# Frankia [NiFe] uptake hydrogenases and genome reduction: different lineages of loss

Katharina Pawlowski, Daniel Wibberg, Sara Mehrabi, Nadia Binte Obaid, András Patyi, Fede Berckx, Han Nguyen, Michelle Hagen, Daniel Lundin, Andreas Brachmann, et al.

► To cite this version:

Katharina Pawlowski, Daniel Wibberg, Sara Mehrabi, Nadia Binte Obaid, András Patyi, et al. Frankia [NiFe] uptake hydrogenases and genome reduction: different lineages of loss. FEMS Microbiology Ecology, 2024, 100 (12), pp. fiae147. 10.1093/femsec/fiae147. hal-04842363

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# Frankia [NiFe] uptake hydrogenases and genome reduction: different lineages of loss

Katharina Pawlowski<sup>1,*,</sup> Daniel Wibberg<sup>2</sup>, Sara Mehrabi<sup>1,3</sup>, Nadia Binte Obaid<sup>1</sup>, András Patyi<sup>1</sup>, Fede Berckx<sup>1,4</sup>, Han Nguyen<sup>5</sup>, Michelle Hagen<sup>1</sup>, Daniel Lundin<sup>6</sup>, Andreas Brachmann<sup>7</sup>, Jochen Blom<sup>1,8</sup>, Aude Herrera-Belaroussi<sup>9</sup>, Danis Abrouk<sup>1,9</sup>, Petar Pujic<sup>9</sup>, Ann-Sofi Hahlin<sup>10</sup>, Jörn Kalinowski<sup>1,2</sup>, Philippe Normand<sup>9</sup>, Anita Sellstedt<sup>5,*</sup>

## Abstract

Uptake hydrogenase (Hup) recycles H₂ formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup⁻, while only one Hup⁻ Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a, and 3b according to Søndergaard, Pedersen, and Greening (HydDB: a web tool for hydrogenase classification and analysis. Sci Rep 2016;6:34212. https://doi.org/10.1038/srep34212.); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h, and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H₂ formed by nitrogenase while group 1h and group 3b are not.

**Keywords:** root nodules; actinorhiza; Frankia; nitrogen fixation; uptake hydrogenase

## Introduction

The reduction of atmospheric dinitrogen to ammonia during biological nitrogen fixation (BNF) via the activity of the metalloenzyme complex nitrogenase represents one of the most important evolutionary innovations; its evolution preceded that of oxygenic photosynthesis. Phylogenetic analyses indicate that BNF most probably evolved in an anaerobic thermophilic ancestor of hydrogenotrophic methanogenic archaea and later diversified via lateral gene transfer into anaerobic, and eventually also into aerobic bacteria (Mus et al. 2019). Consistent with its origin in an anoxic environment, nitrogenase is extremely oxygen sensitive. The nitrogenase reaction involves the reductive elimination of two [Fe–H–Fe] bridging hydrides to form H₂; i.e. hydrogen evolution is part of the reduction of dinitrogen to ammonia (Khadka et al. 2017). Since hydrogen evolution represents a loss of energy, it is not surprising that many nitrogen-fixing organisms contain an uptake hydrogenase, which recycles the hydrogen produced in N₂ fixation (Schubert and Evans 1976). Hydrogen metabolism is

of considerable ecological importance given the prevalence of hydrogen in the environment (Piché-Choquette and Constant 2019); the activities of H₂-producing and -consuming microbes shape the global H₂ cycle that is linked to the global cycling of other elements. Accordingly, different types of hydrogenase enzymes exist that fulfil different functions (Vignais et al. 2001, Vignais and Billaud 2007). Søndergaard et al. (2016) used the amino acid sequences to infer an expanded classification scheme predicting biological function.

In any case, the question arises why some nitrogen fixers have lost their uptake hydrogenase. This question is of particular importance for symbiotic nitrogen fixers. Two groups of nitrogen-fixing soil bacteria can enter intracellular symbioses with plants where they fix nitrogen while hosted within plant cells in special organs, the root nodules: rhizobia (alpha- or beta-*Pseudomonadota*) with legumes and one nonlegume genus, *Parasponia*, and *Actinomycetota* of the genus Frankia with a group of dicotyledonous plants from eight different families belonging to three orders,

<footnote n="1"><sup>1</sup>Department of Ecology, Environment and Plant Sciences, Stockholm University, 10691 Stockholm, Sweden</footnote>

<footnote n="2"><sup>2</sup>Center for Biotechnology (CeBiTec), Bielefeld University, 33594 Bielefeld, Germany</footnote>

<footnote n="3"><sup>3</sup>Department of Ecology and Genetics, Evolutionary Biology, Uppsala University, 75236 Uppsala, Sweden</footnote>

<footnote n="4"><sup>4</sup>Department of Crop Production Ecology, Swedish University of Agricultural Sciences, 75651 Uppsala, Sweden</footnote>

<footnote n="5"><sup>5</sup>Department of Plant Physiology, UPSC, Umeå University, 90187 Umeå, Sweden</footnote>

<footnote n="6"><sup>6</sup>Department of Biology and Environmental Science, Linnaeus University, 39182 Kalmar, Sweden</footnote>

<footnote n="7"><sup>7</sup>Biocenter of the LMU Munich, Genetics Section, Grosshadner Str. 2-4, D-82152 Planegg-Martinsried, Germany</footnote>

<footnote n="8"><sup>8</sup>Bioinformatics and Systems Biology, Justus-Liebig Universität Giessen, 35392 Giessen, Germany</footnote>

<footnote n="9"><sup>9</sup>Université de Lyon, Université Lyon 1, CNRS, UMR5557, Ecologie Microbienne, INRA, UMR 1418, 43 bd du 11 novembre 1918, 69622 Villeurbanne, France</footnote>

<footnote n="10"><sup>10</sup>Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden</footnote>

<footnote>*Corresponding authors. Katharina Pawlowski, Department of Ecology, Environment and Plant Sciences, Stockholm University, 10691 Stockholm, Sweden. E-mail: katharina.pawlowski@su.se; Anita Sellstedt, Department of Plant Physiology, UPSC, Umeå University, 90187 Umeå, Sweden. E-mail: anita.sellstedt@umu.se</footnote>

<footnote>Editor: [Paolina Garbeva]</footnote>
Fagales, Rosales, and Cucurbitales, collectively called actinorhizal plants (Pawlowski and Bisseling 1996). Many symbiotic nitrogen fixers examined can form an uptake hydrogenase (i.e. they are Hup⁺), and it could be shown that the recycling of H₂ significantly increased the yield in agricultural legumes (Dixon 1972, Schubert and Evans 1976, Albrecht et al. 1979). Nevertheless, among surveyed legume symbioses Hup⁺ symbioses are in the minority (Annan et al. 2012).

Actinorhizal symbioses are a more complex field. The microsymbionts, *Frankia* strains, are very diverse, with genome sizes ranging from 4.3 to 10.5 MB, and can be grouped into four phylogenetic clades called clusters, three of which are symbiotic and represent host specificity groups; the fourth cluster contains nonsymbiotic strains that cannot fix nitrogen (Normand et al. 1996, Nguyen and Pawlowski 2017). Cluster-1 strains nodulate actinorhizal Fagales (cluster-1c strains nodulate Casuarinaceae, while other cluster-1 strains nodulate actinorhizal Betulaceae and Myricaceae) except for members of two outlier genera. Cluster-3 strains nodulate most actinorhizal members within the Rosales families Elaeagnaceae and Rhamnaceae except for one outlier genus, and can nodulate the two outlier genera of the Fagales, *Gymnostoma* and *Morella*. Strains of cluster-2, the earliest divergent symbiotic cluster of *Frankia* (Sen et al. 2014, Gtari et al. 2015, Persson et al. 2015) nodulate all actinorhizal Cucurbitales, actinorhizal Rosaceae, and members of the outlier genus of the Rhamnaceae, *Ceanothus*. Cluster-2 strains can be separated in two divergent lineages, the continental lineage which encompasses strains from mainland Eurasia, Japan, and North America, and the island lineage encompassing strains from the Southern hemisphere, the Philippines, and Taiwan (Berckx et al. 2022). A phylogenetic tree including all *Frankia* species published up to the end of 2022 is shown in Fig. S1.

An early study examined different actinorhizal plants—*Alnus rubra* and *Myrica californica* (nodulated by cluster-a strains), *Purshia tridentata* (nodulated by cluster-2), and *Elaeagnus angustifolia* (nodulated by cluster-3)—and found a RE of nitrogenase between 0.97 and 0.99, indicating the activity of uptake hydrogenase (Schubert and Evans 1976). All actinorhizal nodules induced by cluster-1 strains analysed since then displayed Hup activity (Tjepkema et al. 1986, Winship et al. 1987, Pesce et al. 2017), with one exception: nodules induced on roots of *Alnus incana* (Betulaceae and Fagales) by the so-called 'local source' from Umeå in northern Sweden, an inoculum that could not be cultured (Sellstedt et al. 1986, Huss-Danell 1991). The 'local source' has another unusual feature in that here, *Frankia* can form sporangia within host plant cells, not just during saprotrophic growth. Meanwhile, more *Frankia* strains that sporulate in nodules have been found, particularly among strains that nodulate alder (Cotin-Galvan et al. 2016, Schwob et al. 2018) but also among some strains that nodulate *Myrica* sp. or *Comptonia* sp. (Kashanski and Schwintzer 1987), i.e. other host plants of cluster-1 *Frankia*. So far, no *Frankia* strains that sporulate in nodules could be cultured; sequencing showed strong genome reduction, which would be consistent with the loss of saprotrophic potential (4.3–5 MB genome size compared to 7.5–8 MB genome size in other cluster-1a *Frankia*; Herrera-Belaroussi et al. 2020, Pozzi et al. 2020).

*Frankia* strains contain [NiFe] hydrogenases (Mattsson et al. 2001, Richau et al. 2013), heterodimeric metalloenzymes consisting of a large subunit of ca. 60 kDa (HupL) and a small subunit of ca. 30 kDa (HupS). Six maturation proteins (HypA–HypF) are required for the biosynthesis of the NiFe(CN)₂CO catalytic cluster and its insertion into HupL (Peters et al. 2015). HypE and HypF synthesize the thiocyanate, while the delivery of cyanide to

iron occurs at the HypC–HypD complex. HypA and HypB are involved in acquisition and insertion of Ni²⁺ ions into HupL after the Fe(CN)₂CO moiety has been added. In the final step, a peptide on the C-terminus of HupL is processed by a maturation endopeptidase (HupD), causing a conformational change which enables enzyme activity. After *Frankia* genome sequences began to become available in 2007 (Normand et al. 2007), it turned out that genomes of *Frankia* strains from clusters-1 and 3 contained two sets of uptake hydrogenase genes with associated genes of maturation proteins, organized in syntons-1 and -2 (Leul et al. 2007, 2009, Richau et al. 2013). Synton-1 was expressed at a higher level in culture, while synton-2 was expressed at higher levels in nodules (Leul et al. 2007, Richau et al. 2013). Proteins encoded by genes from synton-1 and synton-2, respectively, showed different phylogenies, which was interpreted to suggest HGT from other soil actinobacteria to *Frankia* (Leul et al. 2009).

Given that many more genomes from *Frankia* and other actinobacterial strains have been sequenced since 2009, our views on the uptake hydrogenase gene situation in *Frankia* strains should be reassessed. Moreover, the question has to be answered how hydrogenase activity was lost in one or more cluster-1a strain(s) that sporulate(s) in nodules; which genes/syntons were lost here? It also has to be considered that so far, only one cluster-3 genome and no cluster-2 genomes were analysed with regard to uptake hydrogenase syntons (Leul et al. 2007, 2009), and only nodules from one host plant of each cluster have been examined for Hup activity (Schubert and Evans 1976). What is the uptake hydrogenase gene/synton situation in *Frankia* strains from cluster-2 which, like the cluster-1a strains sporulating in nodules, show genome reduction (5–6 MB; Nguyen and Pawlowski 2017)?

Therefore, we analysed the Hup synton situation and phylogeny of the large and small subunits and several accessory proteins based on representatives of all *Frankia* species described before 2023. We also examined hydrogenase activity and transcription in nodules and, if available, cultures for a strain from *Frankia* cluster-1 (*Frankia alni* ACN14aT; Normand et al. 2007), and for strains from cluster-1 that sporulate in nodules: the type strains *Candidatus Frankia alpina* AiOr and *Candidatus Frankia nodulisporulans* AgTrsT and two representative of the 'local source', the original inoculum and Ca. *F. nodulisporulans* UmASH1 (Sellstedt et al. 1986, Pozzi et al. 2020, Herrera-Belaroussi et al. 2020). Additionally, we examined a strain from the continental lineage of cluster-2 (*Candidatus Frankia californiensis* Dg2; Normand et al. 2017) and a strain from cluster-3, Hr75.2.1 that was isolated by André Moiroud in 1991 from a nodule from *Hippophae rhamnoides* growing in the Isère department in the village of Evrieu, France (Bautista-Guerrero et al. 2011), the genome of which was sequenced for this study. Sequence analysis [average nucleotide identity (ANI), core genome phylogeny] showed that Hr75.2.1 is a representative of *Frankia soli* (Gtari et al. 2020; Fig. S2). A summary of all hydrogenase syntons/types and the corresponding genes is presented in Table 1.

## Experimental procedures

### Plant and bacterial material and growth conditions

Seeds of *Alnus glutinosa* (L.) Gaertn were obtained from Svenska Skogplantor (Bålsta, Sweden), while seeds of *A. incana* (L.) Moench were obtained from Levinsen (Gørløse, Denmark). Seeds were germinated on coarse-grained sand (1.2–2 mm diameter; Rådasand, Lidköping, Sweden) in a greenhouse at a light/dark rhythm of 16 h
<table><caption>**Table 1.** All hydrogenase types/syntons analysed in this study. When one gene is missing, it is mentioned by name; when more than one gene is missing, the synton is called 'incomplete'. Not shown, no sequence from this synton is discussed in this manuscript.</caption><thead><tr><th rowspan="2">Cluster</th><th rowspan="2">Species, strain</th><th colspan="4">NiFe hydrogenase synton/type</th></tr><tr><th>hupL1 (group 1 h)<br>hypABCDEFhupDSL<sup>a</sup></th><th>hupL2 (group 2a)<br>hypABCDEFGhupDSL<sup>b</sup></th><th>hupL3 (group1f)<br>hypABCDEFhupCDSL<sup>a,c</sup></th><th>hupL4 (group 3b)<br>hyhGBhupSLD</th></tr></thead><tbody><tr><td rowspan="10">Cluster-1</td><td>Frankia alni ACN14a</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>Frankia canadensis ARgP5</td><td>Yes</td><td>Yes, hypF2 missing</td><td>Only hypF3</td><td>No</td></tr><tr><td>Frankia torreyi Cp11S</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td></tr><tr><td>Frankia torreyi ACN1ag</td><td>Yes (not shown)</td><td>Yes (not shown)</td><td>No</td><td>Yes</td></tr><tr><td>Frankia nodulisporulans AgTrS</td><td>No</td><td>hypF2 incomplete</td><td>No</td><td>No</td></tr><tr><td>Frankia nodulisporulans UmASH1</td><td>No</td><td>hypF2 incomplete</td><td>No</td><td>No</td></tr><tr><td>Frankia nodulisporulans LS</td><td>No</td><td>hypF2 incomplete</td><td>No</td><td>No</td></tr><tr><td>Frankia alpina AiOr</td><td>Incomplete</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>Frankia casuarinae CcI3</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td></tr><tr><td>Frankia casuarinae KB5</td><td>Yes (not shown)</td><td>Yes (not shown)</td><td>No</td><td>Yes</td></tr><tr><td rowspan="5">Cluster-2</td><td>Frankia daviscae Dg1</td><td>No</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>Frankia coriariae BMG5.1</td><td>No</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>Frankia californiensis Dg2</td><td>No</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>Frankia meridionalis Cppng1</td><td>No</td><td>No</td><td>Yes</td><td>No</td></tr><tr><td>Frankia sp. CiP3</td><td>No</td><td>No</td><td>Yes</td><td>No</td></tr><tr><td rowspan="6">Cluster-3</td><td>Frankia elaeagni BMG5.12</td><td>Incomplete<sup>d</sup></td><td>hypF2-hypD2-hypE2</td><td>Yes</td><td>Yes</td></tr><tr><td>Frankia discariae BCU110501</td><td>Yes, hypF1 missing</td><td>Only hypF2</td><td>Yes</td><td>No</td></tr><tr><td>Frankia soli NRRL B 16 219</td><td>Yes, hypF1 missing</td><td>Only hypF2</td><td>Yes</td><td>Yes</td></tr><tr><td>Frankia soli Hr75.2.1</td><td>Yes, hypF1 missing</td><td>Only hypF2</td><td>Yes (not shown)</td><td>Yes</td></tr><tr><td>Frankia soli EAN1pec</td><td>Yes, hypF1 missing</td><td>Only hypF2</td><td>Yes</td><td>No</td></tr><tr><td>Frankia irregularis DSM45899</td><td>Yes, hypF1 missing</td><td>Only hypF2</td><td>Yes</td><td>No</td></tr><tr><td rowspan="5">Cluster-4</td><td>Frankia saprophytica CN3</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td></tr><tr><td>Frankia asymbiotica NRRL B 16 386</td><td>Yes</td><td>No</td><td>No</td><td>No</td></tr><tr><td>Frankia inefficax EuI1c</td><td>Yes, hypF1 missing</td><td>No</td><td>Only hypF3</td><td>No</td></tr><tr><td>Frankia sp. EUN1h</td><td>Yes (not shown)</td><td>No</td><td>No</td><td>Yes</td></tr></tbody></table>

light/8 h dark, with 23°C during the light and 20°C during the dark phase. After germination, seedlings were transferred to larger pots with sand, and after 4 weeks, plantlets were transferred to experimental pots (13 cm diameter) containing a 1:1 mixture of autoclaved sand (1.2–2 mm quartz, Rådasand) and autoclaved germination soil (Såjord, Hasselfors, Sweden), and inoculum was added. Inocula were for prepared for eight plants each (four A. glutinosa and four A. incana): a culture of Frankia alni ACN14a grown in BAP medium without N source (Benoist et al. 1992), washed twice with sterile milliQ H₂O, and resuspended in sterile milliQ H₂O; nodules containing Candidatus Frankia nodulisporulans UmASH1 (Herrera-Belaroussi et al. 2020), nodules containing Ca. F. nodulisporulans AgTrS (Herrera-Belaroussi et al. 2020), nodules containing the original 'local source' (Sellstedt 1986) or nodules containing Candidatus F. alpina AiOr (Pozzi et al. 2020) crushed in 10 ml sterile milliQ H₂O per sample, respectively. The nodules representing the inocula for UmASH1, AgTrS, 'local source' and AiOr had been formed on greenhouse plants grown on autoclaved sand inoculated with surface-sterilized crushed nodules, and therefore were used without a new round of surface sterilization. Plants were watered with deionized H₂O on alternate days and with one-fourth strength Hoagland's medium (Hoagland and Arnon 1938) every 2 weeks. Before inoculation, one-fourth strength Hoagland's with 10 mM KNO₃ was used; after inoculation, one-fourth strength Hoagland's with 1 mM KNO₃ was used. Nodules were harvested for RNA isolation 6 months after inoculation; whole root systems were used for acetylene reduction assays

(ARA) and H₂ evolution measurements ca. 23 weeks after infection, in case of 'local source' inoculum ca. 9 months after infection.

Datisca glomerata (C. Presl) Baill. seeds originating from plants growing at Gates Canyon in Vacaville, CA, USA, had been propagated in the greenhouse since 1997. Seeds were germinated on sand and transferred to a 1/1 (v/v) mixture of sand and germination soil (Såjord) after 3–6 weeks. Plants were nodulated with the Dg2 inoculum (Nguyen et al. 2016) 3 months after germination. Before inoculation, plants were watered with deionized H₂O on alternate days and with one-fourth Hoagland's medium with 10 mM KNO₃, after inoculation with the same medium but with 1 mM KNO₃ every 2 weeks. Temperature and light conditions were as described for alders. Plants were used for uptake hydrogenase assays 13 months after inoculation in August 2021. Nodules for RNA isolation were collected from D. glomerata plants inoculated with Dg2 6 months after inoculation; ARA and hydrogen evolution were measured ca. 9 months after inoculation.

Seeds of H. rhamnoides L. were obtained from Saflax (www.saflax.de), surface-sterilized with 3% sodium hypochlorite and 0.01% Tween 20 for 20 min, washed extensively with autoclaved milliQ H₂O, and kept in autoclaved milliQ H₂O at 4°C for 3 days. Then, they were sown onto moistened filter paper in Petri dishes covered with aluminium foil. Three weeks later, seedlings were transferred to small pots containing a mix of sterilized gravel and sand; 50% of the plants were inoculated with Frankia sp. Hr75.2.1 grown in PUM medium without nitrogen (Mattsson and Sellstedt

<footnote><sup>a</sup>Two hypC1 and hypC3 copies in cluster-3.</footnote>

<footnote><sup>b</sup>Two hypC2 copies in cluster-2.</footnote>

<footnote><sup>c</sup>hypB3 and hypF3 missing in cluster-3.</footnote>

<footnote><sup>d</sup>hypD1E1F1, hypA1B1 missing.</footnote>
2000) and washed twice with sterile milliQ H₂O, while 50% were
were left uninoculated. The plants were grown at 18 h light/6 h
dark with 22°C during the light and 18°C during the dark phase,
and 60% humidity. They were watered with Evan's medium with
5 mM NH₄NO₃ once per week for 4 weeks, and afterwards with
Evan's medium without nitrogen once per week (Evans et al. 1972),
otherwise with tap water. Nodules were harvested for RNA iso-
lation ca. 6 months after inoculation; ARA and hydrogen evolu-
tion measurements were performed ca. 1 year after inoculation.
In each case, temperatures were maintained through the entire
growth period and during ARA and hydrogen evolution measure-
ments as temperature affects the RE of nitrogenase (Walsh and
Layzell 1986).

RNA isolation and reverse
transcription-quantitative polymerase chain
reaction

RNA was isolated from cultures and nodules as described by Nguyen et al. (2019) and Berckx et al. (2020). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed as described by Zdyb et al. (2018). *InfC* (encoding the translation initiation factor IF-3) was used as reference gene (Alloisio et al. 2010). Primers used in this study are listed in Table S1. Statistical evaluation was performed in RStudio (RStudio Team 2022) using one-way ANOVA with *post hoc* Tukey HSD test.

Analysis of hypF2 in Ca. Frankia nodulisporulans AgTrS, UmASH1, and ‘local source’

In order to find out whether the appearance of a broken hypF2
gene (one operon ending with a 3'-truncated version, another
operon beginning with a 5'-truncated version) was an assembly
error, DNA was isolated from nodules induced by AgTrS, UmASH1,
and the 'local source' on A. glutinosa as described by Berckx et al.
(2022). Polymerase Chain Reaction (PCR) was performed for hypF2
using DreamTaq DNA Polymerase (Thermo Fisher) and three dif-
ferent primer pairs from the 5' and 3' part (Table S1) with the pro-
gram 95°C 5 min; 35 cycles of 95°C 30 s, 63°C or 64°C 30 s, 72°C
1 min; and 72°C 10 min. To further check the completeness of the
hypF2 gene, the raw assembly data in *.ace format were imported
into Consed (Gordon et al. 1998). The region encoding hypF2 was
identified and manually inspected.

Hr75.2.1 and ‘local source’ genome sequencing
and assembly

DNA was isolated from a Hr75.2.1 culture grown in basic alka-
line propionate (BAP) liquid medium without nitrogen (Benoist
et al. 1992) according to Nguyen et al. (2019). DNA was used for
next generation sequencing (NGS) using Illumina technology. Raw
sequencing data of 1.11 GB in size was obtained and used for
genome assembly. The assembled genome was then annotated
on the MaGe genomic Platform (https://image.genoscope.cns.fr/
microscope/home/index.php). Genome data were deposited at
GenBank, BioProject PRJEB60167. The genome of Hr75.2.1 is
8.269 105 MB in size with 71.59% GC.

For sequencing the genome of the original 'local source'
from Umeå (Sweden), a representative of *Candidatus* Frankia
nodulisporulans (Herrera-Belaroussi et al. 2020), nodules of the
original inoculum, (Sellstedt et al. 1986) were used which had been
stored at -80°C. DNA was isolated and sequenced as described
by Berckx et al. (2022). Genome data were deposited at NCBI,
accession numbers CAWVPV010000001-CAWVPV010000515. The

genome of the 'local source' (LS) is 4.238 073 MB in size with
71.68% GC.

Organism phylogeny and ANI

ANI comparisons were performed in order to determine which
species the *H. rhamnoides*-nodulating strain Hr75.2.1 belonged to,
using the usually applied ANI threshold range of 95% for species
delineation (Goris et al. 2007), the EDGAR platform (Blom et al.
2009, 2016) and the type strains of all *Frankia* species available thus
far. On the same platform, a core genome phylogenetic tree was
inferred using the FastTree software (http://www.microbesonline.
org/fasttree/; Price et al. 2009, 2010).

Protein phylogeny

Hyp and Hup trees were constructed as described previously
with some modifications (Wibberg et al. 2020). Briefly, multiple
alignments were generated for all selected protein sequences us-
ing MUSCLE (Edgar 2004). All amino acid alignments were sub-
jected to automatic elimination of poorly aligned positions using
the Castresana Lab Gblocks standalone application with default
parameters (version 0.91b; Castresana 2000); the ‘with-hal’ gap
treatment was applied and the minimum block length was set
to 5 (Talavera and Castresana 2007). Phylogenetic trees based on
curated alignments using the maximum likelihood method with
1000 bootstrap resampling were calculated using MEGA version
11 (Tamura et al. 2021).

Hydrogenase operon analysis

*Frankia* [NiFe] hydrogenase genes were identified via Blast
searches on NCBI and JGI (https://img.jgi.doe.gov/cgi-bin/w/main.
cgi). Plots of the hydrogenase regions were created by a modified
in-house script as described before (Eikmeyer et al. 2012).

16S profiles

DNA was isolated from nodules induced by UmASH1 and the
original 'local source' inoculum, respectively, on A. glutinosa as
described by Berckx et al. (2022). DNA quality was assessed by
gel electrophoresis and photometrically using a NanoDrop®
ND-2000c UV-Vis spectrophotometer (NanoDrop Technologies).
The V3-V4 region of the 16 s rRNA gene was amplified using
barcoded versions of the universal primer set 341F (5'-CCT
AYG GGR BGC ASC AG-3') and 806R (5'-GGA CTA CNN GGG
TAT CTA AT-3'). Amplicon quality was assessed by agarose gel
electrophoresis prior to library preparation. Library quality was
controlled using a Qubit 2.0 Fluorometer (Thermo Fisher Sci-
entific) and an Agilent Bioanalyzer 2100 system. Libraries were
sequenced on an Illumina NovaSeq 6000 platform employing
250 bp paired-end reads at Novogene Europe (Cambridge, UK).
Processing of raw data entailed: data split (paired-end reads
was assigned to samples based on their unique barcode and
truncated by cutting off the barcode and primer sequence),
sequence assembly [paired-end reads were merged using FLASH
(V1.2. 1 1; http://ccb.jhu.edu/software/FLASH/) (Magoč and
Salzberg 2011), a highly efficient tool designed to merge paired-
end reads by utilizing the overlap between reads generated
from opposite ends of the same DNA fragment; this process
results in the creation of sequences known as raw tags; data
filtration [quality filtering on the raw tags were performed us-
ing the fastp (Version 0.23.1) software to obtain high-quality
Clean Tags (Bokulich et al. 2013)]; chimera removal [the tags
were compared with the reference database [Silva database
(16S/18S), https://www.arb-silva.de/; Unite Database (ITS),
https://unite.ut.ee/ to detect chimera sequences, and the effective tags were obtained by removing the chimera sequences with the vsearch package (V2.16.0, https://github.com/torognes/vsearch) (Edgar et al. 2011).

## Distribution of hydrogenase types in the class Actinomyceta

The hidden Markov model (HMM) of HupL was identified using the HupL1 sequence from F. alni ACN14a (CAJ61042.1) for a search on www.uniprot.org/blast. The raw HMM was downloaded from http://pfam.xfam.org/family/PF00374. In order to run an hmmsearch of PF00374 with HMMER (Eddy 2011), a database was created out of the amino acid fasta files of the PROKKA (version 1.14.6; Seeman 2014) annotations of all Actinobacteriota species representative genomes in the Genome Taxonomy Database (GTDB) (release R07-RS207; Parks et al. 2019). A ranking of the HMMER output per sequence was created using 'hmmrank' by Daniel Lundin (https://github.com/erikrikarddaniel/hmmrank). The best-ranked sequences were used to identify the hydrogenase type on hydDB (https://services.birc.au.dk/hyddb/) (Søndergaard et al. 2016). A set of counting tables for each taxonomic rank was created out of the hydDB output using RStudio (version 2022.07.1, R version 4.2.0; RStudio Team 2022) with the tidyverse package (https://www.tidyverse.org/) (Wickham et al. 2019).

In order to create a family tree for all Actinomyceta, the bacterial GTDB reference tree was used (https://data.gtdb.ecogenomic.org/releases/release207/207.0/bac120_r207.tree), subset to c_Actinomyceta and pruned to family level using Dendroscope (version 3.8.3) (https://uni-tuebingen.de/fakultaeten/mathematisch-naturwissenschaftliche-fakultaet/fachbereiche/informatik/lehrstuehle/algorithms-in-bioinformatics/software/dendroscope/) (Huson and Scornavacca 2012).

## Determination of nitrogenase and uptake hydrogenase activities

Roots of intact whole-plant systems were incubated in gas-tight cuvettes, which were sealed with suba seals (Sigma-Aldrich, Sweden) and left in the greenhouse for 1 h. Measurement of hydrogen was performed by taking out 1 ml samples from each cuvette with a gas-tight syringe and injecting them into a GC-8AIT gas chromatograph with a TCD detector (Thermal Conductivity Detector; Shimadzu Scientific Instruments, Columbia, USA) (Mattsson and Sellstedt 2000). The TCD-GC was equipped with a column packed with Molecular Sieve 5A with argon as carrier gas, and run at 80°C column temperature and 120°C detector temperature. In the cases where hydrogen evolution could not be detected after 1 h, a second measurement was made after 24 h.

For measurements of nitrogenase activities, the ARA was used. Plants roots of intact whole-plant systems incubated in the above-mentioned gas-tight cuvettes were vented for 2 h; then, 10% (v/v) of the gas phase in the cuvette were exchanged with 99.5% (v/v) acetylene; then the cuvettes were kept in the greenhouse for 1 h. Measurement of ethylene was performed by taking 1 ml samples from each cuvette with a gas-tight syringe and injecting into a GC-8AIT gas chromatograph with a FID detector (Flame Ionization Detector; Shimadzu Scientific Instruments) (Mattsson and Sellstedt 2000, Richau et al. 2017). The FID-GC was equipped with a column packed with Porapak T, and run at 90°C column temperature and 220°C detector temperature, with N₂ as carrier gas.

The relative efficiency (RE) of nitrogenase is used to describe the extent of hydrogen loss during N₂ fixation (Schubert and Evans 1976). RE is a coefficient describing the electron allocation to nitrogenase.

Divergence of the coefficient indicates the presence of other interacting enzyme systems, such as for example uptake hydrogenase.

$$ RE = 1 - (\text{rate of H}_2 \text{ evolution in air}) / (\text{rate of C}_2\text{H}_2 \text{ reduction}). $$

According to Burns and Hardy (1975) and Schubert and Evans (1976), the rate of hydrogen evolution in the absence of other reducible substrates should depict the total electron flux to the nitrogenase system. Therefore, in the presence of acetylene at a saturating amount of 10% (v/v), no hydrogen is formed and the total electron flow to nitrogenase is most likely used to reduce acetylene (Dart and Day 1971). Consequently, the RE of electron transfer to nitrogen via nitrogenase can be expressed by the ratio defined the RE, and an RE above 0.75 indicates uptake hydrogenase activity (Schubert and Evans 1976).

## Results and discussion

### Four different types of [NiFe] hydrogenase can be found in the Frankia strain genomes

[NiFe] hydrogenases can be grouped into 29 classes based on the amino acid sequences of their large subunits (Søndergaard et al. 2016). Using BlastP searches on NCBI with different large subunits of hydrogenases from Streptomyces, two groups of HupL proteins were found to be encoded by Frankia genomes, a large group of sequences of more than 500 amino acids, and a smaller group of proteins of ca. 430 amino acids. The proteins representing the 500+ amino acid large subunit of [NiFe] uptake hydrogenase, HupL, were used to build a phylogenetic tree. The results (Fig. 1A) clearly show three distinct forms of HupL, corresponding to HupL1 and HupL2 from the two syntons previously identified in representatives of Frankia cluster 1, and one form that was previously identified in a strain from cluster-3 but not distinguished from HupL2 (Leul et al. 2009). The latter, now termed HupL3, was found in all genomes of cluster-3 strains examined, in one strain of the nonsymbiotic cluster-4 and in all strains from the island lineage of cluster-2 (Nguyen et al. 2019, Berckx et al. 2022, Fig. 1A). The phylogeny of the proteins representing the small submit of [NiFe] uptake hydrogenases, HupS, was similar to that of HupL (Fig. S3).

HupL1 represents a ca. 595 amino acid large subunit of [NiFe] hydrogenase group 1 h (Søndergaard et al. 2016). HupL2 represents a ca. 535 amino acid large subunit of [NiFe] hydrogenase of group 2a, and HupL3 a ca. 573 amino acid large subunit of group 1f. All three types—1 h, 2a, and 1f—are classified as 'plasma membrane associated?' in HydDB (https://services.birc.au.dk/hyddb/). Also the small subunits (HupS) encoded by the three different syntons had distinct sizes: 350–360 amino acids for HupS from synton-1, 320–323 amino acids for the version from synton-2, and 410–420 amino acids for the version from synton-3 (Table S2).

The smaller version of HupL (409–451 amino acids), termed HupL4 in this manuscript and present in several strains, represents a type 3b cytosolic [NiFe] hydrogenase (Søndergaard et al. 2016; Table S2).

### The phylogeny of the hydrogenase structural proteins of syntons 1, 2, and 3 follows that of Frankia

Within the Frankia genus, cluster-1 representatives contain synton-1 and synton-2, cluster-3 representatives contain synton-1 and synton-3, and the representatives of the earliest branching clade, cluster-2, either contain synton-1 (continental lineage) or
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Phylogenies of [NiFe] hydrogenase large subunit and accessory proteins. Colouring is according to synton (synton-1, group 1 h; synton-2, group 2a; synton-3, group 1f; and synton-4, group 3b). (A) *Frankia* hydrogenase large subunit phylogeny. HupL proteins of *Frankia* species fall in four phylogenetic groups, HupL1 (synton-1), HupL2 (synton-2), HupL3 (synton-3), and HupL4 (synton-4). Numbering of synton-1 and synton-2 is based on the previously identified hydrogenase syntons published by Leul et al. (2009). (B) Phylogeny of HypF proteins of *Frankia* species. Protein numbering is based the HupL protein of the same synton; the genes encoding the HypF1al proteins from cluster-3 species are not linked to any synton in the genome (al, alone); the numbers were assigned because these genomes contain synton-1 without a hypF gene (and synton-3 with a hypF gene), nevertheless, they group with synton-2 HypF proteins. The numbering of *Frankia* inefficax Eul1c HypF3 is doubtful in so far that the corresponding gene is not linked to the only synton present in the genome, synton-1), but based on sequence, it represents a member of synton-3. *Frankia canadensis* ARg5P HypF2 was annotated as such since ARg5P contains synton-1 (which contains hypF1) and synton-2 (which does not contain hypF2); 'hypF2' is not linked to either synton and the phylogenetic tree clearly shows it encodes a HypF3 protein. In this figure, F. soli EAN1pec was used instead of the type strain NRRL B-19216 because the latter contains two additional copies of hypF3 (unlinked to any hup operon), which is atypical. (C) Phylogeny of HypD proteins of *Frankia* species. Protein numbering is based on the HupL protein of the same synton. The HypD3 (synton-3, group 1f) proteins of the island lineage of cluster-2 clearly represent synton-1 (group 1 h) proteins based on their sequence. The size bar shows the number of changes. Bootstrap values are included. Please note that F. alpina, F. nodulisporulans, F. datiscae, F. californiensis, and *Frankia* sp. (novel species described by Berckx et al. 2022) are *Candidatus* species.</figcaption></figure>

synton-3 (island lineage) [Fig. 1A; see Berckx et al. (2022) for the two lineages of *Frankia* cluster-2]. The corresponding genes could have evolved by duplication and modification within *Frankia* or could have been acquired by horizontal gene transfer (HGT). Based on the comparison of the phylogeny of HupL1-4 (Fig. 1A) and HupS1-4 (Fig. S3) and the core genome phylogeny of the *Frankia*

genus (Fig. S1), no HGT needs to be invoked; HupL1-3 and HupS1-3 clearly coevolved and their phylogeny more or less follows the core genome phylogeny.

Synton-4 (group 3b) was not found in any sequenced genome of *Frankia* cluster-2. It was found in two strains of the nonsymbiotic *Frankia* clade, cluster-4, *Frankia saprophytica* CN3 and *Frankia*
sp. EUN1h; however, two genes in the EUN1h operon are pseudo-
genized, so this strain cannot form a group 3b [NiFe] hydrogenase
(Table S2). Synton-4 was found in some representatives of cluster-
3, namely in *Frankia elaeagni* BMG5.12 and in some strains of F. soli,
the type strain NRRL B-16219 (but two genes in the operon are
pseudogenes, so a type 3b hydrogenase cannot be formed) and
in Hr75.2.1 (operon intact), but not in EAN1pec (for the represen-
tatives of F. soli, see Fig. S2). In cluster-1, it was found among the
alder-infective strains in both representatives of *Frankia torreyi*, but
not at all in F. alni, *Ca*. Frankia alpina or *Ca*. Frankia nodulisporu-
lans. However, it is present—and intact—in all 13 genomes avail-
able from *Casuarina*-infective strains (shown for the type strain
Ccl3 and for strain KB5 in Fig. 1A; see also Table S2). The phylogeny
of the two subunits of group 3b [NiFe] hydrogenase, HupL4 and
HupS4, does not follow *Frankia* species phylogeny (Fig. 1A; Fig. S3).

The phylogeny of the maturation proteins HypD and HypE, and of HypF, differs from the phylogeny of the structural hydrogenase proteins

The fact that both lineages of sporulating strains of cluster-1a
have retained genes from different syntons, combined with the
fact that synton-3 lacks hypB3 in most cases examined, raises the
question whether the phylogeny of the maturation protein genes
follows the phylogeny of the structural genes, or whether we can
find evidence for the exchange of maturation proteins between
syntons. Therefore, the phylogeny of the largest maturation pro-
teins, HypD, HypE, and HypF, was examined. For HypF, phylogeny
clearly separated the HypF proteins of the different syntons, and
within these group mostly followed *Frankia* phylogeny (Fig. 1B).
However, the position of cluster-3 HypF proteins was one inter-
esting exception. While syntons-3 of cluster-3 contain a hypF gene,
syntons-1 of this cluster do not. However, each genome contains a
hypF gene that is unlinked to any synton, termed ‘hypF1alone’. The
corresponding proteins (HypF1al) group with the HypF proteins
from synton-2 (Fig. 1B), suggesting that they represent leftovers
of a lost synton-2. Furthermore, the HypF2 protein of the cluster-
1 strain *Frankia canadenis* ARgP5—encoded by a gene that is not
linked to synton-2, but synton-1 contains a hypF gene and synton-
2 does not—clearly represents a HypF3 protein (Fig. 1B); thus, it
could be a leftover of a lost synton-3. Similarly, the cluster-4 strain
*Frankia inefficax* Eu11c contains a synton-1 (group 1 h) hydrogenase
and a synton-3 (group 1f) HypF protein, possibly a remnant of a
lost synton-3.

HypD and HypE clearly coevolved (Fig. 1C and Fig. S4). They
can be grouped into five different clades, which neither repre-
sent genes from the same synton, nor follow *Frankia* phylogeny
(A–E; Fig. 1C for HypD; for HypE see Fig. S4). The synton-2 HypD/E
proteins of cluster-1 and of the continental lineage of cluster-2
are separated, the latter group with synton-3 HypD/E proteins of
cluster-3 and of the cluster-4 species *F. inefficax* Eu11c. The synton-
3 HypD/E proteins of cluster-3 and of the island lineage of cluster-2
are separated in that the latter group with synton-1 HypD/E pro-
teins of two of the cluster-3 species examined. Thus, the phyloge-
netic analysis suggests that HypDE of the continental lineage of
cluster-2 are remnants of a lost synton-3, and that HypDE of the
island lineage of cluster-2 are remnants of a lost synton-2.

Altogether, these data show that the hydrogenase matura-
tion protein genes evolved independently of the corresponding
structural protein genes, and can be exchanged between syn-
tons 1-3/[NiFe] hydrogenase groups 1 h, 1f and 2a. Given that
HypE and HypF interact during hydrogenase maturation, while
HypD interacts with HypC (Peters et al. 2015), it is surprising that

HypE and HypD coevolved while HypF seems to have evolved in-
dependently (since HypC proteins of groups 1 h, 1f, and 2a are
less than 100 amino acids in length, their phylogeny was not
analysed).

Organization of the four different [NiFe] uptake
hydrogenase syntons in the genus Frankia

The configuration of the three large syntons differs (Fig. 2A). Two
versions of synton-1 (group 1 h) were found. In synton-1 of cluster-
1 strains, all genes have the same orientation. It starts with hypA1-
hypB1, which may represent a separate operon, followed by an
operon encompassing a gene encoding a hypothetical protein
of unknown function, the maturation protease (maturase) gene,
hupS1-hupL1, a gene encoding the FeS cluster biogenesis protein
NfuA, followed by three genes encoding hypothetical proteins and
ending with hypF1-hypC1-hypD1-hypE1. The nfuA gene is missing
in the genome of *Frankia casuarinae* Ccl3. In contrast, synton-1
of cluster-3 strains begins with hypC1a, followed by hupS1, hupL1,
four ORFs encoding hypothetical protein, the maturation protease
gene, hypC1b, hypD1, hypF1, hypA1, hypB1, and then hypF1 but in the
opposite orientation. The cluster-4 strain *F. inefficax* Eu11c has a
similar configuration of synton-1, but with hypB1 and hypF1 miss-
ing (data not shown); the cluster-4 strains *Frankia saprotrophica*
CN3 and *Frankia asymbiotica* NRRL B-16386 have the cluster-1 con-
figuration of synton-1.

Synton-2 (group 2a) consists of two operons in divergent ori-
entation, one representing hypA2-hypB2 and the large one repre-
senting hupS2-hupL2-hupD2, followed by a gene encoding a hy-
pothetical protein and a gene encoding an NHL repeat contain-
ing protein, followed by hypF2-hypC2-hypD2-hypE2. In cluster-2
strains of the continental lineage, the structural genes are not
part of the larger operon, and the arrangement of genes down-
stream of hupD2 is different: hypF2-hypC2b, followed by a hypo-
thetical protein gene and gmhA, then hypE2-hypC2a-hypD2. The
gmhA gene found between hypC2b and hypE2 (FsymDg_2476 in
*Candidatus* Frankia datiscae Dg1; see Fig. 2A) encodes a D-glycero-
beta-D-manno-heptose 1-phosphate adenylyltransferase, an en-
zyme involved in the synthesis of the lipopolysaccharide pre-
cursor ADP-L-glycero-β-D-manno-heptose (ADP-L-β-D-heptose) in
Gram-negative bacteria and GDP-D-α-D-heptose in Gram-positive
bacteria (Valvano et al. 2002, Kneidinger et al. 2002, Karan et al.
2020). GmhA is often found in group 1 h and group 2a hydrogenase
operons (Islam 2020); however, gmhA is not present in synton-2 of
cluster-1 *Frankia* strains nor in synton-1 (group 1 h) of any *Frankia*
cluster.

Also synton-3 was represented by two versions. The first ver-
sion of synton-3 was found in cluster-3 genomes. It consists of
one large operon, hypC3b-hupS3-hypL3, followed by four genes for
hypothetical proteins, followed by the maturation protease gene
and then by hypC3a-hypE3-hypD3-hypA3 (and in *Frankia irregu-
laris*, by hypB3 at the end; in the other cluster-3 species, hypB3
was missing). The second version was found in the island lin-
eage of cluster-2. Here, synton-3 consists of two operons in diver-
gent orientation, one encompassing the maturation protein genes
hypC3-hypD3-hypE3-hypF3 and the other containing hupS3-hupL3,
a gene encoding a [NiFe] hydrogenase cytochrome subunit (hupC),
a gene encoding the maturation endopeptidase (maturase) hupD,
and hypA3.

In summary, the operon organization and the order of the mat-
uration protein genes hypC, hypD, hypE, and hypF differ between
the three syntons. No hypB3 gene could be identified in synton-3
in most of the cluster-3 genomes available, while the genomes of
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Complete operons of the four syntons and erosion of syntons-1 and -2. (A) Operon structure of the four different syntons in *Frankia* spp. Genes with known functions with regard to [NiFe] hydrogenase are depicted in the same colour for each synten. Genes encoding structural proteins are surrounded by dotted lines. Syntons-1, -2, and -3 have different configurations in different *Frankia* clusters. (B) Operon structure of eroding syntons in cluster-1a and cluster-2. The remnant of synten-1 in *Candidatus* *Frankia* alpina *AiOr* and its superficially intact synten-2 are shown. The two parts of synten-2 of *Candidatus* *Frankia* nodulisporulans *AgTrS* with the break in *hypF2* show that no Hup activity can be expected in this species. Erosion of synten-2 was most advanced in the 'local source' inoculum stored since 1986. The directly sequenced 'local source' inoculum (*Ca*. *Frankia* nodulisporulans LS) showed even further erosion of synten-2 in that the 3'-part of *hypF2* and the 5'- and 3'-end of *hypE* were missing (data not shown). Size bars denote 1 kb.</figcaption></figure>
cluster-2 strains of the island lineage (Berckx et al. 2022), which only contain synton-3, lack both *hypA3* and *hypB3*.

Synton-4 did not contain any maturation protein genes besides the one encoding the maturation protease. It consists, as described in HydDB (Søndergaard et al. 2016), of the genes of the diphorase module [*hyhG*, encoding the iron-sulfur binding subunit (F. *casuarinae* Ccl3: Franccci3_4500), followed by *hyhB* (Franccci3_4498), followed by the genes encoding the hydrogenase module with the [NiFe] center, *hyhS* (*hupS4*; Franccci3_4497) encoding the small subunit and *hyhL* (*hupL4*; Franccci3_4496) encoding the large subunit, followed by the maturation protease gene (Franccci3_4495)]. However, in *Frankia*, between the two genes for the diaphorase module the gene for a small transcriptional regulator with a cyclic nucleotide binding domain (Franccci3_4499) is present. In *F. casuarinae*, synton-4 is closely linked (ca. 7 kb distance) to the *nifHDK* operon.

## Genome analysis shows loss of hydrogenase-related genes in cluster-1a spore formers and in all cluster-2 strains

Figure 2(B) shows the configuration of the four different syntons in lineages of genome reduction. In representatives of the continental lineage of cluster-2, *Frankia coriariae* and *Ca. Frankia datsicae*, synton-2 had been retained but is divided into two operons; the structural protein genes *hupS2-hupL2*, followed by the ferredoxin subunit gene, are not on the same operon as the maturation protein genes; instead, *HupS2-hupL2* had been transferred to another position in the chromosome. In *Ca. F. datsicae* Dg1, the only completely assembled genome of a cluster-2 strain, the maturation protein gene operon is located in position 2941034-2934745, while the structural protein gene operon in 488967-488785. The structural protein genes are followed by a gene encoding the ferredoxin subunit of nitrite reductase (F_syrmDg_0417 in Fig. 2B). In representatives of the island lineage of cluster-2 (Berckx et al. 2022), neither synton-1 nor synton-2 was found. Instead, synton-3 is present, but is lacking *hypB3*. While *hypB3* is also missing in the genomes of most cluster-3 strains examined, cluster-3 strains contain a complete synton-1 including *hypB1*.

In the two species of cluster-1 strains sporulating in nodules, *Ca. F. alpina* and *Ca. F. nodulisporulans*, (most of) synton-1 could not be found (Fig. 2B). *Ca. F. alpina* AiOr has retained the complete synton-2, i.e. the *hypA2-hypB2* operon and a larger operon representing *hupS2-hupL2-maturation protein gene-two genes encoding hypothetical proteins-hypF2-hypC2-hypD2-hypE2*. The AiOr genome also contains part of synton-1: it begins with a truncated version of *hupL1*, followed by four genes encoding hypothetical proteins, then *hypF1*, *hypC1*, a complete and a truncated version of *hypD1*, and *hypE1*. In the four representatives of *Ca. F. nodulisporulans* the genomes of which were sequenced, no remnants of synton-1 were found. The genome of the type strain *AgTrS* contains a truncated version of synton-2 consisting of *hypA2* and *hupS2-hupL2-hupD2* (maturation protease gene)-hypothetical protein gene, ending with a 3'-truncated version of *hypF2*. Another operon, *hypF2'-hypC2-hypD2-hypE2*, was also found, as was a *hypB2* gene. The genomes of the two representatives of the Swedish 'local source', strains *AgUmASt1* and *AgUmASH1*, contained a complete *hypA2-hypB2* operon, while the larger operon was distributed over two contigs as in *AgTrS*. The genome of *AgUmASH1* contains a hypothetical protein gene after the NHL repeat protein gene in the larger operon (Herrera-Belaroussi et al. 2020). Interestingly, the erosion of synton-2 is most advanced in the genome sequenced from nodules of the

original 'local source' that were stored at -80°C since the 1980s. Here, only a truncated version of *hypE2* is left of the *hypF2'-hypC2-hypD2-hypE2* operon present in the three other genomes of *Ca. F. nodulisporulans* strains (Fig. 2B).

In summary, the cluster-1 strains sporulating in nodules have lost all or most of synton-1; all members of *Ca. F. nodulisporulans* have lost all of synton-1. Furthermore, the genomes of all four representatives of *Ca. F. nodulisporulans* contain no intact *hypF*; instead, *AgTrS*, *UmASH1*, and *UmASt1* contained two truncated copies of *hypF2*, a 3'-truncated one in the operon with the structural genes, and a 5'-truncated one in the operon with the maturation protein genes. This should exclude any [NiFe] uptake hydrogenase activity. In order to check whether an assembly error had occurred and the strains actually contained a complete copy of *hypF2*, RT-PCR was performed with a 5'-primer derived from the 3'-truncated version and a 3'-primer derived from the 5'-truncated version of the gene. However, no product was obtained (data not shown). Lastly, the raw data of the *AgTrS* genome were analysed for the presence of overlapping reads between the two parts of *hypF2*, but none were found; on the contrary, the raw data showed that there were at least 300 bp of repetitive sequences between the two parts of *hypF2* (data not shown). Thus, *Ca. F. nodulisporulans* should not be able to form an active [NiFe] hydrogenase, while *Ca. F. alpina* might.

## Exchangeability of maturation proteins between groups of [NiFe] hydrogenases

[NiFe] hydrogenase group 1f (synton-3) is consistently lacking *hypB* in all *Frankia* strains studied, including in cluster-3 strains of the island lineage where synton-3 encodes the only [NiFe] hydrogenase. It has been known for some time that accessory proteins for the incorporation of Ni can be exchanged between different systems, e.g. de Reuse et al. (2013) summarized that in *Heliobacter pylori* HypA/HypB contribute to the incorporation of Ni in urease, another enzyme that contains Ni as the active centre. Thus, the lack of a *hypB* gene in all but one representatives of synton-3 strongly suggests that HypB can be substituted for by the corresponding Ni incorporation protein from the other Hup synton (synton-1, group 1 h). However, that does not explain whether, and if yes, how the lack of *hypB3* in cluster-2 strains of the island lineage is compensated for.

Phylogenetic analysis of HypF proteins showed that in contrast with the structural proteins of [NiFe] hydrogenase, HypF phylogeny did not always follow *Frankia* phylogeny (Fig. 1B). HypF phylogeny did, however, show a separation between HypF3 and HypF1/2, with the exception that *Frankia canadensis* ARgP5 *hypF2*, which is not part of any Hup synton and was termed *hypF2* since synton-1 of ARgP5 does contain a *hypF* gene while synton-2 does not, grouped with HypF3. Furthermore, extant cluster-3 strains do not contain synton-2 (group 2a), but their syntons-1 lack *hypF1*, while their genomes contain unlinked *hypF* genes that encode HypF2 proteins (Fig. 1B). These data not only support a scenario where the ancestral *Frankia* strain had all three [NiFe] uptake hydrogenase syntons (groups 1f, 1 h, and 2a), but also suggest that accessory proteins can be exchanged between [NiFe] hydrogenases of different groups.

## How did *Frankia* end up with four types of hydrogenases: evolution of hydrogenases in *Actinomyceta*

Phylogenetic data indicate that BNF evolved in an anaerobic, thermophilic ancestor of hydrogenotrophic methanogens and later
spread via lateral gene transfer (HGT) into anaerobic bacteria, and eventually into aerobic bacteria including Cyanobacteria (Mus et al. 2019). At one point, the ancestor of *Frankia* must have acquired nitrogenase genes and thus the capacity to fix nitrogen while producing hydrogen gas as a by-product. Thus, the ancestor of *Frankia* could profit from the acquisition of uptake hydrogenase: first, to reabsorb the H₂ produced by nitrogenase, second, to produce ATP via the oxyhydrogen (Knallgas) reaction; third, to remove oxygen from the oxygen-sensitive nitrogenase complex; and fourth, to provide reduction equivalents (Zhang et al. 2014). Or did the *Frankia* ancestor already contain hydrogenase genes, and if yes, for which type(s) of hydrogenase? In this context, it is interesting that in strains of the nonsymbiotic *Frankia* cluster-4, nitrogen fixation (nif) genes are missing while genes encoding [NiFe] uptake hydrogenase group 1 h (synton-1) are commonly found. Therefore, we set out to study the occurrence of different types of hydrogenase in the phylogenetic environment of *Frankia*.

According to the GTDB taxonomy (Chaumeil et al. 2019, Parks et al. 2022), Frankiaceae are part of the order Mycobacteriales in the class Actinomycetia in the phylum Actinobacteriota. We analysed the presence of different types of [NiFe] hydrogenases in the families that make up the Actinomycetia class (Fig. 3). It is obvious that several types of [NiFe] hydrogenases are present in different families of the Actinomycetia, specifically of the Mycobacteriales. Within the Mycobacteriales, two other families besides Frankiaceae contain the four types of hydrogenases present in *Frankia* spp.; these families (Mycobacteriaceae and Pseudonocardiaceae) contain also hydrogenases from two (Pseudonocardiaceae) or three (Mycobacteriaceae) other groups. Within the entire class Actinomycetia, altogether four families (Streptosporangiaceae, Streptomycetaceae, Mycobacteriaceae, and Pseudonocardiaceae) contain hydrogenases of (at least) the same four groups that *Frankia* spp. contain.

This means it is parsimonious to assume that the *Frankia* ancestor contained different types of [NiFe] hydrogenases. Furthermore, the phylogeny of the maturation protein HypF in *Frankia* shows evidence that the common ancestor of the four *Frankia* clades contained syntons 1–4, i.e. hydrogenases of the groups 1 h, 2a, 1f, and 3b, and that at least some maturation proteins can be exchanged between these different groups of [NiFe] hydrogenases. The exchangeability of maturation proteins between the groups 1 h, 2a, and 1f can be extended to group 3b hydrogenases (synton-4), given that synton-4 does not contain any genes encoding the proteins required for the insertion of the Fe(CN)₂CO cofactor, or Ni, in the large subunit of [NiFe] hydrogenases. In this context, it is interesting that so far, synton-4 was never the only hydrogenase synton found in a *Frankia* genome.

Altogether, the assumption that the common ancestor of *Frankia* contained all four [NiFe] hydrogenase syntons (groups 1f, 1 h, 2a, and 3b) found in different extant strains, seems the most parsimonious.

## Acetylene reduction and uptake hydrogenase activity in intact nodules

Uptake hydrogenase activity can be deduced when nitrogenase activity, as determined by the ARA, is compared with hydrogen evolution (Schubert and Evans 1976, Sellstedt et al. 1986). The results of measurements performed on all symbiotic systems covered in this study are shown in Table 2(A). Mature nodules of *D. glomerata* infected by *Ca. F. californiensis* Dg2, and of *H. rhamnoides* infected by *F. soli* Hr75.2.1, showed Hup activity in that no hydrogen evolution could be detected while nitrogen fixation took place.

The data depicted in Table 2(B) show that *F. soli* Hr75.2.1 displayed uptake hydrogenase activity also during nitrogen fixation under aerobic conditions in culture.

Nodules of *A. glutinosa* or *A. incana* induced by *F. alni* ACN14a also showed no detectable H₂ production, i.e. due to Hup activity. In contrast, alder nodules induced by *Ca. F. alpina* AiOr or one of the three representatives of *Ca. F. nodulisporulans* used in this study, showed hydrogen evolution during nitrogen fixation. In nodules induced by *Ca. F. alpina* AiOr, which has retained the complete synton-2, the RE of nitrogenase was 0.60–0.94. Based on the stoichiometry of the nitrogenase reaction and the RE calculations, a RE below 0.75 means the strain lacks uptake hydrogenase activity (Schubert and Evans 1976, Sellstedt et al. 1986, Huss-Danell 1991). Thus, while no synton-2 gene was lost in AiOr, a promoter or one or more of the genes must have acquired mutations; the strain has lost synton-1 (group 1 h) and is in the process of losing synton-2 (group 2a).

Nodules induced by the type strain of *Ca. F. nodulisporulans*, AgTrS, displayed an RE of 0.70–0.76, i.e. barely any uptake hydrogenase activity. Nodules induced by the two representatives of the 'local source', the original inoculum and UmASH1, showed RE values of 0.56–0.77 and 0.26–0.47, respectively (Table 2A). In the original study, nodules induced by the 'local source' had shown an RE of 0.57, i.e. they did not display uptake hydrogenase activity (Sellstedt et al. 1986). In a later study by Huss-Danell (1991), several species of *Alnus* sp. were inoculated with the 'local source' of *Frankia*, and the RE ranged between 0.48 and 0.75. Thus, the RE values measured for the two representatives of the 'local source' analysed are consistent with previous publications for the 'local source' of *Frankia*, and all representatives of *Ca. F. nodulisporulans* lack Hup activity, consistent with the fact that based on the genome sequence, all representatives of *Ca. F. nodulisporulans* should be equally lacking in [NiFe] hydrogenase activity since they do not contain an intact hypF gene. However, the differences between the RE values of the different intrastrains are surprising.

## The 'local source' (*Ca. F. nodulisporulans*) is represented by four inocula in this study which behave differently with regard to nitrogenase RE and nodule morphology

The 'local source' inoculum, which recently was shown to be a member of *Ca. Frankia nodulisporulans* (Herrera-Belaroussi et al. 2020) has been maintained since the 1980s in the form of nodules stored at –80°C. Occasionally, fractions were used to inoculate plants growing on a mix of autoclaved soil and autoclaved gravel, like the plant which was inoculated in 2014 and maintained in a growth chamber, from which the nodules were harvested in 2019 that were used to isolate and sequence the UmASH1 genome (Herrera-Belaroussi et al. 2020). Later, nodules were collected from the same plant and propagated on an *A. glutinosa* plant to be used as inoculum in this study. The differences between the genomes of the 'local source', UmASH1, UmASt1, and the type strain AgTrS are consistent with the observation made earlier for the equally uncultivable cluster-2 strains, namely that inocula represent mixtures of strains and different representatives will end up inducing nodules (Berckx et al. 2022). Thus, it cannot be excluded that the different inocula contained a minor contribution of strains with a complete synton-2, leading to the occasional occurrence of nodule lobes containing *Frankia* with uptake hydrogenase activity.

In independent experiments performed in Umeå and Stockholm, nodules induced by UmASH1 on roots of *A. glutinosa* and *A. incana* consistently did not resemble those induced by the 'local
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Distribution of [NiFe] hydrogenase types in Actinomycetia. To see whether the occurrence of four different types of hydrogenase in *Frankia* spp. is likely to have involved HGT, the occurrence of all hydrogenase types was examined using the HydDB tool (Søndergaard et al. 2016) in the entire class Actinomycetia. The phylogeny of Actinomycetia was taken from GTDB (Parks et al. 2019, 2022). The origin of the order Mycobacteriales, which contains the genus *Frankia* is indicated in the phylogenetic tree by an orange circle; families belonging to the Mycobacteriales are labelled by asterisks. The groups of hydrogenases that occur in *Frankia* are given in color and bold print for the entire class—1 h (synton-1), 2a (synton-2), 1f (synton-3), and 3b (synton-4)—, the other groups are given in grey.</figcaption></figure>
<table><caption>Table 2A. Acetylene reduction and H₂ evolution in nodules.</caption><thead><tr><th rowspan="2">Plant species</th><th rowspan="2">Frankia strain</th><th colspan="2">Plant height<sup>a</sup></th><th rowspan="2">ARA (µmol H<sub>2</sub> h<sup>-1</sup>)</th><th rowspan="2">RE</th><th rowspan="2">Average</th><th rowspan="2">S<sub>DEV</sub></th><th rowspan="2">Nodule weight (g)</th><th rowspan="2">Average</th><th rowspan="2">S<sub>DEV</sub></th><th rowspan="2">dpi</th><th rowspan="2">Season</th></tr><tr><th>H<sub>2</sub> (µmol h<sup>-1</sup>)</th><th>C<sub>2</sub>H<sub>4</sub> h<sup>-1</sup></th></tr></thead><tbody><tr><td>A. incana</td><td>ACN14a</td><td>42.5</td><td>0</td><td>9.38</td><td>1.0</td><td>1.0</td><td>0</td><td>0.118</td><td>0.194</td><td>0.053</td><td>159</td><td>Spring 2021-05-15</td></tr><tr><td>A. incana</td><td>ACN14a</td><td>55.0</td><td>0</td><td>7.36</td><td>1.0</td><td></td><td></td><td>0.210</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>ACN14a</td><td>46.5</td><td>0</td><td>8.48</td><td>1.0</td><td></td><td></td><td>0.209</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>ACN14a</td><td>49.5</td><td>0</td><td>9.28</td><td>1.0</td><td></td><td></td><td>0.24</td><td></td><td></td><td></td><td></td></tr><tr><td>A. incana</td><td>AiOr</td><td>54.0</td><td>0.92</td><td>14.37</td><td>0.94</td><td>0.81</td><td>0.15</td><td>0.410</td><td>0.303</td><td>0.081</td><td>162</td><td>Spring 2021-05-18</td></tr><tr><td>A. incana</td><td>AiOr</td><td>47.5</td><td>1.80</td><td>13.47</td><td>0.87</td><td></td><td></td><td>0.231</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>AiOr</td><td>38.5</td><td>2.19</td><td>12.00</td><td>0.83</td><td></td><td></td><td>0.252</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>AiOr</td><td>40.5</td><td>4.46</td><td>10.98</td><td>0.60</td><td></td><td></td><td>0.320</td><td></td><td></td><td></td><td></td></tr><tr><td>A. incana</td><td>AgTrS</td><td>31.5</td><td>2.58</td><td>8.97</td><td>0.71</td><td>0.72</td><td>0.027</td><td>0.267</td><td>0.251</td><td>0.096</td><td>163</td><td>Spring 2021-05-19</td></tr><tr><td>A. incana</td><td>AgTrS</td><td>43.5</td><td>3.43</td><td>14.72</td><td>0.76</td><td></td><td></td><td>0.365</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>AgTrS</td><td>53.0</td><td>3.98</td><td>13.73</td><td>0.71</td><td></td><td></td><td>0.131</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>AgTrS</td><td>37.5</td><td>3.90</td><td>11.46</td><td>0.70</td><td></td><td></td><td>0.240</td><td></td><td></td><td></td><td></td></tr><tr><td>A. incana</td><td>UmASH1</td><td>44.0</td><td>7.6</td><td>13.51</td><td>0.43</td><td>0.41</td><td>0.1</td><td>0.266</td><td>0.203</td><td>0.084</td><td>161</td><td>Spring 2021-05-17</td></tr><tr><td>A. incana</td><td>UmASH1</td><td>46.5</td><td>7.76</td><td>10.48</td><td>0.26</td><td></td><td></td><td>0.253</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>UmASH1</td><td>38.0</td><td>7.48</td><td>14.04</td><td>0.47</td><td></td><td></td><td>0.212</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>UmASH1</td><td>56.0</td><td>6.97</td><td>12.98</td><td>0.46</td><td></td><td></td><td>0.082</td><td></td><td></td><td></td><td></td></tr><tr><td>A. incana</td><td>Local source</td><td>59.0</td><td>4.59</td><td>14.30</td><td>0.68</td><td>0.63</td><td>0.09</td><td>0.432</td><td>0.351</td><td>0.128</td><td>259</td><td>Summer 2021-08-23</td></tr><tr><td>A. Incana</td><td>Local source</td><td>77.0</td><td>5.22</td><td>12.95</td><td>0.60</td><td></td><td></td><td>0.421</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>Local source</td><td>74.0</td><td>2.83</td><td>12.41</td><td>0.77</td><td></td><td></td><td>0.312</td><td></td><td></td><td></td><td></td></tr><tr><td>A. glutinosa</td><td>Local source</td><td>38.5</td><td>7.66</td><td>17.36</td><td>0.56</td><td></td><td></td><td>0.157</td><td></td><td></td><td></td><td></td></tr><tr><td>D. glomerata</td><td>Dg2</td><td>20.5</td><td>0</td><td>1.99</td><td>1.0</td><td>1.0</td><td>0</td><td>0.361</td><td>0.470</td><td>0.153</td><td>267</td><td>Summer 2021-08-31</td></tr><tr><td>D. glomerata</td><td>Dg2</td><td>18.5</td><td>0</td><td>2.40</td><td>1.0</td><td></td><td></td><td>0.578</td><td></td><td></td><td></td><td></td></tr><tr><td>H. rhamnoides</td><td>Hr75.2</td><td>45.0</td><td>0</td><td>11.32</td><td>1.0</td><td>1.0</td><td>0</td><td>0.826</td><td>0.478</td><td>0.491</td><td>260</td><td>Summer 2021-08-24</td></tr><tr><td>H. rhamnoides</td><td>Hr75.2</td><td>30.0</td><td>0</td><td>2.13</td><td>1.0</td><td></td><td></td><td>0.132</td><td></td><td></td><td></td><td></td></tr><tr><td>H. rhamnoides</td><td>Hr75.2</td><td>31.5</td><td>0</td><td>0.99</td><td>1.0</td><td>1.0</td><td>0</td><td>0.0926</td><td>0.091</td><td>0.002</td><td>161</td><td>Winter 2022-02-08</td></tr><tr><td>H. rhamnoides</td><td>Hr75.2</td><td>29.0</td><td>0</td><td>1.38</td><td>1.0</td><td></td><td></td><td>0.0895</td><td></td><td></td><td></td><td></td></tr></tbody></table>

S<sub>DEV</sub>, standard deviation.

<footnote><sup>a</sup>The H. rhamnoides plants had three to four branches; the length of the longest one is given under 'plant height'.</footnote>
<table><caption>**Table 2B.** Acetylene reduction and H₂ evolution in culture.</caption><thead><tr><th>Frankia strain</th><th>N source</th><th>H₂ (nmol H₂ mg prot<sup>-1</sup>)</th><th>ARA (µmol C₂H₄ mg prot<sup>-1</sup>)</th><th>StDEV</th></tr></thead><tbody><tr><td>Hr75.2</td><td>Yes</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Hr75.2</td><td>No</td><td>0</td><td>0.44</td><td>0.07</td></tr><tr><td>Hr75.2</td><td>Yes</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Hr75.2</td><td>No</td><td>0</td><td>0.14</td><td>0.02</td></tr></tbody></table>

Averages of four replicates are shown. StDEV, standard deviation.

source' (Ca. F. nodulisporulans LS). While based on the biomass acquisition of the nodulated plants compared to control plants and on nitrogenase activity (Table 2), both inocula contained effective microsymbionts (data not shown), nodule phenotypes were strikingly different. Nodules induced by the 'local source' had much larger lobes than those induced by UmASH1 or by any of the other alder-compatible inocula used in this study, and this large-lobe phenotype has been stable since the 1980s (data not shown; A. Sellstedt, personal observations). In order to address the difference in nodule structure and nodule uptake hydrogenase activity, DNA was isolated from nodules induced by UmASH1 and the original 'local source' on A. glutinosa, and 16S profiles were generated. The results are depicted in Fig. S5. The profiles were rather similar but showed differences in some minor components in so far that the 'local source' nodules contained *Acidothermus*, *Asticcacaulus*, and *Variovorax* while the UmASH1 nodules contained *Pseudolabrys*, *Haliangium*, and *Reyranella*.

In the current study, all Ca. F. nodulisporulans strains behaved differently with regard to RE. The type strain AgTrS showed an RE of 0.7-0.76. The 'local source' had an RE of 0.56-0.77 while UmASH1, which is derived from nodules induced by the 'local source' showed an RE of 0.26-0.47, which is lower than should be permitted by the stoichiometry of the nitrogenase reaction in the absence of [NiFe] hydrogenase (Fig. 4). Thus, UmASH1 nodules produced more H₂ than the nitrogenase of the dominant *Frankia* strain. A comparison of the 16S profiles of nodules from nodules induced by UmASH1 and nodules induced directly by the 'local source' showed that when *Frankia* sequences were excluded, the rest of the microbiomes were dominated by pseudomonads with streptomycetes the next largest group; rhizobia were found as well. Streptomycese include strains with [NiFe] hydrogenases (Constant et al. 2011). Furthermore, both microbiomes contain *Novosphingobium* spp. strains of which contain a group 2a [NiFe] hydrogenase and/or display nitrogenase activity (Islam et al. 2020, Bizjak et al. 2023). Furthermore, fermentative hydrogen production as found in the soil bacterium *Mycobacterium smegmatis* (Berney et al. 2014), might be performed by members of the nodule microbiome. Altogether, it is likely that the non-Frankia nodule microbiome contributes to H₂ evolution and H₂ uptake. Similarly, the nodule morphology phenotype might be due to gibberellin production by (a) member(s) of the non-Frankia nodule microbiome since in legume nodules, gibberellin increases nodule size (McAdam et al. 2018).

## Expression of genes from the different syntons in nodules and in culture: is the expression of different syntons linked to different life styles?

Expression of one gene from each synton (for synton-2, two genes representing different operons were used) was analysed in nodules, and, for F. alni ACN14a and F. soli Hr75.2.1, also in nitrogen-fixing cultures. The results are shown in Fig. 4. Consistent with previous results (Leul et al. 2007, 2009), in ACN14a, both synton-1 and synton-2 were expressed in nodules and in nitrogen-fixing

cultures. In cultures containing ammonium, synton-1 expression levels were significantly higher than those of synton-2, the expression of which was barely detectable. In nodules, expression of synton-2 was significantly higher than that of synton-1, suggesting that group 1 h hydrogenase does not play a great role during symbiosis. In the three strains representing Ca. F. nodulisporulans, AgTrS, UmASH1, and LS, synton-2 was expressed. Expression levels of the operon with the structural genes and the *hypA2B2* operon were not significantly different, but expression levels of the *hypF2'-hypC2-hypD2-hypE2* operon were higher in UmASH1 and LS than in the type strain AgTrS (Figs 2 and 4). However, the lack of an intact HypF protein should not allow uptake hydrogenase activity in any Ca. F. nodulisporulans strain (Fig. 2). In summary, we can say that the erosion of synton-2 in Ca. F. nodulisporulans does not seem to involve the promoters.

Both operons of synton-2 were expressed in both cluster-2 species examined, Ca. F. datiscae Dg1 and Ca. F. californiensis Dg2, consistent with the uptake hydrogenase activity shown by the corresponding nodules (Table 2A, Fig. 4). All three syntons, synton-1, synton-3, and synton-4, were expressed in nodules induced by the cluster-3 strain F. soli Hr75.2.1, as well as in nitrogen-fixing and non-nitrogen-fixing cultures. For synton-4, expression levels were significantly higher under nitrogen-fixing conditions (nodules and culture) than during growth on ammonium as nitrogen source, while for synton-1, expression levels were significantly higher in nitrogen-fixing cultures than in nodules.

## Can the erosion of particular [NiFe] hydrogenase syntons in specific subgroups of *Frankia* strains provide information about their function?

*Frankia* strain genome sizes show an unusually large variation between 4.3 MB and more than 10 MB (Normand et al. 2007, Nguyen and Pawlowski 2017, Herrera-Belaroussi et al. 2020). There are three lineages of genome reduction within the genus: cluster-1 strain genomes are 7.5-8 MB in size while cluster-1c strains have genomes of 5-6 MB and cluster-1 strains that sporulate in nodules have genomes of 4.2-5 MB (Pozzi et al. 2020, Herrera-Belaroussi et al. 2020; this study). *Frankia* cluster-2 strains have genome sizes between 5 and 6 MB (Nguyen and Pawlowski 2017, Nguyen et al. 2019, Berckx et al. 2022). Strains of these lineages have limited saprotrophic capabilities. Cluster-1c strains apparently depend on the presence of their host plant to grow saprotrophically in soil (Samant et al. 2016), cluster-2 strains as well as cluster-1 strains that sporulate in nodules have lost some saprotrophic capabilities as exemplified by the fact that with two exceptions in cluster-2 (Gtari et al. 2015, Gueddou et al. 2019), they could never be grown in culture.

However, while cluster-2 strains as well as cluster-1 strains that sporulate in nodules have lost [NiFe] hydrogenase synton-1, members of Ca. F. nodulisporulans have also lost functionality of synton-2 in that the hypF gene has broken up in two parts. The loss of synton-2 did not take place in the type strain of the other lineage that sporulates in nodules, Ca. *Frankia* alpina AiOr
<figure><img src="image_5.png" /><figcaption>**Figure 4.** RT-qPCR to analyse the expression levels of different syntons. Expression levels of all available [NiFe] hydrogenase syntons were analysed: F. alni (synton-1 and synton-2), three strains representing *Candidatus* Frankia nodulisporulans (synton-2), the two cluster-2 species *Candidatus* Frankia datiscae Dg1 and *Candidatus* Frankia californiensis Dg2 (synton-2), and the cluster-3 species F. soli Hr75.2.1 (synton-1, synton-3, and synton-4). Expression was analysed in nodules and, if the strains were cultivable, in cultures without nitrogen (nitrogen-fixing conditions) and with nitrogen, respectively. Each column represents the average of three biological replicates; error bars show standard error. Bars labelled with the same letter are not statistically different.</figcaption></figure>

(Pozzi et al. 2020); however, the efficiency of uptake hydrogenase in AiOr was insufficient to reassimilate all hydrogen released during nitrogen fixation, suggesting that at least one of the genes of synton-2 had accumulated mutations. In summary, cluster-1c strains still have both synton-1 and synton-2, cluster-2 strains have lost synton-1, while cluster-1 strains that sporulate in nodules lost synton-1 (*Ca. F. nodulisporulans*) or are losing synton-1 (*Ca. F. alpina*), while the erosion of synton-2 is still in progress in both species.

The fact that synton-1 (group 1 h) was lost in species that show genome reduction and reduced saprotrophic potential indicates that while also expressed in nodules when present, synton-1 is not required for the symbiotic life style. On the contrary, the fact that cluster-2 *Frankia* strains, which show a reduced saprotrophic potential have lost synton-1 (group 1 h) and have retained either synton-2 (group 2a; continental lineage) or synton-3 (group 1f; island lineage), indicates that either hydrogenase group is well suited for the symbiotic lifestyle, and it seems to have been a mat-

ter of chance whether synton-2 (group 2a) or synton-3 (group 1f) was retained during the loss of saprotrophic potential. As stated above, Søndergaard et al. (2016) linked both hydrogenase group 1f and 2a to the recycling of H₂ produced during nitrogen fixation, which is consistent with the RE of 1 for nodules of *D. glomerata* (only group 2a present) and the RE of 1 for nodules of *H. rhamnoides* induced by Hr75.2.1 (group 2a and 1f present).

The role of syntons-2 and -3 (groups 2a and 1f) in symbiosis is consistent with the earlier hypothesis, based on the relative expression levels of synton-1 and synton-2/3 genes under different growth conditions, that syntons-2 and 3 (groups 2a and 1f) are more important for symbiosis and synton-1 (group 1 h) is more important for growth in soil (Leul et al. 2007, 2009). However, group 1 h (synton-1) has not been linked to the recycling of H₂ produced by nitrogenase by Søndergaard et al. (2016), but to hydrogenotrophic respiration using O₂ as terminal electron acceptor; the enzyme is supposed to scavenge electrons from atmospheric H₂ to fuel the respiratory chain during carbon-starvation.
The suggestion that the role of synton-1 (group 1 h) in *Frankia* is not linked to nitrogen fixation is consistent with the fact that all strains examined of the nonsymbiotic cluster-4 have lost the *nif* genes, but retained synton-1 (group 1 h). Thus, the data available thus far suggest that hydrogenases groups 1f and 2a are linked to the recycling of H₂ produced by nitrogenase, while hydrogenase group 1 h is not.

Synton-4 encodes a cytosolic bidirectional [NiFe] hydrogenase of group 3b that according to Søndergaard et al. (2016) can couple the oxidation of NADP to the fermentative evolution of H₂. Its pattern of occurrence is interesting: synton-4 was only found in two genomes of nonsymbiotic *Frankia* species (and is defective in one of them), was not found in any genome of cluster-2 *Frankia*, and its presence in genomes from the other symbiotic clusters, 1 and 3, is patchy (Table S2). However, there is one exception: all genomes of *Casuarina*-infective strains (F. casuarinae, cluster 1c) contain synton-4 (data not shown). One feature that distinguishes these strains from all other symbiotic *Frankia* strains is their environment in symbiosis: microaerobic conditions are established in the infected cells of nodules of (Allo-)Casuarina spp., abolishing the need for the differentiation of *Frankia* vesicles as places of nitrogen fixation; here, nitrogen fixation takes place in hyphae (Zhang and Torrey 1985, Berg and McDowell 1987, 1988, Schubert et al. 2013). It therefore seems plausible that group 3b [NiFe] hydrogenase is important for effective carbon metabolism of *Frankia* under microaerobic conditions. However, group 3b [NiFe] hydrogenases have been described as oxygen tolerant (Søndergaard et al. 2016), and another unusual feature of F. casuarinae is its comparatively fast growth on solid medium (Bassi and Benson 2007). In summary, further studies are required to elucidate the function of group 3b [NiFe] hydrogenase in *Frankia*.

## Symbiotic nitrogen fixation and uptake hydrogenase

Most rhizobial strains examined cannot fix nitrogen *ex planta* and do not possess uptake hydrogenase (Annan et al. 2012, Brito et al. 2005, Schubert and Evans 1976). While the reabsorption of the H₂ produced by nitrogenase would save energy, rhizobia in nodules are provided with carbon sources by the host plant, i.e. they live in an energy-rich environment. The low occurrence of rhizobial uptake hydrogenase might indicate that the host plants can afford the energy loss, and/or that the release of hydrogen gas in the rhizosphere might even be advantageous for the host plant with regard to selection of the rhizosphere microbiome. The results of Greening et al. (2016) suggest that H₂ is a widely utilized energy source for microbial growth and survival; thus, H₂ evolution could be important for rhizosphere conditioning. Consistent with this hypothesis, Li et al. (2018) report that hydrogen treatment of rhizosphere soil samples of *Medicago sativa* plots influenced the composition of microbial communities.

However, symbiotic *Frankia* strains can fix nitrogen *ex planta*; therefore, here the selection for maintaining uptake hydrogenase might also depend on its being advantageous during saprotrophic growth. On the other hand, nitrogen fixation does not seem to be a benefit during saprotrophic growth of *Frankia* based on the fact that the nonsymbiotic strains, cluster-4, have lost the capacity for nitrogen fixation. Then why does the vast majority of symbiotic *Frankia* strains display uptake hydrogenase activity, while the vast majority of symbiotic rhizobia does not? It should be noted that the legumes analysed for uptake hydrogenase activity were herbaceous, while actinorhizal plants, with one exception, represent trees or woody shrubs with perennial nodules. This is likely to

affect the need, or lack of it, for continuous rhizosphere conditioning. It is tempting to speculate that for annual plants, the benefits of quick rhizosphere conditioning by hydrogen evolution outweigh the loss of energy, while for perennial plants, rhizosphere conditioning is predominantly based on root exudation and energy preservation is of larger importance. Plant selection for Hup⁺ or Hup⁻ microsymbionts would explain the differences of distribution of uptake hydrogenase in symbiotic rhizobia versus symbiotic *Frankia* strains.

The functionality of [NiFe] hydrogenases is well examined in bacterial species that, in contrast with *Frankia* spp., are user-friendly for biotechnological approaches, *Escherichia coli* and *Cupriavidus necator* (Lamont and Sargent 2017). While the heterologous expression of *Frankia* genes in Gram-negative bacteria is not an option due to the high GC content of *Frankia* genomes and the consequently different codon preference (Kucho et al. 2013), the use of synthetic genes is a promising future option for detailed analyses.

## Conclusions

*Frankia* strains can contain four different [NiFe] hydrogenase synons representing groups 1f, 1 h, 2a, and 3b according to Søndergaard et al. (2016); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1 h, and 2a follows *Frankia* phylogeny while the phylogeny of accessory proteins does not necessarily. Evidence suggests that accessory proteins can be exchanged between different groups of [NiFe] hydrogenases, which would change the selection pressure on them. An analysis of different [NiFe] hydrogenase types in *Actinomycetia* showed that under the most parsimonious assumption, all four types were present in the ancestral *Frankia* strain. Based on Hup activities analysed and the losses of synons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H₂ formed by nitrogenase while group 1 h is not. We postulate that the difference in selection for Hup⁺ microsymbionts between legumes and actinorhizal plants is due to the fact that actinorhizal plants are trees or woody shrubs while the legumes analysed are herbaceous, a difference which is likely to affect the importance of short-term rhizosphere conditioning via release of H₂.

## Author contributions

Katharina Pawlowski (Conceptualization, Formal analysis, Investigation, Project administration, Supervision, Writing – original draft, Writing – review & editing), Daniel Wibberg (Formal analysis, Visualization, Writing – review & editing), Sara Mehrabi (Investigation, Writing – review & editing), Nadia Binte Obaid (Investigation, Supervision, Visualization, Writing – review & editing), András Patyi (Investigation, Writing – review & editing), Fede Berckx (Supervision, Writing – review & editing), Han Nguyen (Investigation, Writing – review & editing), Michelle Hagen (Formal analysis, Visualization, Writing – review & editing), Daniel Lundin (Formal analysis, Supervision, Writing – review & editing), Andreas Brachmann (Investigation, Writing – review & editing), Jochen Blom (Software, Writing – review & editing), Aude Herrera-Belaroussi (Resources, Writing – review & editing), Danis Abrouk (Formal analysis, Writing – review & editing), Petar Pujic (Formal analysis, Investigation, Writing – review & editing), Ann-Sofi Hahlin (Investigation, Writing – review & editing), Jörn Kalinowski (Supervision, Writing – review & editing), Philippe Normand (Conceptualization, Resources, Supervision, Writing – review & editing), and
Anita Sellstedt (Conceptualization, Investigation, Resources, Supervision, Writing – original draft, Writing – review & editing)

## Supplementary data

Supplementary data is available at *FEMSEC Journal* online.

Conflict of interest: None declared.

## Funding

Open access funding provided by Stockholm University. This project was supported by two grants from the Swedish Research Council Vetenskapsradet (VR 2012–03061 and 2019–05540) to K.P. Funding to ASt from FORMAS (2007) and Department of Plant Physiology Emeriti Funds, Umeå University (Sweden) is greatly acknowledged. The bioinformatics support of the BMBF-funded project 'Bielefeld-Giesen Center for Microbial Bioinformatics' (BiGi) and the BMBF grant FKZ 031A533 within the German Network for Bioinformatics Infrastructure (de.NBI) are gratefully acknowledged. The analysis of hydrogenases in Actinomycetia was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) partially funded by the Swedish Research Council through grant agreement number 2018–05973.

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