Diverse non-canonical electron bifurcating [FeFe]-hydrogenases of separate evolutionary origins in Hydrogenedentota

ABSTRACT Hydrogenedentota, a globally distributed bacterial phylum-level lineage, is poorly understood. Here, we established a comprehensive genomic catalog of Hydrogenedentota, including a total of seven clades (or families) with 179 genomes, and explored the metabolic potential and evolutionary history of these organisms. We show that a single genome, especially those belonging to Clade 6, often encodes multiple hydrogenases with genomes in Clade 2, which rarely encode hydrogenases being the exception. Notably, most members of Hydrogenedentota contain a group A3 [FeFe]-hydrogenase (BfuABC) with a non-canonical electron bifurcation mechanism, in addition to substrate-level phosphorylation and electron transport-linked phosphorylation pathways, in energy conservation. Furthermore, we show that BfuABC from Hydrogenedentota fall into five sub-types. Phylogenetic analysis reveals five independent routes for the evolution of BfuABC homologs in Hydrogenedentota. We speculate that the five sub-types of BfuABC might be acquired from Bacillota (synonym Firmicutes) through separate horizontal gene transfer events. These data shed light on the diversity and evolution of bifurcating [FeFe]-hydrogenases and provide insight into the strategy of Hydrogenedentota to adapt to survival in various habitats. IMPORTANCE The phylum Hydrogenedentota is widely distributed in various environments. However, their physiology, ecology, and evolutionary history remain unknown, primarily due to the limited availability of the genomes and the lack of cultured representatives of the phylum. Our results have increased the knowledge of the genetic and metabolic diversity of these organisms and shed light on their diverse energy conservation strategies, especially those involving electron bifurcation with a non-canonical mechanism, which are likely responsible for their wide distribution. Besides, the organization and phylogenetic relationships of gene clusters coding for BfuABC in Hydrogenedentota provide valuable clues to the evolutionary history of group A3 electron bifurcating [FeFe]-hydrogenases.


Identification and phylogenetic analysis of hydrogenases. A reference set of amino acid sequences corresponding to 3,265 hydrogenases was downloaded from the HydDB database (3).
A local hydrogenase database was built using only 1,941 [NiFe]-and 1,220 [FeFe]-hydrogenase sequences (Data Set S1, Sheets 8 and 9), which contain conserved cysteine residues required to ligate H2-binding metal centers, to reduce the risk of mis-annotation resulting from the presence of various protein families containing hydrogenase homologs unable to metabolize H2 (4).

Supplementary Note 1: Motility of Hydrogenedentota
All the members of Hydrogenedentota contain pilus assembly-encoding genes, and 72% genomes contain flagellar assembly-encoding genes (Fig. 2).Except for genomes from Clade 6, chemotaxis is prevalent in Hydrogenedentota, as the possession of two component system proteins and methyl-accepting chemotaxis proteins were detected in at least 54 and 44 nonredundant strains, respectively, from the other six clades (Fig. 2).

Supplementary Note 2: Metabolic potential of Hydrogenedentota
The breakdown of glucose to pyruvate, can be achieved through EMP (Embden-Meyerhof-Parnas pathway, usually called glycolysis), and almost all Hydrogenedentota genomes in this study have a complete or near-complete EMP pathway.Specifically, genes in the EMP pathway (M00001 module) are annotated in 98 of the 100 non-redundant genomes, and at least 65 genomes are predicted to have a complete EMP pathway, showing glycolysis is a main process for breaking down glucose to pyruvate (Fig. 2; Data Set S1, Sheets 4 and 5).And those genomes lacking a complete EMP pathway may acquire pyruvate via the ED (Entner-Doudoroff) pathway as an alternative since a gene encoding the key enzyme of 2-dehydro-3-deoxyphosphogluconate aldolase (eda) was found in over 80% Hydrogenedentota genomes (Data Set S1, Sheet 4 and Fig. 2).And over 82% of the Hydrogenedentota genomes may have the pentose phosphate pathway (PPP), which provides an alternative to glycolysis for glucose oxidation and essential substrates (e.g., NADPH and pentose phosphate) for the synthesis of nucleotides, amino acids, cofactors and vitamins.The pyruvate oxidation (M00307 module) pathway, a connector that links glycolysis to TCA by providing the fuel of acetyl Coenzyme A (acetyl CoA), was observed in over 98% of the genomes (Fig. 2).Coupled with these metabolic features, the possession of TCA cycle in at least 89% of the genomes further indicates that Hydrogenedentota most likely live a heterotrophic lifestyle.29 non-redundant genomes in Clade 1 are predicted to have the acetate fermentation ability, which has seldom been detected in other clades.Ethanol fermentation appears to be more widely distributed in Hydrogenedentota than acetate production since 47 non-redundant genomes from all the seven clades might produce ethanol as byproduct in anaerobic conditions (Fig. 2).The continuation of glycolysis depends upon the availability of the oxidized form of the electron carrier, NAD + .Thus, NADH must be continuously oxidized back into NAD + to keep glycolysis going under anaerobic conditions.In addition to the above-mentioned possible fermentation processes, an alternate solution might be realized by L-lactate dehydrogenase (LDH, ldh; [EC:1.1.1.27]),catalyzing the reduction of pyruvate to lactate and regenerate NAD + .However, the LDH encoding gene has only been detected in 14% of the non-redundant genomes mainly from Clade 1 (Fig. 2).Alternatively, pyruvate formed via glycolysis could be fermented to lactate by one of the two lactate dehydrogenases (K00016 or K03778), producing NADH, and the lactate excreted from the cell.Notably, the balance of NAD + and NADH might also be temporarily adjusted by malate dehydrogenase (MDH, l-malate:NAD oxidoreductase, EC 1.1.1.37)for its ability to catalyze the NAD + /NADH-dependent interconversion between malate and oxaloacetate, and it may function in almost 80% of the genomes of Hydrogenedentota (Data Set S1, Sheets 4 and 5).Based on these data, we speculate that members of Hydrogenedentota are heterotroph and able to produce ATP via substrate level phosphorylation under anaerobic conditions.
In addition, the glyoxylate cycle, a special variant of the tricarboxylic cycle (TCA) that allows utilization of two carbons compounds in the absence of glucose, are complete in about 26% of the non-redundant genomes (Fig. 2).Instead of converting isocitrate to α-ketoglutarate in TCA cycle, the glyoxylate cycle enzyme isocitrate lyase (EC 4.1.3.1)catalyzes the conversion of isocitrate (C6) into glyoxylate (C2) and succinate (C4).Subsequently, malate synthase (EC 2.3.3.9)catalyzes the condensation of glyoxylate with acetyl-CoA (C2) to produce malate (C4) and a free CoA molecule.Malate can be further oxidized into oxaloacetate, an important precursor for gluconeogenic biosynthesis of glucose and other sugars.However, no genomes containing all of the three key enzymes of gluconeogenesis:phosphoenol pyruvate carboxykinase (pckA, K01596, K01610), fructose-1,6-bisphosphatase (fbp, K02446) and glucokinase (glk, K12407, K25026).And the proportion of genomes containing at least one of these three ratelimiting enzymes is about 82%, 1% and 96%, respectively.Obviously, the lack of fructose-1,6bisphosphatase in most members of Hydrogenedentota blocks the pathway of gluconeogenesis.
Moreover, two genes encoding starch synthase [EC:2.4.1.21]and 1,4-alpha-glucan branching enzyme, which are responsibly for starch or glycogen synthesis, were detected together only in 12 non-redundant genomes.And trehalose, known as a protective agent that help cells adapt to cold and high-pressure habitats, is only synthesized by fewer than 10% of the members of Hydrogenedentota.Therefore, the process of gluconeogenesis storing sufficient energy in glucose, followed by the synthesis of more complex compounds (e.g., glycogen, starch and trehalose), may not occur in Hydrogenedentota.
Group 3b [NiFe]-hydrogenases were annotated in 15 genomes, which belong to Clade I (8), Clade II (1), Clade IV (5) and Clade VII (1) (Data Set S1, Sheet 12 and Fig. 2).Flanking the genes encoding group 3b [NiFe]-hydrogenase in one Hydrogenedentota genome (GCA_016182645.1) is a gene encoding a pyruvate-ferredoxin/flavodoxin oxidoreductase (K03737, por/nifJ), which generates reduced ferredoxin during the conversion of pyruvate to acetyl-coA.The resulting reduced ferredoxin might be electron donor used by group 3b [NiFe]hydrogenase to produce H2.Two genes encoding a NAD(P)-binding subunit and hydrogenase small subunit, respectively, are adjacently located (Data Set S1, Sheet 12 and Fig. 2), suggesting that NAD(P) may be used by group 3b [NiFe]-hydrogenases as an electron acceptor.It appears that this type of hydrogenases may play an important role in conserving energy in the form of NAD(P)H for anabolism.

Fig. S7 .
Fig. S7.Schematic diagram indicating the extent of conservation in residues comprising