Introduction

Legume root and stem nodules fix atmospheric dinitrogen (N₂) yielding anabolic-N, which augments growth and reproduction of host plants. In these nodules, the biochemical conversion of N₂ to ammonium is owed to endosymbiotic rhizobia (bacteroids) who carry the N₂ fixation genes encoding the dinitrogenase complex. Whether N₂ fixation occurs in legume nodules [1] or in pure cultures of diazotrophic (able to use N₂ as sole N-source) bacteria [2], hydrogen gas (H₂) is then co-produced. From subsequent mechanistic studies of dinitrogenase activity, H₂ co-production is both stoichiometric and requires 2 ATP per H₂ formed [3]–[4]. Yet in agronomic surveys, many legume nodules typically evolve H₂ at high levels, and such H₂ evolution rates correlate with N₂ fixation rates [5]. However, in certain symbiotic legume nodules, bacteroids avidly fix N₂ yet reproducibly evolve little or no H₂ [1]. As this endogenous H₂ production consumes metabolic energy, H₂ recycling, which recoups that energy, allows increased efficiency of N₂ fixation and, in principle, increased plant biomass yields [6]–[7]. This symbiotic nodule H₂ recycling capability correlates with specific bacteroid strains, although host legume cultivars also contribute to H₂ recycling and yield [8], [9]. Indeed, in biochemical assays, bacteroids isolated from H₂ recycling (non-evolving) nodules show high levels of respiratory uptake hydrogenase activity [10], [11].

H₂ recycling during N₂ fixation was first observed with the aerobe Azotobacter vinelandii, a diazotroph but not a legume symbiont. In pure culture, A. vinelandii induces a particulate (respiratory) hydrogenase activity which oxidizes H₂ at the expense of and tolerant of O₂ [2]. In following studies with legume nodule bacteroids, such uptake hydrogenase activity was also affirmed [10]. In the ensuing forty years, hydrogenases, extensively studied, have proven both biochemically diverse and broadly distributed across bacteria and archaea [12]. Among aerobes and microaerophiles able to use H₂ as energy source, uptake hydrogenase activities are typically classified as group I: heterodimeric, globular, hydrophilic proteins carrying a heteronuclear Ni,Fe-catalytic center; group I hydrogenases are generally O₂ tolerant [12]. In cellular terms, the group I, Ni,Fe uptake hydrogenases are tightly associated with respiratory membranes via integral diheme b-type cytochromes, required for physiological activity [13], [14] [Bernhard]. As the group I cell membrane-peripheral complexes face the periplasm, or cell exterior [15], they may be termed exo-hydrogenases.

Azorhizobium caulinodans, a microaerophilic α-proteobacterium originally isolated as nodule endosymbiont of the host legume Sesbania rostrata, is capable of N₂ fixation both in planta and in pure diazotrophic culture [16]. Recently, we discovered in A. caulinodans a second, novel respiratory hydrogenase encoded by the seven-gene hyq operon [17]. The inferred Hyq hydrogenase includes six different structural proteins, including a heterodimeric Ni,Fe-catalytic center hydrogenase conserved with group I enzymes. From bioinformatic analyses, the remaining four Hyq proteins are all membrane-integral [17]. Because all six Hyq hydrogenase subunits are NADH:quinone oxidoreductase (respiratory complex I) homologs [18], the Hyq complex is classified with the reversible group IV hydrogenases [12]. Given structural and functional homology to respiratory complex I [18], the Ni,Fe-catalytic center heterodimers of group IV complexes associated with respiratory membranes presumably face the cell-interior and thus may be termed endo-hydrogenases.

Results

In symbiotic legume nodules both exo- and endo-hydrogenases recycle H₂ produced by N₂ fixation

To assess physiological roles for both bacteroid Hup exo- and Hyq endo-hydrogenases in symbiotic legume nodules fixing N₂ and recycling H₂, A. caulinodans haploid derivatives carrying precise (to the nucleotide pair) in-frame deletions of hup and hyq structural genes encoding the conserved catalytic subunits of, respectively, exo- and endo-hydrogenases were constructed and verified by nucleotide sequencing of mutant loci [17]. Specifically, A. caulinodans exo-hydrogenase null mutants carried in-frame, precise, complete hupSL deletions; endo-hydrogenase null mutants comprised precise, complete hyqRBCEFGI operon deletions. As well, haploid recombinant double-mutants carrying both exo- and endo-hydrogenase null alleles were also constructed (Methods). Pure A. caulinodans cultures were used to inoculate both stems and roots of S. rostrata seedlings aseptically germinated and individually cultivated under N-limitation (Methods). In S. rostrata, symbiotic nodules are developmentally determinate, not meristematic. While both stem- and root-nodules subsequently developed on inoculated plants only, as they are invariably absent on uninoculated plants, stem nodules were chosen for further study. Three week-old and five week-old determinate stem nodules were excised from inoculated plants and individually tested for N₂ fixation activity, assaying acetylene-dependent ethylene production by gas chromatography with flame ionization detection (Methods). Excised stem nodules all showed similar (±15%) high levels of acetylene reduction activity when normalized per fresh nodule biomass (Table 1). Accordingly, all A. caulinodans strains tested were assigned both nodulation-competent (Nod⁺) and N₂ fixation-competent (Fix⁺) phenotypes.

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Table 1. N₂ fixation and H₂ recycling in S. rostrata–A. caulinodans stem nodules.

https://doi.org/10.1371/journal.pone.0012094.t001

Additional excised nodules from these S. rostrata plants were simultaneously tested under air for H₂ evolution activity using gas chromatography coupled to a reducing-compound photometric detector (Methods). In kinetic studies with excised nodules elicited by A. caulinodans strains 61305R (parental), 66081 (exo-hydrogenase mutant) and 66132 (endo-hydrogenase mutant), H₂ evolution was nonexistent (Figs. 1a,b). Whereas, nodules elicited by double (exo- and endo-hydrogenase) mutant 66204 evolved H₂ at very high rates (Fig. 1a) comparable to those measured for acetylene reduction (Table 1). Thus, H₂ evolution by double-mutant 66204-elicited nodules was quantitatively owed to N₂ fixation (dinitrogenase) activity. Results with five week-old determinate nodules from additional S. rostrata plants entirely corroborated results with three week-old nodules (data not presented). Pure bacterial cultures were reestablished from aseptically crushed nodules and strain identities verified by nucleotide sequencing of hup and hyq loci (Methods). In conclusion, A. caulinodans bacteroids in S. rostrata nodules employ both exo- and endo-hydrogenases to recycle endogenous H₂ produced by N₂ fixation. Moreover, H₂ recycling is quantitative, entirely accounting for N₂ fixation activities. Yet as measured by H₂ evolution rates, bacteroid exo- and endo-hydrogenase are interchangeable and individually are fully competent to handle endogenous H₂ recycling in symbiotic S. rostrata nodules.

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Figure 1. Hydrogen (H₂) evolution by excised S. rostrata stem nodules elicited by indicated A. caulinodans strains as endosymbiont.

(A) 70 µmol scale; (B) expanded ordinate, 1 µmol scale; evolved H₂ measured as µmol g⁻¹ (fresh biomass).

https://doi.org/10.1371/journal.pone.0012094.g001

Discussion

Among legume-Rhizobium symbioses, H₂ recycling was first reported in Pisum sativum (garden pea) nodules elicited by specific Rhizobium leguminosarum bv. viciae strains [2]. Genetic studies were subsequently undertaken with [Brady]Rhizobium japonicum strains able to recycle H₂ in Glycine max (soy) nodules [21], [22]. Many subsequent studies with H₂ recycling legume nodules all presumed uptake hydrogenase activity a single entity. These studies include combined genetic and physiological analyses which might have challenged this assertion. For the case of A. caulinodans, single mutants W58, U58 as well as hupSL impaired strain ORS571.2 all were reported to suffer substantial to complete loss of uptake hydrogenase activity [23], [24], [25]. Such conclusions are incompatible with the present finding: A. caulinodans employs two structurally and functionally distinct, genetically-independent, respiratory hydrogenases to recycle endogenous H₂ produced by N₂ fixation.

Whereas, early on the investigative timeline, B. japonicum single mutants unable to be cultured autotrophically on exogenous H₂ yet still able to recycle endogenous H₂ in soy nodules were identified [26]. As these strains showed induction of uptake hydrogenase activity in cultures shifted to O₂ limitation (≤11 µM DOT), they were perhaps understandably considered transcriptional control mutants hypersensitive to O₂. With the benefit of hindsight, this phenotype is precisely that expected of true loss-of-function point mutants affecting hup operon structural genes encoding Hup exo-hydrogenase activity, were the observed limiting-DOT uptake hydrogenase activity in fact owed to Hyq endo-hydrogenase. In A. caulinodans, hyq operon expression requires NifA as transactivator [17], and the pnifA⁺ promoter is in turn strongly transactivated by Fnr, which process requires physiological O₂ limitation in diazotrophic culture [27]. In principle, both exo- and endo-hydrogenase gene-sets, despite being encoded at disparate loci in all organisms identified, might nevertheless share a common genetic predisposition, allowing strategic single mutations to convey dual loss-of-function. However, as strains carrying complete hyq operon deletions still possess wild-type Hup exo-hydrogenase activity, and vice versa, evidence for any genetic, post-transcriptional interaction or interdependence between the two gene-sets is entirely lacking.

As shown previously, in pure diazotrophic (N₂ as sole N-source) cultures, A. caulinodans exo-hydrogenase knockout mutants grow as wild-type, whereas endo-hydrogenase knockout mutants exhibit slow growth [17]. Are exo- and endo-hydrogenase H₂ recycling efficiencies in pure culture and in legume nodules then demonstrably different? Or, do diazotrophic phenotypes imply additional endo-hydrogenase function(s), e.g. chemiosmotic work associated with membrane ion translocation [28] not undertaken by exo-hydrogenase? Obviously, effective exo- and endo-hydrogenase cellular concentrations and/or distributions might be dissimilar in legume nodules and in pure diazotrophic cultures, even though both hup⁺/hyp⁺ (exo-hydrogenase) and hyq⁺ (endo-hydrogenase) gene-sets are then strongly transcribed [17], [25], [29]. Because hup mutants suffer loss of chemoautotrophy with exogenous H₂ as energy substrate [17], [26], exo-hydrogenase kinetic behavior may constitute simple diffusion control. Because hyq mutants do not adversely impact chemoautotrophy with exogenous H₂, endo-hydrogenase kinetic behavior might not constitute simple diffusion control. A critical test of this hypothesis is still lacking. Diazotrophic liquid batch cultures typically employ constant sparging with relatively high gas-phase exhaust rates (0.5 min⁻¹), complicating kinetic behavior and analysis of cellular processes with gaseous substrate(s) subject to simple diffusion control. In such pure liquid diazotrophic batch cultures bacterial densities typically reach 10⁸ cc⁻¹, whereas in determinate S. rostrata nodules, bacteroid densities approach 10¹¹ cc⁻¹, the latter obviously more conducive to endogenous H₂ recycling under simple diffusion control. Notwithstanding, given their apparent co-selection in N₂ fixing micoaerophilic α-proteobacteria capable of H₂ recycling, exo- and endo-hydrogenases likely possess additional, distinctive functionalities yet to be elucidated.

Methods

Acknowledgments

The authors thank Henk Vreman (Stanford University), Steve Hartman and Alex Lowe (Peak Laboratories LLC) for technical assistance with gas chromatographic H₂ analyses.