energy conservation Under methanogenic conditions, the degradation of amino acids and proteinaceous materials inevitably generates fatty acids as byproducts (26). Fatty acid-oxidizing bacteria and methanogens are known to form syntrophic interactions in order to accomplish the endergonic oxidation of these fatty acids (9,19,26). Although the biochemical pathways and genes involved in the syntrophic degradation of short-chain fatty acids (SCFA; e.g., propionate and butyrate) have already been described, they have not yet been elucidated for branched-chain fatty acids (BCFAs; e.g., isobutyrate, isovalerate, and 2-methylbutyrate) derived from branched-chain amino acids (13,26). Syntrophic BCFA degradation to acetate and propionate has been observed in isolates and mixed cultures (14,26,34,35,41). Only three strains of the family Syntrophomonadaceae are currently known to syntrophically degrade 2-methylbutyrate ("Syntrophomonas wolfei subsp. methylbutyratica" strain JCM 14075 T (=4J5 T ) and S. bryantii strain CuCal T ) and isobutyrate (Syntrophothermus lipocalidus strain TGB-C1 T ) (29,33,40). These Syntrophomonadaceae species are considered to be important for fatty acid degradation in anaerobic ecosystems, including the sludge digestion process (19), rice paddy fields (12), and the termite gut (42). Furthermore, an uncultivated Syntrophaceae member has been proposed to degrade BCFA syntrophically in a metha-nogenic bioreactor through metagenomic and metatranscriptomic approaches (21). However, the key catabolic enzymes and energy conservation systems necessary to drive thermodynamically unfavorable BCFA and SCFA degradation remain unclear.
In the present study, the genomes of strains 4J5 (20) and TGB-C1 (4) were investigated in order to identify the metabolic pathways for 2-methylbutyrate and isobutyrate catabolism and energy conservation systems for syntrophic metabolism. A comparative genomic analysis between BCFAand non-BCFA-degrading syntrophs within the family Syntrophomonadaceae (i.e., S. wolfei subsp. wolfei strain Göttingen T [30]) provides genomic insights into the degradation of BCFA in methanogenic ecosystems.

Genome sequencing and annotation
This study analyzed the "S. wolfei subsp. methylbutyratica" strain JCM 14075 T (=4J5 T ) draft genome (DDBJ/GenBank/EMBL accession: BBQT01000001-BBQT01000092) (20), S. lipocalidus strain TGB-C1 T complete genome (CP002048) (4), and S. wolfei strain Göttingen complete genome (CP000448) (30). As reported previously (20), the genomic DNA of strain 4J5 was provided by the RIKEN BRC through the National Bio-Resource Project of MEXT, Japan, and sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) at FASMAC (Atsugi, Japan). Briefly, we constructed and sequenced a 300-bp paired-end library totaling ca. 2.2 Gb of MiSeq data. Assemblies were performed using SPAdes version 3.1.1 (2). The strain 4J5 draft genome comprises 89 scaffolds and has an estimated genome size of 3.2 Mbp with an average G+C content of 45.55%. The quality of the genome sequence was evaluated using the Check M version 1.0.5 program with a marker gene set of the class Clostridia (23). A total of 2,964 protein coding genes were annotated with Prokka version 1.11 (see Supplemental Information) (28). Basic local alignment search tool (BLAST) (ver. 2.2.30) with a non-redundant protein sequence database (nr) and the protein sequence database of Göttingen and TGB-C1 (11) and BLASTKoala of Kyoto Encyclopedia of Genes and Genomes (KEGG) (8) were used to search for functional domains and characterize potential protein functions. Proteins associated with energy conservation systems were identified by criteria based on the genomic and physiological information of previously reported energy conservation pathways (21). Transport systems were identified using TransportDB (25).
As final products of the predicted β-oxidation pathways, the syntrophic degradation of isobutyrate generates two acetyl-CoA and 2-methylbutyrate produces acetyl-CoA and propionyl-CoA (Fig. 1A). Acetyl-CoA yields ATP through dethiolation to acetate by phosphate acetyltransferase (Swmb_02801 and Slip_0902) and acetate kinase (Swmb_02802 and Slip_0903). Regarding strain 4J5, these enzymes may perform the dethiolation of 2-methylbutyrate-derived propionyl-CoA because the active site structure of previously known propionate kinase resembles those of acetate kinase and butyrate kinase (7). AMP-dependent acyl-CoA synthetases found in the 4J5 and TGB-C1 genomes (Swmb_02363 and Swmb_02710; Slip_0475, Slip_0583, and Slip_1686) potentially serve as an alternative acyl-CoA degradation pathway, as suggested by McInerney et al. (17). However, Swmb_02710 and Slip_1686 have high identities (>64% by amino acid sequences) to that of strain Göttingen (Swol_1180), which has been predicted to function in biosynthesis (30). The other homolog found in 4J5 (Swmb_02363) may be involved in poly-β-hydroxybutyrate metabolism due to an association with the poly-β-hydroxybutyrate polymerase gene, as observed in strain Göttingen (Swol_1144). The remaining TGB-C1 homologs (Slip_0475 and Slip_0583) have low amino acid sequence identities (<32%) with the biosynthesis-associated acyl-CoA synthetase (Table S4), implying that these acyl-CoA synthetase genes are responsible for the production of acetate from acetyl-CoA through the degradation of isobutyrate.
A nitrogenase gene cluster (NifBDEHIK) was detected in the genome of strain TGB-C1 (Slip_2124-2130) along with a molybdate transporter (ModABC, Slip_2121-2123) and ammonia transporter (AmtB, Slip_2119) (Fig. 2C, Table S7). A previous microbial genome survey revealed that nitrogen fixation-related proteins (Nif) are distributed in phylogenetically diverse microbes including some fermentative bacteria and syntrophic substrate metabolizers (5). The nitrogenase activity of the fermentative H 2 -producing organism Clostridium butyricum strain CWBI1009 has been proposed to generate H 2 and enhance tolerance to acidification through the consumption of protons in the reaction, which produces ammonia as a base (3). We observed high amino acid sequence similarities (up to 98%) between the nitrogenase, molybdate trans-porter, and ammonia transporter genes of TGB-C1 and CWBI1009, and also moderate similarities (up to 68%) with two other Nif-encoding syntrophs (Thermacetogenium phaeum strain PB and Syntrophobacter fumaroxidans strain MPOB) (Fig. 3, Table S7). Among syntrophs, nitrogen fixation may serve as a mechanism to tolerate acidification and provide hydrogen and ammonia for partner hydrogenotrophic methanogens to survive under hydrogen/ammonia-limited conditions.