# Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa

Philippe Normand, Petar Pujic, Danis Abrouk, Spandana Vemulapally, Trina

Guerra, Camila Carlos-Shanley, Dittmar Hahn

► To cite this version:

Philippe Normand, Petar Pujic, Danis Abrouk, Spandana Vemulapally, Trina Guerra, et al. Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa. Journal of Genomics, 2022, 10, pp.49-56. 10.7150/jgen.74788. hal-04305218

HAL Id: hal-04305218

https://hal.science/hal-04305218v1

Submitted on 24 Nov 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_2.png" /></figure>

IVYSPRING
INTERNATIONAL PUBLISHER

Journal of Genomics

2022; 10: 49-56. doi: 10.7150/jgen.74788

Research Paper

# Draft Genomes of Nitrogen-fixing *Frankia* Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of *Alnus* Glutinosa

Philippe Normand¹✉, Petar Pujic¹, Danis Abrouk¹, Spandana Vemulapally², Trina Guerra², Camila Carlos-Shanley², Dittmar Hahn²

1. Université Claude-Bernard Lyon 1, Université de Lyon, UMR 5557 CNRS Ecologie Microbienne, Villeurbanne, France.

2. Texas State University, Department of Biology, 601 University Drive, San Marcos, TX 78666, USA.

✉ Corresponding author: Philippe Normand, Université Claude-Bernard Lyon 1, Université de Lyon, UMR 5557 CNRS Ecologie Microbienne, Villeurbanne, France. E-mail: philippe.normand@univ-lyon1.fr.

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2022.05.06; Accepted: 2022.05.26; Published: 2022.06.06

## Abstract

The genomes of two nitrogen-fixing *Frankia* strains, Ag45/Mut15 and AgPM24, isolated from root nodules of *Alnus glutinosa* are described as representatives of a novel candidate species. Phylogenomic and ANI analyses confirmed that both strains are related to cluster 1 frankiae, and that both strains belong to a novel species. At 6.4 - 6.7 Mb, their genomes were smaller than those of other cultivated *Alnus*-infective cluster 1 strains but larger than that of the non-cultivated *Alnus*-infective cluster 1 Sp+ strain AgTrS that was their closest neighbor as assessed by ANI. Comparative genomic analyses identified genes essential for nitrogen-fixation, gene composition as regards COGs, secondary metabolites clusters and transcriptional regulators typical of those from *Alnus*-infective cluster 1 cultivated strains in both genomes. There were 459 genes present in other cultivated *Alnus*-infective strains lost in the two genomes, spread over the whole of the genome, which indicates genome erosion is taking place in these two strains.

Key words: *Frankia*, Actinorhizal symbiosis, genome, nitrogen-fixing frankiae, biosynthetic gene clusters

## Introduction

The genus *Frankia* consists of nitrogen- and non-nitrogen-fixing actinobacteria that can occur in root nodules in symbiosis with a variety of woody plants [1, 2], and in soil [3]. Root nodule formation is host plant-specific, with host infection groups, i.e. the *Alnus* and *Casuarina* host infection group, the Rosaceae/Coriariaceae/Datiscaceae host infection group and the Elaeagnaceae/Rhamnaceae host infection group, respectively, largely represented by *Frankia* clusters 1, 2 and 3. These clusters were established by comparative analyses of ribosomal RNA gene sequences [4] and represent nitrogen-fixing frankiae, while cluster 4 frankiae are typically unable to fix N₂, with one exception, and are often not able to form root nodules [4, 5]. Within clusters, assignment of strains to sub-clusters, OTUs, groups and

genomospecies have been used to further describe diversity within the genus [4, 6-9].

Whole genome sequence analyses resulted in the description of several species within the genus *Frankia*. These analyses include isolates deposited as type strains in culture collections, as well as uncultured *Frankia* populations in root nodules of specific host plants described as candidate species [10]. As summarized by Normand and Fernandez [10], cluster 1 is currently the most extensively described cluster with four species and two candidate species described, while one species and three candidate species are identified in cluster 2. Four species belong to cluster 3, and three species to cluster 4, with genomes of two additional potential species published recently [11]. For cluster 1, comparative
sequence analyses of amplicons of an actinobacteria-
specific insertion in the 23S rRNA genes of frankiae
identified several strains clustering together but
distinct from type strains of cluster 1 [12]. These
strains included strains Ag45/Mut15 and AgPM24
isolated from root nodules of *Alnus glutinosa* from two
lake shores, one in Germany and one in The
Netherlands about 500 km apart, i.e. Grossensee
(53.631031, 10.359319) [13], and Hoogmade
(52.162016, 4.591356) [7], respectively. The goal of this
study was to use whole genome sequence analyses to
assess the viability of our previous amplicon-based
analysis, and thus affirm the potential of these strains
for the description of a new species.

**Materials and Methods**

Sample preparation

*Frankia* strains Ag45/Mut15 and AgPM24 that
were previously identified as members of cluster 1,
representing a subcluster designated as subgroup II
(14) or cluster 1b (12) were from a stock frozen at -20
°C in Defined Propionate Medium (DPM) containing
propionate and NH₄Cl as C and N source,
respectively (15), at 30 °C for two weeks. Cells were
harvested by centrifugation (15,000 × g, 5 min) and
aggregates homogenized by brief sonication (10 s at
20% output in a S-450 sonifier, Branson Ultrasonics,
Danbury, CT) (16). After an additional centrifugation,
DNA was extracted from cell pellets using the
SurePrep™ Soil DNA Isolation Kit (Fisher Scientific,
Houston, TX) (17). DNA concentrations were
measured with a Qubit® 2.0 Fluorometer (Life
Technologies, Carlsbad, USA), and DNA sent to the
Microbial Genomics Sequencing Center, Pittsburgh,
PA, USA for library preparation and sequencing
using the Illumina tagmentation protocol and the
NextSeq Illumina platform (2 × 150 bp) using
standard protocols.

Genome assembly

Sequence reads were filtered and trimmed using
the default settings of fastp (18), and reads with
average %GC<54 were removed using bbduk
(https://jgi.doe.gov/data-and-tools/bbtools/bb-tool
s-user-guide/). Genomes were assembled using
SPAdes 3.13.0 [19] and QUAST to check the quality of
the assembled genomes [20]. Their completeness was
estimated using the lineage workflow (lineage_set)
CheckM v1.0.18 [21] with default values.

Comparative genomic analysis

Average Nucleotide Identity (ANI) comparisons
(22) were performed for all *Frankia* genomes of type
strains of described species and other selected

genomes using the pyani platform with the b (Blast)
setting ([23]; https://pyani.readthedocs.io). The
genomes were compared to *Frankia* genomes of type
strains of isolates on the Mage platform [24] to
compute clusters of orthologous genes or COGs [25],
to identify secondary metabolites clusters through
antiSMASH [26], and identify genes specific to the
new genomes or lost in the two genomes. A
phylogenetic tree was reconstructed using a MASH
distance matrix [27] and the tree computed
dynamically directly in the Mage browser using a
rapid neighbour joining algorithm [28].

Results

Sequence data

CheckM analyses showed that the assembled
genomes for strains Ag45/Mut15 and AgPM24 were
complete with scores of 98.09 for both strains. The
number of contigs was 113 and 181 for Ag45/Mut15
and AgPM24, respectively. The largest contig was
550369 and 296366 for Ag45/Mut15 and AgPM24,
respectively. The strain contamination index
(CheckM) was 1.09 and 0.55 for Ag45/Mut15 and
AgPM24, respectively.

Phylogenetic analysis of *Frankia* spp. Isolates

The two genomes were similar in size with about
6.4 Mb and 6.7 Mb, respectively, which is about 1 Mb
smaller than those of other *Alnus*-infective cluster 1
cultivated strains (Table 1). They were also similar in
DNA G+C% content at 71.35-71.37, which is 1% lower
than values for other *Alnus*-infective cluster 1
cultivated strains. A phylogenetic tree generated from
the MASH matrix with *Frankia* genomes of type
strains revealed that the closest strains to
Ag45/Mut15 and AgPM24 were members of cluster 1
(Figure 1). Average nucleotide identity (ANI) between
strains Ag45/Mut15 and AgPM24 was 97%,
indicating that they belong to a single genospecies
(Figure 2). An ANI of 97% was also obtained with
strain AgTrS, an uncultured *Frankia* population in
root nodules representing Candidatus *Frankia
nodulisporulans*. Since this strain is an obligate
symbiont with a very different physiology, it will not
be considered further in the present study. ANI
values at or below 80% were obtained for both strains
in comparison with *Frankia* genomes of type strains of
all described species (Figure 2). The ANI values with
other cluster 1 genomes ranged from 78% (Ccl3) to
80% (ACN14a), while 75-76% values were obtained
with cluster 2 genomes, and 76-77% with cluster 3 and
4 genomes (Figure 2).
<table><caption>**Table 1.** Basic genome characteristics of *Frankia* strains Ag45/Mut15 and AgPM24 compared to those of type strains of *Frankia* species in clusters 1 to 4</caption><thead><tr><th rowspan="2">Strain</th><th colspan="4">Cluster 1</th><th colspan="4">Cluster 2</th><th colspan="4">Cluster 3</th><th colspan="4">Cluster 4</th></tr><tr><th>ACN14a<sup>T</sup></th><th>ARgP5<sup>T</sup></th><th>CpI1<sup>T</sup></th><th>QA3</th><th>Ag45/<br>Mut15</th><th>AgPM24</th><th>Ccl3<sup>T</sup></th><th>BMG5.1<sup>T</sup></th><th>BCU11<br>0501<sup>T</sup></th><th>BMG5.12<sup>T</sup></th><th>G2<sup>T</sup></th><th>Cj<sup>T</sup></th><th>EAN1<br>pec</th><th>EUN1f</th><th>M16386<sup>T</sup></th><th>EuI1c<sup>T</sup></th><th>Cn3<sup>T</sup></th><th>DC12</th></tr></thead><tbody><tr><td>Collection</td><td>DSM<br>45986</td><td>DSM<br>45898</td><td>DSM<br>44263</td><td></td><td></td><td></td><td>DSM<br>45818</td><td>DSM<br>100624</td><td>DSM<br>46785</td><td>DSM<br>46783</td><td>DSM<br>45899</td><td>DSM<br>100623</td><td></td><td></td><td>DSM<br>100626</td><td>DSM<br>45817</td><td>DSM<br>105290</td><td></td></tr><tr><td>Frankia<br>species</td><td>alni</td><td>canadensis</td><td>torreyi</td><td></td><td></td><td></td><td>casuarin<br>ae</td><td>coriariae</td><td>discariae</td><td>elaega ni</td><td>irregularis</td><td>soli</td><td></td><td></td><td>asym-<br>biotica</td><td>inefficax</td><td>sapro-<br>phytica</td><td></td></tr><tr><td>Genomic<br>G+C content<br>(mol%)</td><td>72.8</td><td>72.4</td><td>72.4</td><td>72.6</td><td>71.37</td><td>71.35</td><td>70.1</td><td>71.0</td><td>72.3</td><td>71.7</td><td>70.9</td><td>71.1</td><td>70.94</td><td>70.82</td><td>71.93</td><td>72.3</td><td>71.8</td><td>71.93</td></tr><tr><td>Genome<br>length (nt)</td><td>7497934</td><td>7730285</td><td>7624758</td><td>7590853</td><td>6443382</td><td>6672691</td><td>5433628</td><td>5795263</td><td>7891711</td><td>7589313</td><td>9537992</td><td>8061539</td><td>9035218</td><td>9322173</td><td>9435764</td><td>8815781</td><td>9978592</td><td>6884336</td></tr><tr><td># CDS</td><td>6,714</td><td>7,500</td><td>7,201</td><td>7,307</td><td>6,088</td><td>6,370</td><td>5,593</td><td>6,487</td><td>7,567</td><td>6,977</td><td>8,663</td><td>8,108</td><td>9,063</td><td>9,428</td><td>8,884</td><td>8,099</td><td>9,262</td><td>6,630</td></tr><tr><td># secondary<br>metabolite<br>clusters*</td><td>27</td><td>33</td><td>28</td><td>33</td><td>29</td><td>38</td><td>26</td><td>22</td><td>36</td><td>35</td><td>37</td><td>30</td><td>27</td><td>33</td><td>29</td><td>23</td><td>28</td><td>15</td></tr><tr><td>nifH**</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>shc</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td></tr><tr><td>hupL</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sufD</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>celA1</td><td>2</td><td>2</td><td>2</td><td>0</td><td>2</td><td>2</td><td>0</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><td>glxA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><td>bcsA</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>gvpJ</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>sodF</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>geoA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>argG</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>accA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>can</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>2</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>rhbE</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>lac</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><td>phdA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>dctA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>tgsA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td></tr><tr><td>ddnB</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>mopB</td><td>1</td><td>1</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>2</td><td>2</td><td>2</td><td>1</td><td>2</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>qorB</td><td>1</td><td>2</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>glbN</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0</td><td>1</td></tr><tr><td># contigs</td><td>NC_00827</td><td>OESX01</td><td>JYFN00</td><td>WGS</td><td>JALKF</td><td>JALKFW</td><td>CP0002</td><td>JWIO0000</td><td>ARDT0</td><td>ARFH000</td><td>FAOZ000</td><td>MAXA0</td><td>AAII00000</td><td>ADGX000</td><td>MOMC00</td><td>CP0022</td><td>AGJN00</td><td>LANG0</td></tr><tr><td>Accession</td><td>8.1</td><td>000001</td><td>000000</td><td>NZ_AJW</td><td>T00000</td><td>000000</td><td>49.1</td><td>0000</td><td>0000000</td><td>00000</td><td>00000</td><td>00000000.</td><td>000</td><td>00000</td><td>0000000</td><td>99.1</td><td>000000</td><td>1000000</td></tr><tr><td>Reference</td><td>(30)</td><td>(39)</td><td>(40)</td><td>A.1</td><td>0000</td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

* indicates the number of clusters identified by AntiSMASH

** indicates the number of hits (>50%) following a BlastP. nif is nitrogenase, shc is squalene hopene cyclase, hup is hydrogenase uptake, suf is sulfur-iron cluster, cel is cellulase, glx is glucose oxidase, bcs is cellulose synthase,

gvp is gas vesicle cluster, sodF is superoxide dismutase iron, geoA is geosmine synthase, arG is arginine, acc is acetate carboxylase, can is carbonic anhydrase, rhb is rhizobactin, lac is laccase, phd is a phytoene desaturase,

dct is a dicarboxylate transporter, tgs is diacylglycerol O-acyltransferase. ddn is F<sub>420</sub>H(2)-dependent quinone nitroreductase, mop is molybdenum transport, qor is quinone oxydoreductase, glb is hemoglobin.

## Analysis of functional genes in *Frankia* spp. isolates

Most genes associated with the *Frankia*-actinorhizal plant symbiosis were recovered in the two genomes, i.e. *nif*, *hup*, *suf*, *shc*, *cel*, *glx*, *bcsA* (Table 1). All genes that are more abundant in symbiotic lineages (clusters 1, 2 and 3) than in non-symbiotic lineages (cluster 4) (*sodF*, *geoA*, *argF*, *accA*, *rhbE*, *dctA*, *phdA*, *tgsA*, *ddnB*) were also recovered in Ag45/Mut15 and AgPM24 (Table 1). Conversely, *gvp* genes that code for gas vesicle proteins and one of the two *hup* clusters that are found in infective cluster 1 strains were not found in the two genomes.

The COG computation showed a set-up for Ag45/Mut15 and AgPM24 characteristic of other *Alnus*-infective cluster 1 strains with a low number of categories "N" (Cell motility) and "O" (Posttransla-

tional modification, protein turnover, chaperones) (Table 2). These results are similar for the antiSMASH computation that showed Ag45/Mut15 and AgPM24 to have a set-up characteristic of other *Alnus*-infective cluster 1 strains with a high number of T1PKS and NRPS (Table 3) as were transcriptional regulators with a low number of GntR, IclR, LysR regulators (Table 4). A phyloprofile of genes present in Ag45/Mut15 and AgPM24 but without homologs at a threshold of 50% identity in AA and present in a synteny group in *F. alni* ACN14a, *Frankia* sp. QA3, *F. torreyi* CpI1 and *F. canadensis* ARgP5 yielded 1068 hits of which 621 were "proteins of unknown function", 37 "HTH-transcriptional regulators", 15 "acyl-CoA metabolism", 5 "sigma factors", 9 "amidohydrolase", 14 "ABC transporter" and 7 "P450 cytochrome" (Table S1). Two were involved in the metabolism of xylose and xylulose.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Phylogenetic tree of complete genomes using *Micromonospora lupini* (NZ_CAIE00000000.1) as outgroup. Clusters are indicated on the right.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Heatmap matrix of Average Nucleotide Identity (ANI) comparisons (in percent) for the *Frankia* genomes of type strains of described species using the pyani platform with the b (Blast) setting (23); https://pyani.readthedocs.io). The two genomes described in the present study are highlighted in grey.</figcaption></figure>
<table><caption>**Table 2.** COG characteristics of *Frankia* strains Ag45/Mut15 and AgPM24 compared to those of type strains of *Frankia* species in clusters 1 to 4</caption><thead><tr><th rowspan="2">Strain</th><th colspan="10">Cluster 1</th><th colspan="3">Cluster 2</th><th colspan="3">Cluster 3</th><th colspan="3">Cluster 4</th></tr><tr><th>ACN14a<sup>T</sup></th><th>ARgP5<sup>T</sup></th><th>CpI1<sup>T</sup></th><th>QA3</th><th>Ag45/<br>Mut15</th><th>AgPM24</th><th>Ccl3<sup>T</sup></th><th>BMG5.1<sup>T</sup></th><th>BCU110501<sup>T</sup></th><th>BMG5.12<sup>T</sup></th><th>G2<sup>T</sup></th><th>Cj<sup>T</sup></th><th>EAN1<br>pec</th><th>EUN1f</th><th>M16386<sup>T</sup></th><th>Eul1c<sup>T</sup></th><th>Cn3<sup>T</sup></th><th>DC12</th></tr><tr><th>species</th><th>alni</th><th>cana-<br>densis</th><th>torreyi</th><th></th><th></th><th></th><th>casuarinae</th><th>coriariae</th><th>discariae</th><th>elaegni</th><th>irregul<br>aris</th><th>soli</th><th></th><th></th><th>asym-<br>biotica</th><th>in-<br>efficax</th><th>sapro-<br>phytica</th><th></th></tr></thead><tbody><tr><th>Class<sup>1</sup></th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>D</th><td>56</td><td>66</td><td>75</td><td>64</td><td>56</td><td>61</td><td>57</td><td>80</td><td>65</td><td>63</td><td>80</td><td>66</td><td>65</td><td>78</td><td>63</td><td>62</td><td>64</td><td>67</td></tr><tr><th>M</th><td>241</td><td>189</td><td>253</td><td>236</td><td>225</td><td>241</td><td>207</td><td>203</td><td>292</td><td>259</td><td>297</td><td>248</td><td>292</td><td>311</td><td>299</td><td>258</td><td>266</td><td>255</td></tr><tr><th>N</th><td>19</td><td>15</td><td>26</td><td>22</td><td>12</td><td>17</td><td>12</td><td>30</td><td>20</td><td>16</td><td>28</td><td>21</td><td>20</td><td>29</td><td>16</td><td>20</td><td>11</td><td>17</td></tr><tr><th>O</th><td>181</td><td>134</td><td>181</td><td>190</td><td>133</td><td>140</td><td>147</td><td>149</td><td>200</td><td>165</td><td>176</td><td>200</td><td>200</td><td>195</td><td>177</td><td>173</td><td>200</td><td>149</td></tr><tr><th>T</th><td>325</td><td>226</td><td>320</td><td>326</td><td>291</td><td>290</td><td>232</td><td>253</td><td>405</td><td>336</td><td>436</td><td>400</td><td>405</td><td>418</td><td>415</td><td>405</td><td>494</td><td>282</td></tr><tr><th>U</th><td>42</td><td>38</td><td>50</td><td>38</td><td>45</td><td>50</td><td>48</td><td>50</td><td>54</td><td>53</td><td>66</td><td>56</td><td>54</td><td>64</td><td>53</td><td>52</td><td>52</td><td>50</td></tr><tr><th>V</th><td>94</td><td>74</td><td>86</td><td>102</td><td>77</td><td>81</td><td>60</td><td>78</td><td>107</td><td>84</td><td>117</td><td>126</td><td>107</td><td>110</td><td>130</td><td>113</td><td>153</td><td>113</td></tr><tr><th>J</th><td>212</td><td>226</td><td>212</td><td>257</td><td>209</td><td>212</td><td>202</td><td>243</td><td>207</td><td>197</td><td>203</td><td>219</td><td>207</td><td>226</td><td>243</td><td>241</td><td>247</td><td>232</td></tr><tr><th>K</th><td>565</td><td>402</td><td>594</td><td>646</td><td>509</td><td>525</td><td>369</td><td>409</td><td>739</td><td>577</td><td>778</td><td>688</td><td>739</td><td>755</td><td>785</td><td>809</td><td>945</td><td>520</td></tr><tr><th>L</th><td>270</td><td>254</td><td>351</td><td>356</td><td>308</td><td>319</td><td>433</td><td>289</td><td>613</td><td>398</td><td>398</td><td>518</td><td>613</td><td>468</td><td>380</td><td>286</td><td>409</td><td>399</td></tr><tr><th>C</th><td>435</td><td>323</td><td>455</td><td>472</td><td>346</td><td>347</td><td>256</td><td>362</td><td>492</td><td>394</td><td>527</td><td>451</td><td>492</td><td>530</td><td>555</td><td>507</td><td>589</td><td>332</td></tr><tr><th>E</th><td>523</td><td>386</td><td>482</td><td>534</td><td>452</td><td>451</td><td>335</td><td>396</td><td>577</td><td>461</td><td>630</td><td>516</td><td>577</td><td>623</td><td>670</td><td>661</td><td>704</td><td>447</td></tr><tr><th>F</th><td>111</td><td>82</td><td>104</td><td>108</td><td>96</td><td>94</td><td>94</td><td>92</td><td>107</td><td>94</td><td>103</td><td>101</td><td>107</td><td>97</td><td>129</td><td>116</td><td>114</td><td>107</td></tr><tr><th>G</th><td>326</td><td>274</td><td>321</td><td>342</td><td>289</td><td>297</td><td>233</td><td>249</td><td>418</td><td>326</td><td>372</td><td>360</td><td>418</td><td>428</td><td>450</td><td>426</td><td>488</td><td>302</td></tr><tr><th>H</th><td>192</td><td>149</td><td>186</td><td>187</td><td>170</td><td>184</td><td>174</td><td>173</td><td>187</td><td>177</td><td>192</td><td>181</td><td>187</td><td>182</td><td>188</td><td>186</td><td>208</td><td>163</td></tr><tr><th>I</th><td>432</td><td>258</td><td>400</td><td>460</td><td>296</td><td>303</td><td>191</td><td>297</td><td>513</td><td>412</td><td>643</td><td>405</td><td>513</td><td>619</td><td>586</td><td>624</td><td>619</td><td>313</td></tr><tr><th>P</th><td>311</td><td>243</td><td>323</td><td>332</td><td>307</td><td>313</td><td>210</td><td>293</td><td>381</td><td>298</td><td>408</td><td>343</td><td>381</td><td>387</td><td>402</td><td>394</td><td>427</td><td>278</td></tr><tr><th>Q</th><td>376</td><td>226</td><td>368</td><td>371</td><td>304</td><td>339</td><td>197</td><td>320</td><td>488</td><td>369</td><td>565</td><td>417</td><td>488</td><td>550</td><td>531</td><td>534</td><td>569</td><td>256</td></tr><tr><th>R</th><td>1009</td><td>704</td><td>1005</td><td>1059</td><td>814</td><td>836</td><td>619</td><td>682</td><td>1216</td><td>969</td><td>1323</td><td>1064</td><td>1216</td><td>1280</td><td>1343</td><td>1332</td><td>1508</td><td>865</td></tr><tr><th>S</th><td>301</td><td>226</td><td>315</td><td>286</td><td>258</td><td>278</td><td>223</td><td>243</td><td>323</td><td>297</td><td>336</td><td>328</td><td>323</td><td>338</td><td>341</td><td>334</td><td>375</td><td>284</td></tr></tbody></table>

<table><caption>**Table 3.** Number of secondary metabolites clusters (antiSMASH) of *Frankia* strains Ag45/Mut15 and AgPM24 compared to those of cultivated type strains of *Frankia* species in clusters 1 to 4</caption><thead><tr><th rowspan="2">Strain</th><th colspan="10">Cluster 1</th><th colspan="3">Cluster 2</th><th colspan="3">Cluster 3</th><th colspan="3">Cluster 4</th></tr><tr><th>ACN14a<sup>T</sup></th><th>ARgP5<sup>T</sup></th><th>CpI1<sup>T</sup></th><th>QA3</th><th>Ag45/<br>Mut15</th><th>AgPM24</th><th>Ccl3<sup>T</sup></th><th>BMG5.1<sup>T</sup></th><th>BCU110501<sup>T</sup></th><th>BMG5.12<sup>T</sup></th><th>G2<sup>T</sup></th><th>Cj<sup>T</sup></th><th>EAN1<br>pec</th><th>EUN1f</th><th>M16386<sup>T</sup></th><th>Eul1c<sup>T</sup></th><th>Cn3<sup>T</sup></th><th>DC12</th></tr><tr><th>species</th><th>alni</th><th>cana-<br>densis</th><th>torreyi</th><th></th><th></th><th></th><th>casua-<br>rinae</th><th>coriariae</th><th>discariae</th><th>elaegni</th><th>irregu<br>laris</th><th>soli</th><th></th><th></th><th>asym-<br>biotica</th><th>in-<br>efficax</th><th>sapro-<br>phytica</th><th></th></tr></thead><tbody><tr><th>t1PKS<sup>1</sup></th><td>6</td><td>9</td><td>8</td><td>8</td><td>9</td><td>11</td><td>1</td><td>6</td><td>16</td><td>13</td><td>6</td><td>9</td><td>5</td><td>9</td><td>6</td><td>5</td><td>2</td><td>1</td></tr><tr><th>t2PKS</th><td>1</td><td>3</td><td>1</td><td>3</td><td>1</td><td>1</td><td>2</td><td>2</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td><td>2</td><td>1</td><td>2</td><td>1</td><td>1</td></tr><tr><th>t3PKS</th><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>2</td><td>0</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>3</td><td>1</td><td>1</td><td>2</td></tr><tr><th>otherKS</th><td>4</td><td>4</td><td>3</td><td>3</td><td>3</td><td>5</td><td>4</td><td>1</td><td>4</td><td>3</td><td>6</td><td>4</td><td>4</td><td>6</td><td>2</td><td>1</td><td>2</td><td>1</td></tr><tr><th>t1pks-NRPS</th><td>1</td><td>0</td><td>1</td><td>0</td><td>1</td><td>2</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>NRPS</th><td>3</td><td>6</td><td>2</td><td>2</td><td>6</td><td>6</td><td>0</td><td>1</td><td>1</td><td>2</td><td>9</td><td>5</td><td>3</td><td>5</td><td>4</td><td>2</td><td>7</td><td>1</td></tr><tr><th>terpene</th><td>5</td><td>3</td><td>5</td><td>5</td><td>4</td><td>4</td><td>4</td><td>3</td><td>4</td><td>4</td><td>3</td><td>5</td><td>4</td><td>4</td><td>5</td><td>4</td><td>4</td><td>3</td></tr><tr><th>lanthipeptide</th><td>1</td><td>1</td><td>1</td><td>3</td><td>0</td><td>3</td><td>6</td><td>2</td><td>4</td><td>3</td><td>2</td><td>1</td><td>4</td><td>3</td><td>1</td><td>2</td><td>1</td><td>2</td></tr><tr><th>bacteriocin</th><td>2</td><td>1</td><td>2</td><td>2</td><td>1</td><td>2</td><td>1</td><td>1</td><td>2</td><td>2</td><td>2</td><td>2</td><td>3</td><td>0</td><td>3</td><td>1</td><td>2</td><td>0</td></tr><tr><th>siderophore</th><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>lassopeptide</th><td>1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>2</td><td>1</td><td>2</td></tr><tr><th>betalactone</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0</td><td>1</td><td>2</td><td>0</td><td>1</td></tr><tr><th>thiopeptide</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>butyrolactone</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>phosphonate</th><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>arylpolyene</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>nucleoside</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>ladderane</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>oligosaccharide</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td></tr><tr><th>resorcinol</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td></tr><tr><th>LAP</th><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><th>other</th><td>0</td><td>2</td><td>2</td><td>3</td><td>1</td><td>0</td><td>4</td><td>1</td><td>1</td><td>5</td><td>1</td><td>1</td><td>0</td><td>1</td><td>2</td><td>1</td><td>5</td><td>1</td></tr><tr><th>Total/strain</th><td>27</td><td>33</td><td>28</td><td>33</td><td>29</td><td>38</td><td>26</td><td>22</td><td>36</td><td>35</td><td>37</td><td>30</td><td>27</td><td>33</td><td>29</td><td>23</td><td>28</td><td>15</td></tr></tbody></table>

<footnote n="1">¹class: D: Cell cycle control, cell division, chromosome partitioning; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; O: Posttranslational modification, protein turnover, chaperones; T: Signal transduction mechanisms; U: Intracellular trafficking, secretion, and vesicular transport; V: Defense mechanisms; J: Translation, ribosomal structure and biogenesis; K: Transcription; L: Replication, recombination and repair; C: Energy production and conversion; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; G: Carbohydrate transport and metabolism; H: Coenzyme transport and metabolism; I: Lipid transport and metabolism; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism; R: General function prediction only; S: Function unknown.</footnote>

<footnote>1-tnPKS is type "n" PolyKetide Synthase, NRPS is Non Ribosomal Peptide Synthase, LAP is Linear Azole/azoline-containing Peptide.</footnote>
<table><caption>**Table 4.** Number of transcriptional regulators of *Frankia* strains Ag45/Mut15 and AgPM24 compared to those of type strains of *Frankia* species in clusters 1 to 4</caption><thead><tr><th rowspan="2">Strain</th><th colspan="5">Cluster 1</th><th colspan="2">Cluster 2</th><th colspan="2">Cluster 3</th><th colspan="4">Cluster 4</th></tr><tr><th>ACN14a<sup>T</sup></th><th>ARgP5<sup>T</sup></th><th>CpI1<sup>T</sup></th><th>QA3</th><th>Ag45/ Mut15</th><th>Ccl3<sup>T</sup></th><th>BMG5.1<sup>T</sup></th><th>BCU110501<sup>T</sup></th><th>BMG5.12<sup>T</sup></th><th>G2<sup>T</sup></th><th>Cj<sup>T</sup></th><th>EAN1-pec</th><th>M16386<sup>T</sup></th><th>Eul1c<sup>T</sup></th><th>Cn3<sup>T</sup></th><th>DC12</th></tr><tr><th>species</th><th>alni</th><th>canade<br>nsis</th><th>torreyi</th><th></th><th></th><th>casua-<br>rinae</th><th>coriariae</th><th>discariae</th><th>elaeagni</th><th>irregularis</th><th>soli</th><th></th><th>asym-<br>biotica</th><th>inefficax</th><th>sapro-<br>phytica</th><th></th></tr></thead><tbody><tr><td><strong>Class<sup>1</sup></strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AraC</td><td>9</td><td>9</td><td>10</td><td>16</td><td>6</td><td>6</td><td>2</td><td>5</td><td>15</td><td>13</td><td>17</td><td>16</td><td>28</td><td>17</td><td>20</td><td>22</td><td>21</td><td>6</td></tr><tr><td>ArsR</td><td>9</td><td>6</td><td>5</td><td>1</td><td>7</td><td>6</td><td>6</td><td>5</td><td>4</td><td>4</td><td>11</td><td>6</td><td>11</td><td>9</td><td>9</td><td>16</td><td>8</td><td>8</td></tr><tr><td>AsnC</td><td>3</td><td>2</td><td>2</td><td>4</td><td>4</td><td>3</td><td>3</td><td>2</td><td>3</td><td>3</td><td>3</td><td>3</td><td>5</td><td>4</td><td>5</td><td>5</td><td>5</td><td>3</td></tr><tr><td>CRP</td><td>4</td><td>2</td><td>1</td><td>1</td><td>4</td><td>4</td><td>2</td><td>3</td><td>3</td><td>3</td><td>5</td><td>2</td><td>3</td><td>5</td><td>3</td><td>5</td><td>2</td><td>3</td></tr><tr><td>DeoR</td><td>4</td><td>1</td><td>0</td><td>0</td><td>1</td><td>2</td><td>0</td><td>0</td><td>2</td><td>1</td><td>0</td><td>2</td><td>4</td><td>0</td><td>2</td><td>2</td><td>2</td><td>1</td></tr><tr><td>DtxR</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>FurC</td><td>2</td><td>3</td><td>3</td><td>4</td><td>3</td><td>3</td><td>2</td><td>2</td><td>5</td><td>4</td><td>5</td><td>5</td><td>4</td><td>4</td><td>5</td><td>4</td><td>4</td><td>5</td></tr><tr><td>GntR</td><td>25</td><td>19</td><td>10</td><td>20</td><td>7</td><td>5</td><td>6</td><td>8</td><td>21</td><td>12</td><td>19</td><td>19</td><td>20</td><td>24</td><td>27</td><td>35</td><td>30</td><td>11</td></tr><tr><td>IclR</td><td>3</td><td>6</td><td>4</td><td>9</td><td>4</td><td>3</td><td>2</td><td>1</td><td>4</td><td>7</td><td>12</td><td>6</td><td>12</td><td>12</td><td>6</td><td>13</td><td>11</td><td>7</td></tr><tr><td>LuxR</td><td>10</td><td>19</td><td>19</td><td>36</td><td>10</td><td>14</td><td>20</td><td>15</td><td>22</td><td>9</td><td>18</td><td>15</td><td>58</td><td>40</td><td>18</td><td>64</td><td>29</td><td>14</td></tr><tr><td>LysR</td><td>18</td><td>16</td><td>12</td><td>22</td><td>11</td><td>10</td><td>5</td><td>5</td><td>14</td><td>10</td><td>20</td><td>13</td><td>13</td><td>17</td><td>24</td><td>20</td><td>22</td><td>13</td></tr><tr><td>MarR</td><td>21</td><td>19</td><td>13</td><td>33</td><td>16</td><td>15</td><td>15</td><td>23</td><td>18</td><td>20</td><td>31</td><td>25</td><td>27</td><td>30</td><td>32</td><td>33</td><td>35</td><td>19</td></tr><tr><td>MerR</td><td>8</td><td>17</td><td>9</td><td>22</td><td>10</td><td>10</td><td>12</td><td>4</td><td>13</td><td>12</td><td>15</td><td>13</td><td>15</td><td>17</td><td>16</td><td>19</td><td>18</td><td>7</td></tr><tr><td>TetR</td><td>92</td><td>78</td><td>117</td><td>127</td><td>61</td><td>65</td><td>30</td><td>47</td><td>113</td><td>77</td><td>39</td><td>126</td><td>147</td><td>156</td><td>156</td><td>191</td><td>18</td><td>59</td></tr><tr><td>WhiB</td><td>7</td><td>7</td><td>8</td><td>7</td><td>5</td><td>6</td><td>6</td><td>6</td><td>10</td><td>5</td><td>6</td><td>9</td><td>6</td><td>5</td><td>8</td><td>10</td><td>7</td><td>8</td></tr></tbody></table>

A reverse study for genes present in *F. alni* ACN14a, *Frankia* sp. QA3, *F. torreyi* Cpl1 and *F. canadensis* ARgP5 but absent in Ag45/Mut15 and AgPM24 yielded 459 hits among which an xanthine dehydrogenase locus, a CRISPR-locus, a acetyl/ propionyl CoA carboxylase locus, an uptake hydrogenase locus, a dicarboxylate transporter, a hup locus, the GVP locus, several transporters (Table S1).

The genes lost in Ag45/Mut15 and AgPM24 have been mapped on the genome of ACN14a and found to be evenly spread over the whole genome (Supplementary Fig. 1).

## Discussion

Phylogenomic and ANI analyses confirmed that strains Ag45/Mut15 and AgPM24 are related to cluster 1 frankiae, and indicate that both strains isolated from *Alnus glutinosa* belong to a novel species. Genome sizes of both strains were about 6.4 Mb and 6.7 Mb, respectively, and thus smaller than genomes of most cluster 1 and some cluster 3 frankiae (7.5 Mb to 7.9 Mb), though genomes of *Frankia casuarinae* (4.9 to 5.6 Mb) and *F. nodulisporulans* (4.9 Mb), as well as *F. coriariae* as cluster 2 representative (5.8 Mb) were even smaller (see [5] for review). Some cluster 3 frankiae were much larger in size (9.0 to 10.4 Mb) [29-31], similar to many cluster 4 frankiae (8.8 to 10.7 Mb) [11]. Frankiae with larger genomes that often result from duplications of genes involved in substrate transfers into central metabolic pathways (e.g. cluster 1, 3 and 4 frankiae), might have an enhanced potential to exploit a large variety of environments [30, 32], compared to frankiae with

smaller genomes due to genome reductions that are associated to reduced saprotrophic potential, while maintaining their symbiotic potential (e.g. *F. casuarinae*, *F. nodulisporulans* and *F. coriariae*).

Both Ag45/Mut15 and AgPM24 belong to a group of strains within cluster 1 that are able to use leaf litter compounds as carbon resource in addition to root exudates commonly used by other strains of clusters 1, 3 and 4 [16, 33]. Together with cluster 3 frankiae, members of this group have been identified as major populations in soils, with absolute numbers depending on the sampling depth, physicochemical conditions and the vegetation [12, 14, 34, 35]. Young stands of both host trees (e.g. natural stands of *A. glutinosa*) [35], and non-host trees (e.g. young plantations of *Betula nigra*) [14] seem to promote members of this group, as do leaf litter amendments to soils, both for introduced and indigenous populations [36]. Thus, this group with strains Ag45/Mut15 and AgPM24 as representatives could be adapted to carbon resources provided by the decomposition of plant material and represent a group of frankiae characteristic of soils in early stages of plant-mediated organic matter accumulation.

Functional genes typically found in nitrogen-fixing frankiae (i.e. clusters 1, 2 and 3) within the *Frankia*-actinorhizal plant symbiosis were recovered in the genomes of strains Ag45/Mut15 and AgPM24. These strains appear to have lost a large number of genes dispensable for saprotrophic life as is the case in Sp+ lineages [37] or in cl2 lineages [32] where one megabase or more relative to the closest neighbor has been lost. This process of gene erosion is slow with

<footnote n="1">¹class: AraC: arabinose regulator; ArsR: arsenic resistance; AsnC: asparagine synthase regulator; CRP: cyclic AMP receptor protein (catabolite repression); DeoR: deoxyribonucleoside synthesis operon regulator; DtxR: diphtheria toxin repressor; FurC: ferric uptake regulator; GntR: gluconate regulator; IclR: isocitrate lyase regulator; LuxR: quorum-sensing luminescence regulator; LysR: lysine regulator; MarR: Multiple antibiotic resistance regulator; MerR: mercury resistance regulator; TetR: Tetracycline repressor; WhiB: regulation of morphological differentiation.</footnote>
seemingly a conserved possibility of growth under
physiologically demanding conditions [38]. The full
extent of diversity with cluster 1 is slowly emerging
with the description of genomes from new lineages
such as the present one. It appears some lineages such
as *F. torreyi* and even more so *F. casuarinae* have been
isolated repeatedly while others such as QA3, *F.
canadensis* and the two lineages Ag45/Mut15 and
AgPM24 described in the present study have been
more rarely isolated. Two related factors have
probably caused this distortion, one is the
evolutionary success over long eras resulting in a
higher abundance in nature and the other is the
physiological ability to grow relatively fast on a large
range of substrates resulting in a higher isolation
success rate. Their genome composition should now
be analyzed in that light of adaptation to contrasted
biotopes.

## Data Summary

Genomes of the strains sequenced in this study
from Dr. Dittmar Hahn culture collection and were
deposited in the National Center for Biotechnology
Information (NCBI), under BioProject Number
PRJNA680372. Whole Genome Sequencing (WGS)
accession numbers are JALKFT000000000 for strain
Ag45/Mut15, and JALKFW000000000 for strain
AgPM24.

A list of other *Frankia* genomes utilized in this
study can be found in Table 1. All sequences were
downloaded from the NCBI Assembly database.

## Supplementary Material

Supplementary figure and table.

https://www.jgenomics.com/v10p0049s1.pdf

## Acknowledgements

The authors are indebted to the Graduate
College (Doctoral Research Support Fellowship to S.
Vemulapally), and the Department of Biology at
Texas State University for financial support.

## Competing Interests

The authors have declared that no competing
interest exists.

## References

1. Benson DR, Dawson JO. Recent advances in the biogeography and genecology of symbiotic *Frankia* and its host plants. *Physiol Plant*. 2007;130: 318-330.

2. Dawson JO. Actinorhizal plants: their use in forestry and agriculture. Outlook Agricul. 1986;15: 202-208.

3. Chaia EE, Wall LG, Huss-Danell K. Life in soil by the actinorhizal root nodule endophyte *Frankia*. A review. *Symbiosis*. 2010; 51: 201-226.

4. Normand P, Orso S, Cournoyer B, Jeannin P, Chapelon C, et al. Molecular phylogeny of the genus *Frankia* and related genera and emendation of the family Frankiaceae. *Int J Syst Bacteriol*. 1996; 46: 1-9.

5. Gtari M, Nououi I, Sarkar I, Ghodhbane-Gtari F, Tisa LS, et al. An update on the taxonomy of the genus *Frankia* Brunchorst, 1886, 174<sup>AL.</sup> Anton Leeuw. 2019;112: 5-21.

6. Hahn D. Polyphasic taxonomy of the genus *Frankia*. In: Pawlowski K., Newton WE. (Eds.) Nitrogen-fixing actinorhizal symbioses. Springer Verlag, Berlin, Germany, 2007, pp. 25-48.

7. Hahn D, Mirza B, Benagli C, Vogel G, Tonolla M. Typing of nitrogen-fixing *Frankia* strains by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. *Syst Appl Microbiol*. 2011; 34: 63-68.

8. Pozzi AC, Bautista-Guerrero HH, Abby SS, Herrera-Belaroussi A, Abrouk D, et al. Robust *Frankia* phylogeny, species delineation and intra species diversity based on Multi-Locus Sequence Analysis (MLSA) and Single-Locus Strain Typing (SLST) adapted to a large sample size. *Syst Appl Microbiol*. 2018; 41: 311-323.

9. Fernandez MP, Meugnier H, Grimont PAD, Bardin R. Deoxyribonucleic acid relatedness among members of the genus *Frankia*. *Int J Syst Bacteriol*. 1989; 39: 424-429.

10. Normand P, Fernandez MP. *Frankia* Brunchorst 1886, 174<sup>AL.</sup> In: Whitman WB, Rainey FA, Kämpfer P, Trujillo ME, DeVos P, Hedlund B, Dedysh S. (Eds.) Bergey's Manual of Systematics of Archaea and Bacteria, 2019. 10.1002/9781118960608.gbm00042.pub2.

11. Carlos-Shanley C, Guerra T, Hahn D. Draft genomes of non-nitrogen-fixing *Frankia* strains. *J Genomics*. 2021; 9: 68-75.

12. Ben Tekaya S, Ganesan AS, Guerra T, Dawson JO, Forstner MRJ, Hahn D. SybrGreen- and TaqMan-based quantitative PCR approaches allow assessment of the abundance and relative distribution of *Frankia* clusters in soils. *Appl Environ Microb*. 2017; 83(5), e02833-16.

13. Hahn D, Starrenburg MJC, Akkermans ADL. Variable compatibility of cloned *Alnus glutinosa* ecotypes against ineffective *Frankia* strains. *Plant Soil*. 1988; 107: 233-243.

14. Samant S, Huo T, Dawson JO, Hahn D. Abundance and relative distribution of *Frankia* host infection groups under actinorhizal *Alnus glutinosa* and non-actinorhizal *Betula nigra* trees. *Microb Ecol*. 2016; 71: 473-481.

15. Meesters TM, van Genesen ST, Akkermans ADL. Growth, acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in *Frankia* strains Cc1.17 and Cp1.2. *Arch Microbiol*. 1985; 143: 137-142.

16. Mirza BS, Welsh A, Hahn D. Saprophytic growth of inoculated *Frankia* sp. in soil microcosms. *FEMS Microbiol Ecol*. 2007; 62: 280-289.

17. Samant S, Sha Q, Iyer A, Dhabekar P, Hahn D. Quantification of *Frankia* in soils using SYBR Green based qPCR. *Syst Appl Microbiol*. 2012; 35: 191-197.

18. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. *Bioinformatics*. 2018; 34: i884-i890.

19. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes De Novo Assembler. *Curr Protoc Bioinformatics*. 2020; 70: e102.

20. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. *Bioinformatics*. 2013; 29: 1072-1075.

21. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. *Genome Res*. 2015; 25: 1043-1055.

22. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, et al. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. *Int J Syst Evol Microbiol*. 2007; 57: 81-91.

23. Pritchard L, Glover RH, Humphris S, Elphinstone JG, Toth IK. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. *Anal Methods-Uk*. 2016; 8: 12-24.

24. Vallenet D, Calteau A, Cruveiller S, Gachet M, Lajus A, et al. MicroScope in 2017: an expanding and evolving integrated resource for community expertise of microbial genomes. *Nucleic Acids Res*. 2017; 45: D517-D528.

25. Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, et al. The COG database: new developments in phylogenetic classification of proteins from complete genomes. *Nucleic Acids Res*. 2001; 29: 22-28.

26. Medema MH, Blin K, Cimermancic P, de Jager V, Zakrzewski P, et al. antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. *Nucleic Acids Res*. 2011; 39: W339-346.

27. Ondov BD, Treangen TJ, Melsted P, Mallonee AB, Bergman NH, et al. Mash: fast genome and metagenome distance estimation using MinHash. *Genome Biol*. 2016; 17: 132.

28. Simonsen M, Mailund T, Pedersen CNS. Rapid Neighbour-Joining. In: Crandall KA, Lagergren J. (Eds.) WABI 2008: Algorithms in Bioinformatics, Springer Verlag, Heidelberg, 2008, pp. 113-122.

29. Pujic P, Bolotin A, Fournier P, Sorokin A, Lapidus A, et al. Genome sequence of the atypical symbiotic *Frankia* R43 strain, a nitrogen-fixing
and hydrogen-producing actinobacterium. Genome Announc. 2015; 3(6): e01387-15.

30. Normand P, Lapierre P, Tisa LS, Gogarten JP, Alloisio N, et al. Genome characteristics of facultatively symbiotic *Frankia* sp strains reflect host range and host plant biogeography. Genome Res. 2007; 17: 7-15.

31. Nouioui I, Ghodhbane-Gtari F, Rhode M, Sangal V, Klenk HP, et al. *Frankia irregularis* sp nov., an actinobacterium unable to nodulate its original host, *Casuarina equisetifolia*, but effectively nodulates members of the actinorhizal Rhamnales. Int J Syst Evol Microbiol. 2018; 68: 2883-2890.

32. Persson T, Battenberg K, Demina IV, Vigil-Stenman T, Heuvel BV, et al. Candidatus *Frankia datiscae* Dg1, the actinobacterial microsymbiont of *Datisca glomerata*, expresses the canonical nod genes nodABC in symbiosis with its host plant. PLoS One. 2015; 10(5): e0127630.

33. Mirza BS, Welsh AK, Hahn D. Growth of *Frankia* strains in leaf litter-amended soil and the rhizosphere of a non-actinorhizal plant. FEMS Microbiol Ecol. 2009; 70: 132-141.

34. Samant SS, Dawson JO, Hahn D. Growth responses of indigenous *Frankia* populations to edaphic factors in actinorhizal rhizospheres. Syst Appl Microbiol. 2015; 38: 501-505.

35. Samant S, Amann RI, Hahn D. Evaluation of the 23S rRNA gene as target for qPCR based quantification of *Frankia* in soils. Syst Appl Microbiol. 2014; 37: 229-234.

36. Nickel A, Pelz O, Hahn D, Saurer M, Siegwolf R, Zeyer J. Effect of inoculation and leaf litter amendment on establishment of nodule-forming *Frankia* populations in soil. Appl Environ Microbiol. 2001; 67: 2603-2609.

37. Bethencourt L, Vautrin F, Taib N, Dubost A, Castro-Garcia L, et al. Draft genome sequences for three unisolated *Alnus*-infective *Frankia* Sp+ strains, AgTrS, AiOr and AvVan, the first sequenced *Frankia* strains able to sporulate *in-planta*. J Genomics. 2019; 7: 50-55.

38. Gtari M, Ghodhbane-Gtari F, Nouioui I, Ktari A, Hezbri K, et al. Cultivating the uncultured: growing the recalcitrant cluster-2 *Frankia* strains. Sci Rep-Uk. 2015; 5: 13112; doi: 13110.11038/srep13112.

39. Normand P, Nouioui I, Pujic P, Fournier P, Dubost A, et al. *Frankia canadensis* sp nov., isolated from root nodules of *Alnus incana* subspecies *rugosa*. Int J Syst Evol Microbiol. 2018; 68: 3001-3011.

40. Oshone R, Hurst SGt, Abebe-Akele F, Simpson S, Morris K, et al. Permanent draft genome sequences for two variants of *Frankia* sp. strain CpI1, the first *Frankia* strain isolated from root nodules of *Comptonia peregrina*. Genome Announc. 2016; 4(1): e01588-15.

41. Sen A, Beauchemin N, Bruce D, Chain P, Chen A, et al. Draft genome sequence of *Frankia* sp. strain QA3, a nitrogen-fixing actinobacterium isolated from the root nodule of *Alnus nitida*. Genome Announc. 2013; 1: e0010313.

42. Wall LG, Beauchemin N, Cantor MN, Chaia E, Chen A, et al. Draft genome sequence of *Frankia* sp. strain BCU110501, a nitrogen-fixing actinobacterium isolated from nodules of *Discaria trinevis*. Genome Announc. 2013; 1(4): e00505-13.

43. Nouioui I, Beauchemin N, Cantor MN, Chen A, Detter JC, et al. Draft genome sequence of *Frankia* sp. strain BMG5.12, a nitrogen-fixing actinobacterium isolated from Tunisian soils. Genome Announc. 2013; 1(4): e00468-13.

44. Nouioui I, Gtari M, Goker M, Ghodhbane-Gtari F, Tisa LS, et al. Draft genome sequence of *Frankia* strain G2, a nitrogen-fixing actinobacterium isolated from *Casuarina equisetifolia* and able to nodulate actinorhizal plants of the order Rhamnales. Genome Announc. 2016; 4(3): e00437-16.

45. Gtari M, Ghodhbane-Gtari F, Nouioui I. *Frankia soli* sp. nov., an actinobacterium isolated from soil beneath *Ceanothus jepsonii*. Int J Syst Evol Microbiol. 2020; 70: 1203-1209.

46. Tisa LS, Oshone R, Sarkar I, Ktari A, Sen A, et al. Genomic approaches toward understanding the actinorhizal symbiosis: an update on the status of the *Frankia* genomes. Symbiosis. 2016; 70: 5-16.

47. Nouioui I, Gueddou A, Ghodhbane-Gtari F, Rhode M, Gtari M, et al. *Frankia asymbiotica* sp nov., a non-infective actinobacterium isolated from *Morella californica* root nodule. Int J Syst Evol Microbiol. 2017; 67: 4897-4901.