The plant pathogen Pectobacterium atrosepticum contains a functional formate hydrogenlyase-2 complex

Pectobacterium atrosepticum SCRI1043 is a phytopathogenic gram-negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly-understood membrane-bound enzyme termed formate hydrogenlyase-2 (FHL-2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL-2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL-2 complex was shown to comprise a rare example of an active [NiFe]-hydrogenase-4 (Hyd-4) isoenzyme, itself linked to an unusual selenium-free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane domain of FHL-2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL-2 function and structure in particular. Significance Statement Pectobacterium atrospecticum contains the genes for the formate hydrogenlyase-2 enzyme, considered the ancient progenitor of mitochondrial respiratory Complex I. In this study, the harnessing of P. atrosepticum as a new model system for understanding bacterial hydrogen metabolism has accelerated new knowledge in FHL-2 and its component parts. Importantly, those component parts include an unusual selenium-free formate dehydrogenase and a complicated [NiFe]-hydrogenase-4 with a large membrane domain. FHL-2 is established as the major source of molecular hydrogen produced under anaerobic conditions by P. atrospectium, however surprisingly some components of the membrane domain were not essential for this activity.


Introduction
Many members of the -Proteobacteria are facultative anaerobes with the ability to switch their metabolisms to exploit the prevailing environmental conditions. Aerobic or anaerobic respiration is generally preferred, depending on the availability of respiratory electron acceptors. In this phylum, and specifically under anaerobic conditions, the three-carbon product of glycolysis, pyruvate, is often further metabolised by the oxygen-sensitive pyruvate formatelyase enzyme to generate acetyl CoA and the one-carbon compound formic acid (Pinske & Sawers, 2016). Studies of the model bacterium Escherichia coli have established that endogenously-produced formate is initially excreted directly from the cell using a dedicated channel (Lu et al., 2011). Under respiratory conditions this formate would be used as an electron donor through the activity of periplasmic enzymes, but under fermentative conditions the formate accumulates in the extracellular milieu until its rising concentration begins to cause a drop in extracellular pH. This is thought to trigger formate re-uptake, which in turn induces synthesis of formate hydrogenlyase (FHL) activity in the cell (Rossmann et al., 1991, McDowall et al., 2014, Sargent, 2016. FHL activity then proceeds to detoxify the formic acid by disproportionation to carbon dioxide and molecular hydrogen (H2).
While FHL activity has been characterised in E. coli (Sargent, 2016), it is not confined to enteric bacteria and has been reported across the prokaryotic domains, including in hyperthermophilic archaea where it is not only involved in pH homeostasis but also in generating transmembrane ion gradients (Kim et al., 2010, Lim et al., 2014. The ion pumping activity stems from an evolutionary link between FHL and the respiratory NADH dehydrogenase Complex I (Bohm et al., 1990, Batista et al., 2013, Schut et al., 2016. Like Complex I, FHL comprises a cytoplasmic catalytic domain linked to an integral membrane domain. The catalytic domain contains a [NiFe]-hydrogenase of the 'Group 4' type, which is primarily dedicated to H2 production (Greening et al., 2015), and is linked by [Fe-S]-clustercontaining proteins to a molybdenum-dependent formate dehydrogenase (Maia et al., 2015). The FHL membrane domain is predicted to take two different forms allowing the enzyme to be further subclassified as either 'FHL-1' or 'FHL-2' (Sargent, 2016, Finney & Sargent, 2019. The FHL-1 is the predominant archetypal FHL activity of E. coli (McDowall et al., 2014) and comprises [NiFe]hydrogenase-3 (Hyd-3), formate dehydrogenase-H (FdhF), and a relatively small membrane domain compared to Complex I that contains only two proteins ( Figure 1A). Genes for the much less well understood FHL-2 enzyme are also found in E. coli (Andrews et al., 1997). This isoenzyme is predicted to comprise a [NiFe]-hydrogenase-4 (Hyd-4), an as-yet undefined formate dehydrogenase, and a much larger membrane domain than FHL-1, containing at least five individual integral membrane subunits and more closely resembling the Complex I structure ( Figure 1B). Understanding the structure, function and physiological role of E. coli Hyd-4 and FHL-2 has been hindered by poor native expression levels (Skibinski et al., 2002, Self et al., 2004; a missing important accessory gene from the hyf cluster (Sargent, 2016); and a lack of consensus on the appropriate experimental conditions to test (Bagramyan et al., 2001, Mnatsakanyan et al., 2004. In order to bring fresh impetus to understanding and harnessing the FHL-2 complex, it was considered that an appropriate alternative biological model system was required. Pectobacterium atrosepticum SCRI1043 is a phytopathogenic -Proteobacterium that can grow under anaerobic conditions. A transcriptomic study identified a chromosomal locus ( Figure 1C) that was transcribed under anaerobic conditions in this organism (Babujee et al., 2012, Bell et al., 2004. This locus neatly collects together almost all of the known genes for hydrogen metabolism (Figure 1C), including genes for a bidirectional Hyd-2-type [NiFe]-hydrogenase; genes for specialist metallo-cofactor biosynthesis; a putative formate-responsive transcriptional regulator; a predicted formate dehydrogenase gene; and an 11-cistron operon apparently encoding a Hyd-4 isoenzyme and its associated accessory proteins (Babujee et al., 2012).
In this work, a molecular genetic approach was taken to characterise the hydrogen metabolism locus of P. atrosepticum. A bank of un-marked and in-frame gene deletion mutants was constructed and used to demonstrate unequivocally that the unusual FHL-2 is functional in P. atrosepticum and responsible for the majority of H2 production under anaerobic conditions. The complex was shown to contain an active Hyd-4 and, unusually, a version of formate dehydrogenase that does not rely on selenocysteine. Surprisingly, it was shown that many of the genes encoding the large membrane domain of FHL-2 can be removed without adversely affecting H2 production activity. Overall, this work introduces P. atrosepticum as a tractable model system and presents important genetic, biochemical and physiological characterisation of FHL-2 and [NiFe]-hydrogenase-4.

P. atrosepticum produces molecular hydrogen under anaerobic conditions.
P. atrosepticum SCRI1043 (Bell et al., 2004) contains the genes for potentially H2-evolving enzymes (Babujee et al., 2012). Therefore, the initial goal of this study was to establish the growth conditions under which molecular hydrogen could be evolved. First, the SCRI1043 wild-type strain was grown under anaerobic fermentative conditions in a minimal medium supplemented with 0.8% (w/v) glucose. The culture headspace was sampled at periodic intervals and the amount of H2 present quantified by gas chromatography (GC). Under these conditions, H2 evolution activity was found to be temperature dependent, with H2 accumulation in the headspace observed to be maximal when the phytopathogen was incubated at 20 or 24 C (Figure 2A). Taking forward 24 °C as standard incubation temperature, H2 evolution was observed and found to level off after 40 hours incubation ( Figure 2B).
When anaerobic respiratory conditions were tested, comprising 0.5% (v/v) glycerol and 0.4% (w/v) nitrate, H2 production was found to cease with no H2 detectable after 48 hours growth ( Figure  2C). However, replacement of nitrate with 0.4% (w/v) fumarate as a terminal electron acceptor allowed the generation of low, but detectable, levels of H2 ( Figure 2C). Maximal H2 production is observable under fermentative conditions ( Figure 2C).

Hyd-4 is the predominant hydrogen producing enzyme in P. atrosepticum.
To determine the molecular basis of the observed H2 production activity, a molecular genetic approach was taken. Initially, the genes encoding the catalytic subunits of the [NiFe]-hydrogenases were targeted. First, a strain PH001 ( Table 1) was constructed carrying an unmarked in-frame deletion of the hyfG gene, predicted to encode the catalytic subunit of a Hyd-4 isoenzyme. When cultured fermentatively in the presence of glucose, the PH001 (hyfG) strain produced less than 5% of the total H2 accumulated by the wild-type control under the same conditions ( Figure 3A). Next, the gene encoding the catalytic subunit of Hyd-2 (hybC) was tested. Mutant strain PH002 (Table 1) was prepared carrying only a hybC allele and, in this case, H2 evolution under fermentative conditions was essentially indistinguishable from the wild-type strain ( Figure 3A). Finally, a hybC hyfG double mutant (PH003 , Table 1) was constructed and was found to be completely devoid of the ability to produce gaseous H2 ( Figure 3A).
P. atrospeticum can be stably transformed and plasmids encoding either hyfG or hybC were constructed. In the case of PH001 (hyfG) and PH003 (hybC hyfG), H2 evolution could be rescued in the mutant strains by supplying extra copies of hyfG on a plasmid ( Figure 3B).
Taken altogether, the data presented in Figures 2 and 3 demonstrate that Hyd-4 is responsible for the majority of physiological H2 production by P. atrospeticum, and that this activity is present under fermentative conditions at temperate growth temperatures ≤24 C.

P. atrosepticum contains an active FHL-2 with a selenium-free formate dehydrogenase.
Having established that Hyd-4 was active, the next task was to test the hypothesis that Hyd-4 could be part of a wider FHL-2 complex (Figure 1). First, formate-dependence on H2 production was tested by growing the wild-type parental strain, the PH001 (hyfG) strain, and the PH002 (hybC) strain anaerobically in the presence of increasing amounts of exogenous formate ( Figure 4A). A correlation was observed between the amount of H2 produced and the amount of formate added to the growth medium ( Figure 4A), and H2 production remained dependent upon the presence of an active Hyd-4 ( Figure 4A), providing initial evidence for a link between formate and H2 metabolism.
The P. atrosepticum SCRI1043 genome contains a gene encoding a putative formate dehydrogenase close to those for Hyd-4 ( Figure 1C). The gene product shares 85% overall sequence identity with E. coli FdhF but interestingly contains a cysteine residue at position 140 (Supp. Figure  S1), which is occupied by a critical selenocysteine in the E. coli version and other related enzymes (Axley et al., 1991). A mutant strain was therefore constructed (PH004 , Table 1) carrying an fdhF allele. The PH004 (fdhF) strain produced very low levels of H2 gas under fermentative conditions ( Figure 4B). Addition of a hybC allele to the fdhF strain to generate a double mutant (PH005 , Table  1) had no further effect on the amount of H2 that could be produced ( Figure 4B).
Elsewhere on the P. atrosepticum SCRI1043 genome two further homologs of FdhF are encoded. The ECA1507 gene encodes a protein with 65% overall sequence identity with FdhF, and the ECA1964 gene encodes a protein with 22% overall sequence identity with FdhF (Bell et al., 2004). Deletion of the genes encoding ECA1507 or ECA1964 alone ( Table 1) had no influence on the H2 production capability of the bacterium ( Figure 4C). Moreover, when the genes were supplied in multicopy on an expression vector, neither was able to rescue the phenotype of the fdhF mutant ( Figure 4D).
Taken altogether, these data establish that P. atrosepticum SCRI1043 has functional formate hydrogenlyase activity where molecular hydrogen production is clearly linked to both formate availability and the presence of a formate dehydrogenase gene. Importantly, the predominant electron donor for the reaction is an unusual version of formate dehydrogenase that does not require selenocysteine at its active site, and the enzyme responsible for proton reduction is a [NiFe]hydrogenase-4.

The role of the FHL-2 membrane domain in hydrogen production.
One clear defining structural difference between the FHL-1 type formate hydrogenlyase found in E. coli and the FHL-2 type of P. atroscepticum SCRI1043 is the number of genes encoding components of the membrane domains (Figure 1). An FHL-1 enzyme is predicted to contain two different membrane proteins, HycC (related to HyfB in FHL-2) and HycD (related to HyfC) ( Figure 1A). Alternatively, an FHL-2 enzyme is predicted to contain three additional membrane proteins, including HyfE (not present in FHL-1) and two further homologs of HycC/HyfB, namely HyfD and HyfF ( Figure  1B).
To explore the roles of the extra hyfDEF genes located within the FHL-2 locus, mutant strains were constructed (Table 1). First, versions of the hybC strain PH002, lacking either the genes encoding the entire FHL-2 membrane domain (PH007: hybC, hyfBCDEF - Table 1) or lacking the extra membrane components not found in FHL-1 (PH008: hybC, hyfDEF - Table 1) were constructed. In addition, the hybC strain PH002, producing Hyd-4 as the only active hydrogenase, was modified by addition of a 10-His sequence between codons 82 and 83 of the hyfG gene. This new epitope-tagged strain was called PH009 (hybC, hyfG His - Table 1). Finally, versions of PH009 lacking either the genes encoding the entire FHL-2 membrane domain (PH020: hybC, hyfG His , hyfBCDEF - Table 1) or lacking the extra membrane components not found in FHL-1 (PH021: hybC, hyfG His , hyfDEF - Table 1) were constructed.
Deletion of the genes encoding the entire membrane domain reduced the FHL-2-dependent H2 accumulation levels to around 5% of that observed in the parent strain ( Figure 5A). The addition of the 10-His tag to HyfG allowed the Hyd-4 catalytic subunit to be visualised in whole cell extracts by Western immunoblotting (Figure 5B). The polypeptide was clearly detectable when P. atroscepticum was cultured under anaerobic fermentative conditions ( Figure 5C). Interestingly, the amount of cellular HyfG His was seen to increase when the genes encoding the membrane domain were removed ( Figure 5). This is particularly pertinent for the PH020 strain (hybC, hyfBCDEF), which is essentially devoid of FHL-2 activity (Figure 5A), since it can be concluded that genetic removal of the complete membrane domain does not destabilise the Hyd-4 catalytic subunit, but instead leads to a physiologically inactive enzyme. It is also notable that in the absence of the genes encoding membrane proteins that the HyfG His protein migrates as two electrophoretic species during SDS-PAGE ( Figure 5B). This is a common observation for catalytic subunits of [NiFe]-hydrogenases as they are synthesised as precursors that undergo proteolytic processing at the C-terminus following cofactor insertion (Bock et al., 2006). In this case, the faster migrating species was calculated as 56.4 kDa, while the slower migrating species was estimated as 62.6 kDa by SDS-PAGE. The predicted molecular mass of HyfG His prior to proteolytic processing is 67,559 Da, and the predicted molecular weight of the 32-residue Cterminal tail that is removed is 3,821 Da.
Conversely, partial modification of the FHL-2 membrane domain to leave only those subunits present in FHL-1 (hybC, hyfDEF) had no negative effect on hydrogen production levels (Figure 5A), rather a slight increase was observed. This is consistent with a noticeable enhancement of HyfG His levels in the cells upon removal of the hyfDEF genes ( Figure 5B). The available evidence suggests that HyfD, HyfE and HyfF have no essential roles in the biosynthesis and hydrogen production activity of FHL-2.

A requirement for accessory genes in anaerobic hydrogen production.
FHL-2 is a multi-subunit metalloenzyme and assembly of such enzymes is often carefully coordinated by dedicated chaperones, sometimes called accessory proteins or 'maturases'. Maturation of molybdenum-dependent formate dehydrogenases has been reported to require the action of an FdhD protein, which is believed to supply an essential sulfur ligand to the active site metal (Arnoux et al., 2015). In P. atrosecpticum SCRI1043, fdhD (ECA0093) is not part of the FHL-2 locus but is located elsewhere on the chromosome next to a gene encoding superoxide dismutase (sodA or ECA0092) (Bell et al., 2004). Genetic modification of the PH002 strain, containing only Hyd-4 and FHL-2 activity, by the incorporation of a fdhD allele (PH013: hybC, fdhD - Table 1) led to a defect in physiological H2 production under fermentative conditions ( Figure 5D). This phenotype could be rescued by the provision of extra copies of fdhD in trans ( Figure 5D).
Maturation of [NiFe]-hydrogenases requires the activity of a network of proteins involved in metal homeostasis and cofactor maturation and insertion (Sargent, 2016). The P. atrosepticum FHL-2 locus ( Figure 1C) contains a hoxN gene (ECA1252) encoding a putative membrane-bound nickel ion transporter (Eitinger & Mandrand-Berthelot, 2000). Deletion of the hoxN gene in P. atrosepticum SCRI1043 (strain PH011, Table 1) reduced hydrogen evolution levels to around 50% of that observed for the parental strain ( Figure 5D). Note that there is no other homologue of hoxN encoded on the P. atrosepticum SCRI1043 genome, but there are several uncharacterised ABC transporters that could be related to the high-affinity nikA system (Wu et al., 1991), which could account for the continued availability of nickel for hydrogenase biosynthesis in this experiment.
Once inside the cell, nickel is processed into the Ni-Fe-CO-2CNcofactor through the action of several enzymes and chaperones. One key step in the biosynthesis of the cofactor is the first step in the generation of CNfrom carbamoyl phosphate by HypF (Sargent, 2016). Deletion of the hypF gene from P. atrosepticum (PH010 , Table 1), which is located in the hydrogen metabolism gene cluster under investigation here (Figure 1C), led to the complete abolishment of all detectable H2 evolution ( Figure 5D). It is the only mutant strain reported here that produces no detectable H2 during anaerobic fermentation ( Figure 5D). The mutant phenotype could be rescued by supply of hypF in trans, but note that full H2 evolution levels were not restored ( Figure 5D).
Finally, it was observed that a member of the HyfR family of transcriptional regulators was encoded in the hydrogen metabolism gene cluster ( Figure 1C). The HyfR protein is predicted to be related to FhlA, which is a formate-sensing transcriptional activator (Skibinski et al., 2002). A hyfR strain devoid of the HyfR protein has very low formate hydrogenlyase-2 activity ( Figure 5D).
Taken altogether, it can be concluded that all of the genes required for biosynthesis of FHL-2 are functional in P. atroseptocum SCRI1043, which is entirely consistent with the physiological data reported here.

Key differences between FHL-2 and FHL-1
Formate hydrogenlyases can be classified into two structural classes, FHL-1 and FHL-2 (Finney & Sargent, 2019). The most obvious structural difference between an FHL-1, such as the bestcharacterised E. coli enzyme (McDowall et al., 2014, Pinske & Sargent, 2016, and an FHL-2, such as the P. atrospeticum enzyme characterised here, is the predicted size and composition of the membrane domain ( Figure 1B). The genes encoding FHL-1 include only two membrane proteins, which are a single HycD/HyfC-type protein together with a single HycC/HyfB. This is sufficient to anchor the soluble domain close to the membrane and, in the case of Thermococcus onnurineus FHL-1 (Lim et al., 2014) and the related Ech hydrogenase from Methanosarcina mazei (Welte et al., 2010), will also allow generation of an ion gradient. Operons encoding FHL-2 complexes encode at least a three further integral membrane proteins. In P. atrospeticum these are HyfD and HyfF, which are related to HycC/HyfB, and the HyfE protein. Together, all five proteins are expected to form a stable complex in the membrane, as in the structure of the Group 4 [Ni-Fe]-hydrogenase from Pyrococcus furiosus (Yu et al., 2018). Indeed, this large membrane domain is thought to be the ancient progenitor to the ionpumping membrane domain of respiratory Complex I (Yu et al., 2018, Batista et al., 2013. Given the conservation of these genes, it was surprising that removal of all of the extra membrane proteins from FHL-2 had no discernible effect on the physiological activity of the P. atrosepticum system ( Figure 5A). In this experiment, the soluble domain was clearly fully assembled and active in formate-dependent hydrogen production ( Figure 5A), with Western immunoblotting even pointing towards stabilisation or up-regulation of the catalytic subunit in the absence of hyfDEF ( Figure 5B). This again highlights the principle of modularity in metalloenzyme evolution, since it is clear that the HyfDEF module may be added or removed depending on both selective pressure and also the, as yet undefined in terms of hydrogenases, biochemical function of these membrane proteins.
Interestingly, removal of the entire membrane domain of FHL-2 (HyfBCDEF) led to a complete loss in formate-dependent hydrogen production, even though the catalytic subunit was synthesised as normal (Figure 5A and B). A similar observation was made with E. coli FHL-1, where the catalytic domain was found to be inactive both in vivo and in vitro when produced in the absence of the membrane domain (Pinske & Sargent, 2016). These data suggest that interaction between the soluble domain and the membrane domain is a key step in the biosynthesis and activation of the enzyme.
The P. atrosepticum HyfG catalytic subunit from the Hydrogenase-4 component of FHL-2 shares 74% overall sequence identity (85% similarity) with the E. coli HycE protein from Hydrogenase-3/FHL-1. The sequence variation between these two Group 4A hydrogenases is therefore small with only subtle notable differences. For instance, each protein is known or predicted to undergo cleavage during cofactor insertion and maturation leaving a C-terminal arginine residue in the mature form of the proteins. The cleavage sites themselves are slightly differently conserved an FHL-1-type enzyme compared to an FHL-2, for example …R*MTVV… for HycE-like proteins compared to …R*VTLV… for HyfG. This may reflect the need for a different maturation protease for each type of hydrogenase, however this remains to be tested experimentally. In addition, it is notable that both E. coli and P. atrsoepticum hyfG initiate translation with a GUG start codon.
Phylogenetic analysis of the Group 4A [NiFe]-Hydrogenase subunits, including HycE and HyfG, shows that the enzymes associated with FHL-1 separate into a clearly distinct evolutionary clade from those associated with FHL-2, which form their own distinct clade (Supp. Figure S2). Examples of species that encode both FHL-1 and FHL-2 are rare (Supp. Figure S2).

A selenium-free formate dehydrogenase
An in-frame deletion in the fdhF gene located in the FHL-2 gene cluster (Figure 1) resulted in a ~500 times reduction in H2 production (Figure 4), indicating the majority of H2 production from P. atrosepticum is dependent on this formate dehydrogenase engaging with Hydrogenase-4 to form an FHL-2 complex. Arguably one of the best-studied FdhF enzymes is the E. coli version (Boyington et al., 1997, Gladyshev et al., 1994, Axley et al., 1991. The E. coli enzyme contains selenomethionine, which is incorporated co-translationally at a special UGA 'nonsense' codon within the coding sequence (Zinoni et al., 1987). Replacement of selenocysteine with cysteine in the E. coli enzyme resulted in a dramatically reduced turnover number (Axley et al., 1991). One surprising aspect of P. atrosepticum SCRI1043 is that it contains none of the biosynthetic machinery to synthesise selenomethionine (Babujee et al., 2012). The fdhF gene studied in this work contains a cysteine codon where selenocysteine would be encoded in the E. coli enzyme. Certainly the discovery of a highly active FHL-2 with no need for selenocysteine would benefit scientists interested in engineering this activity into other biological systems.
The FdhF formate dehydrogenase from P. atrosepticum shares 65% overall sequence identity (and 85% similarity) with the well-known E. coli enzyme (Supp. Figure S3). Interestingly, phylogenetic analysis suggests that >50% of species that contain FHL genes utilise a cysteine-dependent, rather than selenocysteine-dependent, formate dehydrogenase (Supp. Figure S3). P. atrosepticum ECA1507 and ECA1964 were identified here as two FdhF-like proteins that could potentially interact with Hydrogenase-4 to generate novel FHL-like complexes. Sequence analysis revealed ECA1507 and ECA1964 share 65% and 22% overall sequence identity with FdhF, respectively, and phylogenetic analysis determined that ECA1964 is more similar to E. coli YdeP than any other predicted molybdenum dependent oxidoreductases in P. atrosepticum (Supp. Figure S3). YdeP has a putative role in acid resistance in E. coli. It is clear that addition of extra copies of ECA1964 in the cell could not complement the fdhF, while production of ECA1507 was able to rescue a small amount of FHL-2 activity (Figure 4).

A role for formate metabolism in a plant pathogen
In the potato pathogen P. atrosepticum, FHL-2 activity was found to be expressed at lower growth temperatures (Figure 2). This suggests that FHL-2 may be produced in planta during the infection or colonisation event. Formate is produced endogenously by enteric bacteria under fermentative conditions, but plants and tubers have multiple metabolic pathways that generate and consume formate. Potato tubers produce an NAD + -dependent formate dehydrogenase (FDH), and the levels of this enzyme are boosted under stress conditions (Hourton-Cabassa et al., 1998). Indeed, proteomic experiments have identified FDH as a differentially-produced protein during wound healing in potato tuber slices, with order of magnitude level changes in protein during this process (Chaves et al., 2009). It could be hypothesised that the expression of FDH in the potato tuber could be coordinated with the initial secretion of formate by a fermenting pathogen. Potentially this would generate NADH from formate in stressed or damaged plant tissues. Recently, it was shown that FDH co-ordinates cell death and defence responses to phytopathogens in Capsicum annum (Bell pepper) (Choi et al., 2014). There is also indication that formate and other molecules that lead to the generation of formate, such as methanol and formaldehyde, induce the production of the NAD + -dependent FDH, perhaps suggesting there is a signalling response to these C1 compounds in plants (Hourton-Cabassa et al., 1998).

Concluding remarks
In this work, P. atrosepticum SCRI1043 has been established as a tractable new model organism for studying hydrogen metabolism in general and FHL function in particular. The organism is a rare example of a bacterium with an active Hydrogenase-4-containing FHL-2 complex, however, in the course of this work, Hydrogenase-4 activity was reported in Trabulsiella guamensis, another -Proteobacterium (Lindenstrauss & Pinske, 2019). In P. atrosepticum, the active Hydrogenase-4 enzyme operates in tandem with an unusual selenium-free formate dehydrogenase, which may be more amenable to biotechnological engineering than selenium-dependent isoenzymes. In evolutionary terms, the FHL-2 complex has been discussed as a key intermediate in the evolution of the NADH dehydrogenase (Complex I) from a structurally simpler membrane-bound hydrogenase (Schut et al., 2016). The most obvious difference in the predicted quaternary structures inferred from the genetics is the large membrane domain present in FHL-2 compared to FHL-1, and data presented here points to the extra membrane protein being not essential for formate-dependent hydrogen evolution in vivo. The role of the FHL membrane domain in generating a transmembrane ion gradient remains to be fully explored in enteric bacteria.

Bacterial strains
The parental P. atrosepticum strain used in this study was SCRI1043 (Bell et al., 2004). In-frame deletion and insertion mutants were constructed using pKNG101 suicide vector in E. coli strain CC118λpir (Kaniga et al., 1991, Coulthurst et al., 2006. Briefly, upstream and downstream regions (≥600 bp) of the target gene(s) was amplified and inserted into pKNG101 using a three fragment Gibson assembly reaction (HiFi Assembly, NEB). For the insertion of a deca-His encoding sequence into hyfG, primers were designed using the NEBuilder online tool to include the deca-His encoding sequence in the overlapping region of the two fragments containing the respective 3' and 5' sequences of hyfG. After successfully assembly and sequencing of pKNG101 plasmids, the CC118λpir strain with desired plasmid, a HH26 pNJ5000 helper strain, and the desired P. atrosepticum strain were grown in rich media, with antibiotics as necessary. Equal volumes of the stationary phase cultures were mixed and 30 µL was spotted on a non-selective rich media plate for 24 hours at 24˚C. P. atrosepticum cells with the pKNG101 plasmid were initially selected for on minimal media agar with streptomycin (100 µg/ml). After this, single colonies were re-streaked on the fresh minimal media agar with streptomycin. Co-integrants were then grown to stationary phase in rich medium with no selection before the culture was diluted 1/500 with phosphate buffer. Then 30 µL of this diluted culture was plated on minimal media agar with sucrose. These colonies were patch screened for sensitivity to streptomycin before PCR screens were performed to check for presence of the desired mutation(s).

Plasmids and complementation
All plasmids were cloned using Gibson assembly (HiFi Assembly, NEB) using DNA amplified from P. atrosepticum SCRI1043 genomic DNA (Table 1). Genes were cloned into pSU-PROM (Kan R ), which includes the constitutive tatA promoter from E. coli (Jack et al., 2004). Complementation plasmids were used to transform electrocompetent P. atrosepticum cells using a 2 mm electroporation cuvette (Molecular BioProduct) with application of an electrical pulse (2.5 kV voltage, 25 µF capacitance, 200 Ω resistance and 2mm cuvette length) via a Gene Pulsar Xcell electroporator (BioRad). Post recovery, cells were plated on LB Lennox agar plates with 50 µg/ml kanamycin.

Hydrogen quantification
Hydrogen was directly quantified from 5 mL cultures grown in sealed Hungate tubes (Pinske & Sargent, 2016). Gas-headspace samples were collected using a syringe with Luer lock valve (SGE), Samples were analysed using Gas Chromatography (Shimadzu GC-2014, capillary column, TCD detector). A hydrogen standard curve was used to quantify sample hydrogen content, this was then normalised to optical density (OD600) and culture volume (Pinske & Sargent, 2016).

Western immunoblotting
Proteins samples were first separated by SDS-PAGE using the method of Laemmli (Laemmli, 1970) before transfer to nitrocellulose (Dunn, 1986). Nitrocellulose membranes were challenged with an anti-His-HRP antibody (Alpha Diagnostics) and a GeneGnome instrument (SynGene) was used to visualise immunoreactive bands following addition of ECL reagent (Bio-Rad).

Structure modelling and phylogenetic analysis
Structural modelling of the formate hydrogenlyases complexes was performed using Phyre 2 predictions of respective subunits (Kelley & Sternberg, 2009). Using Chimera (Pettersen et al., 2004) the X-ray crystal structure of Thermus thermophilus Respiratory Complex I (4HEA) and the Cryo-EM structure of Membrane Bound Hydrogenase (6CFW), the individual FHL-2 subunits were manually assembled into a putative complex organisation for FHL-1 and FHL-2. Phylogenetic analysis of E. coli FdhF-like proteins from organisms possessing a Group 4A [NiFe]-hydrogenase utilised the HydDB database (Greening et al., 2015) to collect accession numbers for all [NiFe]-hydrogenase subunits. In each organism the FdhF orthologs were identified before MUSCLE multiple sequence alignment in Jalview (Waterhouse et al., 2009). Through percentage identity tree generation and manual inspection the closest FdhF-like proteins in each organism were identified. FigTree (http://tree.bio.ed.ac.uk/software/figtree) was used to visualise the finalised phylogenetic trees.  Anaerobic hydrogen production is optimal at lower temperatures. The P. atroscpeticum SCRI1043 parent strain was incubated in M9 medium supplemented with 0.8% (w/v) glucose for 168 hours at the temperatures indicated before gaseous H2 accumulation was quantified. (B) A time-course of H2 accumulation. P. atrosepticum SCRI1043 was incubated in M9 medium supplemented with 0.8% (w/v) glucose at 24 °C and gaseous H2 accumulation was measured every 24 hours. (C) P. atrosepticum SCRI1043 was incubated in M9 medium supplemented with either 0.5% (v/v) glycerol and 0.4% (w/v) nitrate ('Gly Nit'); 0.5% (v/v) glycerol and 0.4% (w/v) fumarate ('Gly Fum'); 0.5% (v/v) glycerol only (Gly); or 0.8% (w/v) glucose only ('Glc') at 24 °C for 48 hours. In all cases, the levels of molecular H2 in the culture headspace were quantified by GC and normalised to OD600 and culture volume. Error bars represent SD (n = 3).