J Genomics 2022; 10:49-56. doi:10.7150/jgen.74788 This volume

Research Paper

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

Philippe Normand1 Corresponding address, 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.

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.
Citation:
Normand P, Pujic P, Abrouk D, Vemulapally S, Guerra T, Carlos-Shanley C, Hahn D. Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa. J Genomics 2022; 10:49-56. doi:10.7150/jgen.74788. Available from https://www.jgenomics.com/v10p0049.htm

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Abstract

Graphic 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.

Keywords: 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 SurePrepTM 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-tools-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% (CcI3) 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 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

Cluster 1Cluster 2Cluster 3Cluster 4
StrainACN14aTARgP5TCpI1TQA3Ag45/Mut15AgPM24CcI3TBMG5.1TBCU110501TBMG5.12TG2TCjTEAN1pecEUN1fM16386TEuI1cTCn3TDC12
CollectionDSM 45986DSM 45898DSM 44263DSM 45818DSM 100624DSM
46785
DSM 46783DSM 45899DSM 100623DSM 100626DSM 45817DSM 105290
Frankia speciesalnicanadensistorreyicasuarinaecoriariaediscariaeelaeagniirregularissoliasymbioticainefficaxsaprophytica
Genomic G+C content (mol%)72.872.472.472.671.3771.3570.171.072.371.770.971.170.9470.8271.9372.371.871.93
Genome length (nt)749793477302857624758759085364433826672691543362857952637891711758931395379928061539903521893221739435764881578199785926884336
# CDS6,7147,5007,2017,3076,0886,3705,5936,4877,5676,9778,6638,1089,0639,4288,8848,0999,2626,630
# secondary metabolite clusters*273328332938262236353730273329232815
nifH**111111111111110000
shc222222122222222222
hupL222211211111111111
sufD111111111111111111
celA1222022011011110001
glxA111111011011110001
bcsA111011011001111111
gvpJ111000001111111111
sodF111111111110110000
geoA111111111111101100
argG111111111111110000
accA111111111111111000
can222222222221220100
rhbE111111111111110000
lac111111101111110111
phdA111111111111111000
dctA111111101011110000
tgsA111111101111111010
ddnB111111001111111000
mopB112222112221220000
qorB121100001111110000
glbN111211111111110101
# contigs15681531201131811116207139832891396174121
AccessionNC_008278.1OESX01000001JYFN00000000WGS NZ_AJWA.1JALKFT000000000JALKFW000000000CP000249.1JWIO00000000ARDT00000000ARFH00000000FAOZ00000000MAXA00000000.1AAII00000000ADGX00000000MOMC00000000CP002299.1AGJN00000000LANG01000000
Reference(30)(39)(40)(41)this study(30)(38)(42)(43)(44)(45)(30)(46)(47)(46)(30)(46)

* 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 F420H(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” (Posttranslational 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 1 

Phylogenetic tree of complete genomes using Micromonospora lupini (NZ_CAIE00000000.1) as outgroup. Clusters are indicated on the right.

J Genomics Image

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 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.

J Genomics Image

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 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

StrainCluster 1Cluster 2Cluster 3Cluster 4
ACN14aTARgP5TCpI1TQA3Ag45/Mut15AgPM24CcI3TBMG5.1TBCU110501TBMG5.12TG2TCjTEAN1pecEUN1fM16386TEuI1cTCn3TDC12
speciesalnicanadensistorreyicasuarinaecoriariaediscariaeelaeagniirregularissoliasymbioticainefficaxsaprophytica
Class1
D566675645661578065638066657863626467
M241189253236225241207203292259297248292311299258266255
N191526221217123020162821202916201117
O181134181190133140147149200165176200200195177173200149
T325226320326291290232253405336436400405418415405494282
U423850384550485054536656546453525250
V9474861027781607810784117126107110130113153113
J212226212257209212202243207197203219207226243241247232
K565402594646509525369409739577778688739755785809945520
L270254351356308319433289613398398518613468380286409399
C435323455472346347256362492394527451492530555507589332
E523386482534452451335396577461630516577623670661704447
F11182104108969494921079410310110797129116114107
G326274321342289297233249418326372360418428450426488302
H192149186187170184174173187177192181187182188186208163
I432258400460296303191297513412643405513619586624619313
P311243323332307313210293381298408343381387402394427278
Q376226368371304339197320488369565417488550531534569256
R10097041005105981483661968212169691323106412161280134313321508865
S301226315286258278223243323297336328323338341334375284

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.

 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

StrainCluster 1Cluster 2Cluster 3Cluster 4
ACN14aTARgP5TCpI1TQA3Ag45/Mut15AgPM24CcI3TBMG5.1TBCU110501TBMG5.12TG2TCjTEAN1pecEUN1fM16386TEuI1cTCn3TDC12
speciesalnicanadensistorreyicasuarinaecoriariaediscariaeelaeagniirregularissoliasymbioticainefficaxsaprophytica
t1PKS1698891116161369596521
t2PKS131311221121121211
t3PKS111111020111113112
otherKS443335414364462121
t1pks-NRPS101012101000110000
NRPS362266011295354271
terpene535544434435445443
lanthipeptide111303624321431212
bacteriocin212212112222303120
siderophore111111111111110000
lassopeptide111211010010001212
betalactone000000000020001201
thiopeptide000000100010000000
butyrolactone000000000000000000
phosphonate110000000000000000
arylpolyene000000001000000000
nucleoside000000100000000000
ladderane000000010000000000
oligosaccharide000000000000000010
resorcinol000000000000000010
LAP000001000000000000
other022310411511012151
Total/strain273328332938262236353730273329232815

1-tnPKS is type “n” PolyKetide Synthase, NRPS is Non Ribosomal Peptide Synthase, LAP is Linear Azole/azoline-containing Peptide.

 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

StrainCluster 1Cluster 2Cluster 3Cluster 4
ACN14aTARgP5TCpI1TQA3Ag45/Mut15AgPM24CcI3TBMG5.1TBCU110501TBMG5.12TG2TCjTEAN1pecEUN1fM16386TEuI1cTCn3TDC12
speciesalnicanadensistorreyicasuarinaecoriariaediscariaeelaeagniirregularissoliasymbioticainefficaxsaprophytica
Class1
AraC99101666251513171628172022216
ArsR965176654411611991688
AsnC322443323333545553
CRP421144233352353523
DeoR410012002102402221
DtxR111111111111111111
FurC233433225455445445
GntR25191020756821121919202427353011
IclR36494321471261212613117
LuxR10191936101420152291815584018642914
LysR1816122211105514102013131724202213
MarR211913331615152318203125273032333519
MerR81792210101241312151315171619187
TetR92781171276165304711377391261471561561911859
WhiB77875666105696581078

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.

A reverse study for genes present in F. alni ACN14a, Frankia sp. QA3, F. torreyi CpI1 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 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.

Attachment

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.

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Author contact

Corresponding address Corresponding author: Philippe Normand, Université Claude-Bernard Lyon 1, Université de Lyon, UMR 5557 CNRS Ecologie Microbienne, Villeurbanne, France. E-mail: philippe.normandfr.


Received 2022-5-6
Accepted 2022-5-26
Published 2022-6-6