Ktari et al. Standards in Genomic Sciences (2017) 12:51 DOI 10.1186/s40793-017-0261-3

EXTENDEDGENOMEREPORT Open Access Permanent draft genome sequence of sp. NRRL B-16219 reveals the presence of canonical nod genes, which are highly homologous to those detected in Candidatus Frankia Dg1 genome Amir Ktari1, Imen Nouioui1, Teal Furnholm2, Erik Swanson2, Faten Ghodhbane-Gtari1, Louis S. Tisa2 and Maher Gtari1*

Abstract Frankia sp. NRRL B-16219 was directly isolated from a soil sample obtained from the rhizosphere of Ceanothus jepsonii growing in the USA. Its host range includes members of Elaeagnaceae species. Phylogenetically, strain NRRL B-16219 is closely related to “Frankia discariae” with a 16S rRNA gene similarity of 99.78%. Because of the lack of genetic tools for Frankia, our understanding of the bacterial signals involved during the plant infection process and the development of actinorhizal root nodules is very limited. Since the first three Frankia genomes were sequenced, additional genome sequences covering more diverse strains have helped provide insight into the depth of the pangenome and attempts to identify bacterial signaling molecules like the rhizobial canonical nod genes. The genome sequence of Frankia sp. strain NRRL B-16219 was generated and assembled into 289 contigs containing 8,032,739 bp with 71.7% GC content. Annotation of the genome identified 6211 protein-coding genes, 561 pseudogenes, 1758 hypothetical proteins and 53 RNA genes including 4 rRNA genes. The NRRL B-16219 draft genome contained genes homologous to the rhizobial common nodulation genes clustered in two areas. The first cluster contains nodACIJH genes whereas the second has nodAB and nodH genes in the upstream region. Phylogenetic analysis shows that Frankia nod genes are more deeply rooted than their sister groups from rhizobia. PCR-sequencing suggested the widespread occurrence of highly homologous nodA and nodB genes in microsymbionts of field collected Ceanothus americanus. Keywords: Frankia, Actinorhizal symbiosis, Plant-microbe interactions, Genome, Canonical nod genes, Ceanothus

Introduction conditions [2]. The molecular mechanism for the estab- The symbiosis resulting from members of the lishment of an actinorhizal nitrogen-fixing Frankia interacting with the roots of 8 dicotyledonous remains elusive [3]. Molecular phylogeny of the Frankia plant families (referred to actinorhizal ) is found genus has consistently identified four main clusters worldwide and contributes to the ability of actinorhizal regardless of the typing locus used [1]. These Frankia pioneer plants to grow in poor and marginally fertile clusters also follow and support the host specificity soils [1]. This symbiotic association has drawn interest groups proposed by Baker [4]. Cluster 1 is divided into because of its higher rate of soil nitrogen input and the sub-cluster 1a including F. alni and relatives that are in- ability of the plants to overcome harsh environmental fective on Alnus and Myricaceae and sub-cluster 1b strains that are infective on Allocasuarina, Casuarina * Correspondence: [email protected] and Myricaceae including F. casuarinae [5]. Cluster 2 1Laboratoire Microorganismes et Biomolécules Actives, Université Tunis El Manar (FST) & Université de Carthage (INSAT), 2092 Tunis, Tunisia contains F. coriariae [6] and uncultured microsymbionts Full list of author information is available at the end of the article of Coriariaceae, Datiscaceae, Dryadoideae and

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 2 of 10

Ceanothus, while cluster 3, associated F. elaeagni [5], “F. photosynthetic [35] bradyrhizobia. The only exceptions discariae” [7] and closely related strains are infective on were found in the two Candidatus Frankia genomes, Colletieae, Elaeagnaceae, Gymnostoma and Myricaceae. which contained the canonical nodABC and sulfotrans- Finally, cluster 4 groups a broad range of non-nitrogen- ferase nodH genes [32, 36]. This contradictory situation fixing and infective strains including F. inefficax species justifies additional sequencing of genomes from culti- [8] together with “F. asymbiotica” [9] and other related vated Frankia strains to gain insight into the depth of strains that are unable to establish a symbiosis with acti- the pangenome pool covered. Here we report the first norhizal plants. As has been established for rhizobial proof of the presence of rhizobial homologous canon- and arbuscular mycorrhizal symbioes, the LysM-RLKs ical nodABCH genes within the draft genome of culti- are also involved in the perception of Frankia signal vated Frankia isolate, strain NRRL B-16219 and molecules by the actinorhizal plant [10, 11]. However, widespread occurrence of nodAB in field collected the bacterial signals triggering this symbiosis remain un- Ceanothus americanus microsymbionts. known. At present, more than 30 Frankia genomes from strains in pure culture have been sequenced and anno- Organism information tated [12–30] and two Candidatus genomes were gener- Classification and features ated from nodule metagenomes [31, 32]. Analysis of the Strain NRRL B-16219 metabolizes short fatty acids, Frankia genomes failed to reveal the presence of com- TCA-cycle intermediates and carbohydrates (Table 1). It mon canonical nodABC genes [33] which also appear to is infective on members of Elaeagnaceae and Morella be missing in several photosynthetic [34] and non- cerifera and produces effective root nodules [4, 37]. In

Table 1 Classification and general features of Frankia sp. strain NRRL B-16219 according to MIGS [45] MIGS ID Property Term Evidence codea Classification Domain TAS [46] Phylum TAS [47] Class Actinobacteria TAS [48] Order Frankiales TAS [49] Family Frankiaceae TAS [50, 51] Genus Frankia TAS [52, 53] Species Frankia sp. IDR Strain NRRL B-16219IDA Gram stain Positive IDA Cell shape Filament-shaped IDA Motility Non-motile NAS Sporulation Sporulating NAS Temperature range 25–35 °C TAS [5] Optimum temperature 28 °C TAS [5] pH range; Optimum pH 6.3 – pH 6.8 NAS Carbon source short fatty acids, TCA-cycle intermediates and carbohydrates IDA MIGS-6 Habitat Soil and Host-associated IDA MIGS-6.3 Salinity Not reported MIGS-22 Oxygen requirement Aerobic NAS MIGS-15 Biotic relationship Free-living and Host plant-associated NAS MIGS-14 Pathogenicity Non-pathogen NAS MIGS-4 Geographic location Soil beneath Ceanothus jepsonii, USA IDA MIGS-5 Sample collection 1982 IDA MIGS-4.1 Latitude Not reported - MIGS-4.2 Longitude Not reported - MIGS-4.4 Altitude Not reported - a Evidence codes – IDA INFERRED FROM DIRECT ASSAY, TAS traceable author statement (i.e., a direct report exists in the literature) NAS non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence) Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 3 of 10

coherence with its host range, strain NRRL B-16219 is Genome sequencing information phylogenetically affiliated to cluster 3, known to effectively Genome project history nodulate members of Elaeagnaceae, Rhamnaceae and Because it is one of the rare strains isolated directly from Myricaceae families. Phylogenetic analysis based on 16S the soil, NRRL B-16219 strain was selected as part of an rRNA gene sequence showed that strain NRRL B-16219 effort to gain insight into the depth of the pangenome was most closely related to type strains of “F. discariae” pool and to identify symbiotic signaling molecules. The DSM 46785T (99.78%) and F. elaeagni (98.26%) (Fig. 1). sequencing project was completed in April 2016 and the Frankia sp. strain NRRL B-16219 shows typical generated data was submitted as draft genome to Frankia morphological structures; branched hyphae, ves- Genbank under BioProject PRJNA318440 and the icles, the site of nitrogenase activity, and multilocular accession number MAXA00000000.1. sporangia containing non-motile spores (Fig. 2). Growth conditions and genomic DNA preparation Extended feature descriptions The studied strain was kindly provided by David Labeda, Frankia Strain NRRL B-16219 represents one of the rare ARS USDA bacterial collection, as NRRL B-16219 strain ID. strains directly isolated from soil on plate medium with- Thestrainwasgrownat28°Cinstationaryculturein1-l out passing through plant trapping assay. The strain was bottles containing DPM medium [5], supplemented with isolated from the rhizosphere of Ceanothus jepsonii [37] 0.5 mM NH4Cl as nitrogen source maintained. Biomass following a complex protocol of soil treatment with phe- from 1 month-old culture was harvested by centrifugation at nol (0.7%), sample fractionation through ultracentrifuga- 9000 x g for 15 min, rinsed several times with sterile distilled tion in sucrose density gradient, and plating on solid water. The mycelial mats were broken by repeated passages DPM without nitrogen source. Strain NRRL B-16219 through syringes with progressively smaller diameters (21 g developed unpigmented white colonies after 4 weeks to 27 g). Genomic DNA extraction was performed using growth on DPM medium at 28 °C without shaking. The Plant DNeasy kits (Qiagen, Hilden, Germany) following the strain was phenotyped using GENIII microplates in an recommendation of the manufacturer. Prior to genome se- Omnilog device (BIOLOG Inc., Haywood, USA) as pre- quencing, the quality of the isolated DNA was checked by viously described [5]. It was able to metabolize acetic using the prepared DNA as template for PCR and partial se- acid, citric acid, D-cellobiose, dextrin, D-fructose, D- quences of several housekeeping genes and the 16S rRNA mannitol, D-mannose, fructose-6-phosphate, fusidic gene were generated and analyzed [16]. acid, glucose-6-phosphate, D and L malic acid, p-hy- droxy-phenylacetic acid, propionic acid and D-serine and to grow in presence of 1% sodium lactate and up to Genome sequencing and assembly 1% NaCl. Growth occurred between pH 5.0–6.8. The Sequencing of the draft genome of Frankia sp. NRRL B- strain showed tolerant only to rifamycin. 16219 was performed at the Hubbard Center for

100/100 Frankia asymbiotica DSM 100626T (KY744730) -/99 Frankia inefficax DSM 45817T (KX695197) -/100 92/100 Frankia sp. NRRL B-16219 Frankia discarae DSM 46785T (KX825919) Frankia elaeagni DSM 46783T (KX674377) -/63 Frankia alni DSM 45986T (KX674376) 96/100 Frankia casuarinae DSM 45818T (KX674375) 99/100 Frankia coriariae DSM 100624T (KY066449) Candidatus Frankia datiscae Dg1 (CP002801) Acidothermus cellulolyticus 11BT (CP000481) Jatrophihabitans endophyticus DSM 45627T (JQ346802) Motilibacter peucedani DSM 45328T (FM998003) 87/96 Modestobacter multiseptatus DSM 44406T (Y18646) 100/100 Blastococcus aggregatus DSM 4725T (L40614) 64/- Geodermatophilus obscurus DSM 43160T (CP001867) Nakamurella multipartita DSM 44233T (CP00173) 92/100 Cryptosporangium arvum DSM 44712T (D85465) Fodinicola feengrottensis DSM 19247T (EF490376) Sporichthya polymorpha DSM 43042T (AB025317)

0.01 Fig. 1 Maximum likelihood (ML) phylogenetic tree based on the 16S rRNA gene sequences (1400 nt), showing the relationships between Frankia NRRL B-16219 and Frankia species. The ML tree was inferred using the GTR + GAMMA model and rooted by midpoint-rooting; the branches are scaled in terms of the expected number of substitutions per site. The numbers above the branches are support values when larger than 60% from ML (left) and MP (right) bootstrapping Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 4 of 10

Creek, CA, USA, using similarity search tools. This whole-genome shotgun sequence has been deposited at DDBJ/EMBL/GenBank under the accession number MAXA00000000.1. The version described in this paper is the first version, MAXA00000000.1. A summary of the project information is shown in Table 2.

Genome properties The draft genome of Frankia NRRL B-16219 con- sisted of 289 DNA contigs that correspond to esti- mated genome size of 8,032,739 bp and a GC content of 71.7%. The draft genome contained 6859 total genes, including 6211 protein-encoding genes Fig. 2 Scanning electron micrograph of strain NRRL B-16219 after (90.55%), 561 pseudo genes (8.17%) and 53 RNAs growth for 4 weeks in liquid DPM medium at 28 °C showing hyphae (0.76%) (Table 3). Classification of genes into the (h), vesicles (v) and sporangia (s) COG functional categories is shown in Table 4.

Insights from the genome sequence Genome Studies (University of New Hampshire, Dur- Comparison of genomes from Frankia sp. NRRL B-16219 ham, NH) using Illumina technology [38]. A standard and other Frankia species Illumina shotgun library was constructed and sequenced The Frankia sp. NRRL B-16219 genome was compared using the Illumina HiSeq2500 platform with pair-end to all of the Frankia genomes available at NCBI genome reads of 2 × 250 bp. The Illumina sequence data were database including seven Frankia species including F. trimmed by Trimmonatic version 0.32 [39], and assem- alni, F. casuarinae, F. elaeagni, F. coriariae, “F. discar- bled using Spades version 3.5 [40], and ALLPaths-LG iae”, F. inefficax,and“F. asymbiotica”, two Candidatus version r52488 [41]. Frankia and other Frankia sp. strains. As shown for other closely related strains from cluster 3, strain NRRL Genome annotation B-16219 has one of the largest genome sizes The genome was annotated via the NCBI Prokaryotic (8,032,739 bp) with a high GC content of 71.72%. Genes Genome Annotation Pipeline. Additionally nod gene shown or suggested to be involved in the actinorhizal prediction analysis was done within the Integrated symbiosis were detected. Nitrogenase genes were orga- Microbial Genomes-Expert Review system developed by nized into one operon: nifH-D-K-E-N-X-orf1-orf2-W-Z- the Joint Genome Institute, Walnut Creek, CA, USA B-U and a non-linked nifV gene. Genes encoding the [42] developed by the Joint Genome Institute, Walnut hydrogenase subunits were clustered into two operons.

Table 2 Project information MIGS ID Property Term MIGS 31 Finishing quality Draft genome MIGS-28 Libraries used Illumina Standard library MIGS 29 Sequencing platforms Illumina HiSeq2500 platform MIGS 31.2 Fold coverage 120.5× MIGS 30 Assemblers Spades version 3.5, ALLPaths-LG version r52488 MIGS 32 Gene calling method GeneMarkS+ v3.3 Locus Tag BBK14_RS02460 Genbank ID MAXA00000000.1 Genbank Date of Release October 30, 2016 GOLD ID Gp0153653 BIOPROJECT PRJNA224116 MIGS 13 Source Material Identifier NRRL B-16219 Project relevance Agricultural Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 5 of 10

Table 3 Genome statistics Genes for two different types of truncated hemoglobins, Attribute Value % of Totala HbN and HbO, were also present. Genome size (bp) 8,032,739 100.0 DNA coding (bp) 6,603,166 82.20 Nodulation pathway In rhizobia, the common canonical nodABC genes DNA G + C (bp) 5,760,840 71.72 playing a key role in triggering root nodule formation DNA Contigs289100 .0 in Legumes. These signals are secreted as a reply to Total genes 6859 100.0 host-plant flavonoids perceived by the compatible Protein coding genes 6, 211 91.01 rhizobial strains [43]. The Nod factors perceived by RNA genes 53 0.77 host plant through the LysM-RLKs, and the resulting Pseudo genesb 561 8.18 signal transduction cascade triggers a bacterial invasion of root cortical cells and the genesis of Genes in internal clusters - - functional nodules. Despite the presence of these Genes with function prediction 5046 73.60 LysM-RLKs in the actinorhizal plants [11], none of Genes assigned to COGs 3609 52.64 the Frankia genomes from cultivated strains Genes with Pfam domains 4735 69.06 contained any homologous nod genes [33], but they Genes with signal peptides 176 2.57 arepresentinthetwoCandidatus Frankia genomes Genes with transmembrane helices 296 4.32 [32, 36]. Six nod-like genes were detected in the NRRL B-16219 draft genome (Additional file 1: Table CRISPR repeats 2 - S1) organized into two regions (Fig. 3). The first aThe total is based on either the size of the genome in base pairs or the total genes in the annotated genome cluster contained genes encoding the nodA1, nodC, bPseudo genes may also be counted as protein coding or RNA genes, so is not ABC-2 type transport system ATP-binding protein additive under total gene count (nodJ), ABC-2 transporter efflux protein, DrrB family

Table 4 Number of genes associated with the general COG functional categories Code Value % agea Description J 178 4.27 Translation, ribosomal structure and biogenesis A 1 0.02 RNA processing and modification K 408 9.79 Transcription L 109 2.62 Replication, recombination and repair B 1 0.02 Chromatin structure and dynamics D 32 0.77 Cell cycle control, cell division, chromosome partitioning V 135 3.24 Defense mechanisms T 249 5.98 Signal transduction mechanisms M 173 4.15 Cell wall/membrane biogenesis N 21 0.5 Cell motility U 30 0.72 Intracellular trafficking, secretion, and vesicular transport O 140 3.36 Posttranslational modification, protein turnover, chaperones C 250 6 Energy production and conversion G 207 4.97 Carbohydrate transport and metabolism E 297 7.13 Amino acid transport and metabolism F 94 2.26 Nucleotide transport and metabolism H 262 6.29 Coenzyme transport and metabolism I 351 8.42 Lipid transport and metabolism P 210 5.04 Inorganic ion transport and metabolism Q 256 6.14 Secondary metabolites biosynthesis, transport and catabolism R 508 12.19 General function prediction only S 178 4.27 Function unknown - 3247 47.36 Not in COGs aThe total is based on the total number of protein-coding genes in the genome Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 6 of 10

Fig. 3 Organization of nod genes in Frankia NRRL B-16219 and Candidatus Frankia datiscae Dg1 genomes. Sizes, localization and orientation of the genes are displayed proportionally. These genes are not detectable in any other Frankia genome except Candidatus Frankia Dg2

(nodI) and nodH. The second cluster contained Dg1 and NRRL B-16219, the primer set (forward nodA, nodBandanodH genes. Amino acid sequence primer nodAF 5′-AGCGCGACCCGAGCTCAGGATA similarities between Frankia sp. strain NRRL B-16219 ATCG-3′ and reverse nodBF (5′-CGATCCCACCCGG NodA, B, C, and H predicted proteins ranged from ATGGAGCTGC-3′) was designed in this study. The 86 to 93% and 57–67% with the uncultured Frankia sequenced PCR-products were translated into amino (Dg1 and Dg2) and (α-andβ-) rhizobia, respectively acid sequences to permit the detection of the 23 aa se- (Additional file 2: Table S2). Further phylogenetic quence at the beginning of the 193 aa of the NodA, the analysis (Fig. 4) showed that the Frankia Nod pro- intergenic region (160 nucleotides) and 41 aa at the end teins were positioned at the root of both the α-and of the 230aa of the NodB. Both sequences showed β-rhizobial NodABC proteins as previously reported 100% sequence similarities to their respective homolo- [4, 8]. They were most closely related to plant gous region in NodA (23/193aa) and NodB (41/230aa) nodulating Betaproteobacteria of Burkholderia and protein sequences for Candidatus Frankia Dg1. Paraburkholderia genera. The GC content of Frankia Regardless of their affiliation to cluster 2 or to cluster 3 nod genes ranged from 57.9% for nodA to 66.37% for (Fig. 5), all of the analyzed C. americanus microsym- nodB which is quite similar to that of some rhizobial bionts contained the nodAB genes. In contrast, A. gluti- species including Methylobacterium and Burkoldaria. nosa, C. glauca, E. umbellata and E. angustifolia For both Frankia and rhizobia, GC% of the nod genes microsymbionts failed to amplify the expected PCR was lower than that of total genome sequences. product. This result is in congruence with previous reports claiming that no homologous nod genes are re- Field collected microsymbionts of Ceanothus americanus trievable in sequenced genomes from strains isolated contain nod genes from these actinorhizal plant species [33]. Root nodules from Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia growing in Tunisia and Conclusions Ceanothus americanus and Elaeagnus umbellata grow- We report here the genome sequence of a Frankia strain ing in Durham New Hampshire, USA, were collected. directly isolated from soil rhizosphere. The generated draft The nodA-nodBregionfromC. americanus nodules genome was assembled into 289 contigs corresponding to was PCR-amplified and sequenced. Following the 8,032,739 bp, which falls within the size range of Frankia alignments of the nodAandnodB gene sequences of cluster 3 [33]. Bacterial factors triggering actinorhizal Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 7 of 10

100 NodC Ensifer sp. (WP_018240595) 57.98% (62.3%) 98 NodA Sinorhizobium sp. (WP_018097535) 58.71% ( 62.7%) 100 NodC Ensifer sp. (CAH04369) 58.05% (62.3%) 100 A 57 NodA Sinorhizobium americanum (WP_037390235) 58.54 % (63.0%) C NodC Sinorhizobium meliloti (WP_018097533) 57.98% (62.16%) NodA Microvirga lupini (WP_036353104) 57.36 % (61.6%) 83 NodC Rhizobium undicola (WP_027487437) 55.99% (59.3%) NodA Ensifer sp. (WP_026187645) 59.56% (62.0%) NodC Rhizobium tropici (WP_004125966) 56.14% (61.4%) 99 NodA Mesorhizobium sp. (WP_023678262 ) 59.59% (62.4%) 100 NodC Rhizobium leucaenae (CAA67139) 56.07% (59.4%) 100 NodA Mesorhizobium sp. (WP_023802045) 60.77% (63.1%) NodC Mesorhizobium plurifarium (AFJ42533) 57.92% (63.6%) NodC Mesorhizobium loti (WP_059187590) 56.77% (62.4) 85 NodA Rhizobium gallicum (WP_040114182) 55.49% (59.63% 100 100 100 NodA Rhizobium gallicum ( WP_074070744) 55.57% (60.8%) 99 NodC Mesorhizobium plurifarium (WP_041003406) 56.99% (63.8%) NodA Microvirga vignae (WP_047192745) 59.20% (61.1%) 100 NodC Ensifer sp. (WP_071019928) 57.44% (62.2%) NodA Burkholderia sp. ( WP_026226298) 57.50% (62.9%) NodC Sinorhizobium arboris (WP_028002360) 57.15% (62.0%) 81 100 NodA Paraburkholderia dilworthii (WP_027803811) 57.34% (61.8%) 69 NodC Bradyrhizobium sp. (WP_024510037) 57.98% (62.0%) NodC Bradyrhizobium sp. (WP_065745487) 55.13% (62.3%) NodA Bradyrhizobium sp. (CDY73100) 60.50% (63.8%) 98 100 NodA Bradyrhizobium elkanii (CCC58424) 60.97% (63.7%) 100 NodC Bradyrhizobium elkanii (WP_028351282) 55.13% (63.6%) NodA Paraburkholderia terricola (WP_073430694) 59.84% (63.6%) NodC Paraburkholderia terricola (WP_073430696) 63.34% (63.6%) NodA Frankia sp. NRRL B-16219 (WP_071059923) 57.90% (71.7%) NodC Streptomyces bottropensis (WP_005476197) 68.32% (71.1%) 99 NodA Frankia symbiont of Datisca glomerata (WP_013873450) 58.41% (70.0%) NodC Frankia sp. NRRL B-16219 (WP_071060092) 64.24% (71.7%) 100 100 99 NodA Frankia sp. Dg2 (SBW22889) 59.10% (68.0%) 100 NodC Frankia symbiont of Datisca glomerata (AEH10397) 64.53% (68.0%)

0.05 0.05

54 NodH Methylobacterium nodulans (CAN84687) 53.60% (68.9%) B D NodH Bradyrhizobium sp. (WP_027527032) 55.78% (62.6%) 97 NodB Ensifer sp. (WP_026621871) 58.33% (61..8%) 56 NodH Bradyrhizobium elkanii (WP_028168864) 55.87% (63.8%) 100 NodB Rhizobium sp. (AJW76244) 57.39% (60.7%) 84 NodH Microvirga lotononidis (WP_009489132) 57.56% (63.1) NodB Sinorhizobium arboris (WP_028002359) 60.45% (62.0%) NodH Microvirga lupini (WP_036348737) 57.93% (61.6%) NodB Mesorhizobium sp (WP_066995485) 58.48% (63.3%) NodH Sinorhizobium medicae (WP_011970900) 52.95% (61.2%) NodB Microvirga vignae (WP_047192746) 59.84% (61.1%) NodH Sinorhizobium americanum (WP_037390226) 56.82% (63.0%) NodH Rhizobium gallicum (WP_040114228) 56.09% (59.63%) NodB Microvirga lotononidis (WP_009489136) 59.84% (63.1%) 80 NodB Mesorhizobium sp. (WP_027155850) 58.80% (63.2%) 99 NodH Rhizobium etli (AGS25430) 54.86% (60.82%) 63 NodB Ensifer sp. (WP_069457972) 55.55% (62.2%) NodH Mesorhizobium ciceri (WP_029356619) 55.29% (62.57%) 100 NodB Rhizobium sp (WP_022557358) 55.70% (59.11%) NodH Mesorhizobium plurifarium (WP_052457887) 55.47% (63.8%) 77 NodB Paraburkholderia tuberum (CAC42488) 60.75% (62.8%) NodH Ensifer sp. (WP_026621876) 54.98% (61.8%) 53 NodB Methylobacterium nodulans (CAN84683) 57.12% (68.9%) 83 NodH Ensifer sp. (WP_071019938) 54.31% (62.2) 92 NodB Bradyrhizobium sp. (WP_061851321) 59.40% (63.6%) NodH Methylobacterium sp. (WP_051086545) 50.73 % (71.2%) 100 NodB Bradyrhizobium stylosanthis (WP_063690835) 58.73% (64.6%) NodH Cupriavidus taiwanensis (CAP64014) 52.74% (66.98%) NodB Burkholderia sp. (SIT52076) 54.89% (63.1%) NodH Burkholderia sp. (WP_028362702) 53.84% (63.6%) 83 dNodB Paraburkholderia phymatum (WP_012406750) 54.93% (63.0%) NodH H Burkholderia sp. (SIT52072) 51.78% (63.5%) 100 90 NodH Paraburkholderia phenoliruptrix (WP_015004746) 52.29% (63.0%) 100 NodB Burkholderia sp. (WP_062827050) 55.22% (63.35%) 100 NodB Frankia symbiont of Datisca glomerata (AEH10396 65.22%) 100 NodH Paraburkholderia diazotrophica (SEK09433) 52.32% (63.0%) NodB Frankia sp. NRRL B-16219 (WP_071059924) 66.37% (71.7%) NodH Frankia sp. Dg2 (SBW22905 ) 61.04% (68.0%) NodB Frankia sp. Dg2 (SBW22890) (68.0%) Nod H Frankia sp. Dg2 (SBW22887) 60.64% (68.0%) 100 100 99 NodB Frankia symbiont of Datisca glomerata (AEH09514) 65.22% (68.0%) 68 NifH Frankia sp. NRRL B-16219 (WP_071060089) 62.33% (71.1%) 100

0.1 0.05 Fig. 4 Maximum likelihood phylogeny based on amino acids of nodA(a), nodB(b), nodC(c) and nodH(d). GC-content is provided for nod genes and for genomes (in parenthesis). Bootstrap and probability values larger than 50% are only shown

99 FE57 (AF156743) 57 Microsymbiont in C. americanus nodules 98 NRRL B-16219 (MAXA01000000) 98 Cluster 3 Frankia discariae BCU110501T (ARDT00000000) 95 Frankia elaeagni BMG5.12T (ARFH00000000) Frankia alni ACN14aT (CT573213) Cluster 1 59 94 Frankia casuarinae CcI3T (CP000249) 100 Candidatus Frankia datiscae Dg1 (CP002801) Frankia coriariae BMG5.1T (JWIO00000000) Cluster 2 100 Dg2 (FLUV01002029) FE2 (AF156762) 92 100 Microsymbiont in C. americanus nodules Frankia inefficax EuI1cT (CP002299) Cluster 4

0.02 Fig. 5 Neighbor-Joining phylogenetic tree based on glnA gene sequences. Bootstrap and probability values larger than 50% are only shown. Marked in bold are Frankia strains or microsymbionts with nod genes as present in their genomes or detected by PCR-sequencing analysis Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 8 of 10

symbiosis remain enigmatic since many sequenced Fran- and nodA-B genes. MG drafted the manuscript. All authors read and approved kia genomes have revealed the absence of universal nod- the final manuscript. factors. It was hypothesized that most Frankia strains use Competing interests a novel nod-independent pathway for the infection The authors declare that they have no competing interests. process of actinorhizal plants. In contrast, two Candidatus Frankia Dg1 and Dg2 genomes contain canonical nod Publisher’sNote genes [32, 36]. Here we provide the first proof for the Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. presence of nod genes in the genome of a cultivated Frankia strain. In addition, a PCR-sequencing approach Author details 1 suggested that nod genes are only widespread in C. ameri- Laboratoire Microorganismes et Biomolécules Actives, Université Tunis El Manar (FST) & Université de Carthage (INSAT), 2092 Tunis, Tunisia. canus microsymbionts. This situation is similar to legume 2Department of Molecular, Cellular, and Biomedical Sciences, University of symbionts where two nodulation pathways are described: New Hampshire, 289 Rudman Hall, 46 college Road, Durham, NH the well-studied nod-dependent and an alternative nod- 03824-2617, USA. independent pathway. The majority of rhizobia use the Received: 22 February 2017 Accepted: 22 August 2017 nod-dependent pathway, while some photosynthetic [34] and non-photosynthetic [35] bradyrhizobia use the References alternative nod-independent pathway. Moreover, some 1. Gtari M, Tisa LS, Normand P. Diversity of Frankia strains, actinobacterial rhizobia use both pathways and the use of the nod- symbionts of actinorhizal plants. In: Symbiotic Endophytes. Springer Berlin independent pathway seems to be highly dependent on Heidelberg; 2013. p. 123–48. 2. Dommergues YR. by trees in relation to soil nitrogen host species rather than the presence or absence of nod economy. Fertil Res. 1995;4:215–30. genes in a given bradyrhizobial genome [44]. For Frankia, 3. Ktari A, Gueddou A, Nouioui I, Miotello G, Sarkar I, Ghodhbane-Gtari F, Sen almost all host plants are infected through the nod- A, Armengaud J, Gtari M. Host Plant Compatibility Shapes the Proteogenome of Frankia coriariae. Front Microbiol. 2017;8:720. independent pathway, while the nod-dependent process 4. Baker DD. Relationships among pure cultured strains of Frankia based on may only be present in unstudied actinorhizal species such host specificity. Physiol Plant. 1987;70:245–8. as members of the genus Ceanothus. 5. Nouioui I, Ghodhbane-Gtari F, del Carmen M-CM, Göker M, Meier-Kolthoff JP, Schumann P, Rohde M, Goodfellow M, Fernandez MP, Normand P, Tisa LS, Klenk H-P, Gtari M. Proposal of a type strain for Frankia alni (Woronin Additional files 1866) Von Tubeuf 1895, emended description of Frankia alni, and recognition of Frankia casuarinae sp. nov. and Frankia elaeagni sp. nov. Int J Syst Evol Microbiol. 2016;66:5201–10. Additional file 1: Table S1. Localizations and DNA coordinates for nod 6. Nouioui I, Ghodhbane-Gtari F, Rohde M, Klenk HP, Gtari M. Frankia coriariae genes in NRRL B16219 and Dg1 genomes. (DOCX 12 kb) sp. nov., an infective and effective microsymbiont isolated from Coriaria Additional file 2: Table S2. Percent similarities based on amino acid japonica. Int J Syst Evol Microbiol. 2017;67(5):1266–70. sequence for NodA, B, C and H between Frankia sp. NRRL B-16219, 7. Nouioui I, Del Carmen M-CM, Ghodhbane-Gtari F, Rohde M, Tisa LS, Klenk Candidatus Frankia Dg1 and Dg2, and other rhizobial strains. HP, Gtari M. Frankia discariae sp. nov.: an infective and effective (XLSX 308 kb) microsymbiont isolated from the root nodule of Discaria trinervis. Arch Microbiol. 2017;199(5):641–7. 8. Nouioui I, Ghodhbane-Gtari F, Del Carmen M-CM, Rohde M, Tisa LS, Gtari M, Abbreviations Klenk HP. Frankia inefficax sp. nov., an actinobacterial endophyte inducing DPM: Defined Propionate Medium; glnA: Glutamine synthetase; LysM- ineffective, non nitrogen-fixing, root nodules on its actinorhizal host plants. RLKs: LysM-receptor-like kinases; nifH: Nitrogenase reductase; nodA: Beta-1,4- Antonie Van Leeuwenhoek. 2017;110(3):313–20. N- acetylglucosamine oligosaccharide N-acyltransferase; nodA1: Beta-1,4-N- 9. Nouioui I, Gueddou A, Ghodhbane-Gtari F, Rhode M, Gtari M, Klenk HP. acetylglucosaminyltransferase; nodB: Beta-1,4-N-acettylglucosamine Frankia asymbiotica sp. nov., a non infective actinobacterium isolated from oligosaccharide deacylase; nodC: Beta-1,4-N-acetylglucosaminyltransferase; Morella californica root nodule. Int J Syst Evol Microbiol. 2017. doi:10.1099/ nodH: Beta-1,4-N-acetylglucosamine oligosaccharide 6-O-sulfotransferase ijsem.0.002153. 10. Oldroyd GE. Speak, friend, and enter: signalling systems that promote Acknowledgements beneficial symbiotic associations in plants. Nat Rev Microbiol. 2013;11(4): This work was supported by the Laboratoire Microorganismes & 252–63. Biomolécules Actives, Université Tunis El-Manar, Tunisia (grant LR03ES03) 11. Svistoonoff S, Benabdoun FM, Nambiar-Veetil M, Imanishi L, Vaissayre V, (MG), and the USDA National Institute of Food and Agriculture Hatch 022821 Cesari S, Diagne N, Hocher V, de Billy F, Bonneau J, et al. The independent (LST). Sequencing was performed on an Illumina HiSeq2500 purchased with acquisition of plant root nitrogen-fixing symbiosis in Fabids recruited the an NSF MRI Grant: DBI-1229361 to WK Thomas. The authors would like to same genetic pathway for nodule organogenesis. PLoS One. 2013;8(5): thank Dr. David P. Labeda curator at Agricultural Research Service Culture e64515. Collection for providing Frankia strain and Dr. Manfred Rhode (HZI-Helmholtz 12. D’Angelo T, Oshone R, Abebe-Akele F, Simpson S, Morris K, Thomas WK, Tisa Centre for Infection Research, Germany) for performing scanning electron LS. Permanent Draft Genome Sequence of Frankia sp. Strain BR, a Nitrogen- micrograph for the studied strain. Fixing Actinobacterium Isolated from the Root Nodules of Casuarina equisetifolia. Genome Announc. 2016;4(5). Authors’ contributions 13. D’Angelo T, Oshone R, Abebe-Akele F, Simpson S, Morris K, Thomas WK, Tisa MG conceived and designed the work. LST and ES performed the complete LS. Permanent Draft Genome Sequence for Frankia sp. Strain EI5c, a Single- genome production, including genome assembly and GenBank submission. AK Spore Isolate of a Nitrogen-Fixing Actinobacterium, Isolated from the Root prepared the DNA isolation and the cultivation of Frankia strain NRRL B-16219. Nodules of Elaeagnus angustifolia. Genome Announc. 2016;4(4). TF and MG sampled C. ameracanus and E. umbellata nodules. AK, IN and FGG 14. Ghodhbane-Gtari F, Beauchemin N, Bruce D, Chain P, Chen A, Walston sampled root nodules from A. glutinosa, C. glauca and E. angustifolia, performed Davenport K, Deshpande S, Detter C, Furnholm T, Goodwin L, et al. Draft DNA extraction from all sampled nodules, amplification and sequencing of glnA genome sequence of Frankia sp. strain CN3, an atypical, noninfective (Nod-) Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 9 of 10

ineffective (Fix-) isolate from Coriaria nepalensis. Genome Announc. 2013; 31. Persson T, Benson DR, Normand P, Vanden Heuvel B, Pujic P, Chertkov O, 1(2):e0008513. Teshima H, Bruce DC, Detter C, Tapia R, et al. Genome sequence of 15. Ghodhbane-Gtari F, Hurst SGt, Oshone R, Morris K, Abebe-Akele F, Thomas “Candidatus Frankia datiscae” Dg1, the uncultured microsymbiont from WK, Ktari A, Salem K, Gtari M, Tisa LS. Draft Genome Sequence of Frankia sp. nitrogen-fixing root nodules of the dicot Datisca glomerata. J Bacteriol. Strain BMG5.23, a Salt-Tolerant Nitrogen-Fixing Actinobacterium Isolated 2011;193(24):7017–8. doi:10.1186/s40793-017-0261-3. from the Root Nodules of Casuarina glauca Grown in Tunisia. Genome 32. Nguyen TV, Wibberg D, Battenberg K, Blom J, Vanden Heuvel B, Berry AM, Announc. 2014;2(3). Kalinowski J, Pawlowski K. An assemblage of Frankia Cluster II strains from 16. Gtari M, Ghodhbane-Gtari F, Nouioui I, Ktari A, Hezbri K, Mimouni W, Sbissi I, California contains the canonical nod genes and also the sulfotransferase Ayari A, Yamanaka T, Normand P, et al. Cultivating the uncultured: growing gene nodH. BMC Genomics. 2016;17(1):796. the recalcitrant cluster-2 Frankia strains. Sci Rep. 2015;5:13112. doi:10.1186/ 33. Tisa LS, Oshone R, Sarkar I, Ktari A, Sen A, Gtari M. Genomic approaches s40793-017-0261-3. toward understanding the actinorhizal symbiosis: an update on the status 17. Hurst SGt, Oshone R, Ghodhbane-Gtari F, Morris K, Abebe-Akele F, Thomas of the Frankia genomes. Symbiosis. 2016;70(1-3):5–16. WK, Ktari A, Salem K, Mansour S, Gtari M, et al. Draft Genome Sequence of 34. Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Avarre JC, Jaubert M, Frankia sp. Strain Thr, a Nitrogen-Fixing Actinobacterium Isolated from the Simon D, Cartieaux F, Prin Y, et al. Legumes symbioses: absence of Nod Root Nodules of Casuarina cunninghamiana Grown in Egypt. Genome genes in photosynthetic bradyrhizobia. Science. 2007;316(5829):1307–12. Announc. 2014;2(3). 35. Miche L, Moulin L, Chaintreuil C, Contreras-Jimenez JL, Munive-Hernandez 18. Mansour SR, Oshone R, Hurst SGt, Morris K, Thomas WK, Tisa LS. Draft JA, Del Carmen V-HM, Crozier F, Bena G. Diversity analyses of Genome Sequence of Frankia sp. Strain CcI6, a Salt-Tolerant Nitrogen-Fixing Aeschynomene symbionts in Tropical Africa and Central America reveal that Actinobacterium Isolated from the Root Nodule of Casuarina nod-independent stem nodulation is not restricted to photosynthetic cunninghamiana. Genome Announc. 2014;2(1). bradyrhizobia. Environ Microbiol. 2010;12(8):2152–64. 19. Ngom M, Oshone R, Hurst SGt, Abebe-Akele F, Simpson S, Morris K, Sy MO, 36. Persson T, Battenberg K, Demina IV, Vigil-Stenman T, Vanden Heuvel B, Pujic Champion A, Thomas WK, Tisa LS. Permanent Draft Genome Sequence for P, Facciotti MT, Wilbanks EG, O’Brien A, Fournier P, et al. Candidatus Frankia Frankia sp. Strain CeD, a Nitrogen-Fixing Actinobacterium Isolated from the datiscae Dg1, the Actinobacterial Microsymbiont of Datisca glomerata, Root Nodules of Casuarina equistifolia Grown in Senegal. Genome Expresses the Canonical nod Genes nodABC in Symbiosis with Its Host Announc. 2016;4(2). Plant. PLoS One. 2015;10(5):e0127630. 20. Normand P, Lapierre P, Tisa LS, Gogarten JP, Alloisio N, Bagnarol E, Bassi CA, 37. Baker D, O’Keefe D. A modified sucrose fractionation procedure for the Berry AM, Bickhart DM, Choisne N, et al. Genome characteristics of isolation of frankiae from actinorhizal root nodules and soil samples. Plant facultatively symbiotic Frankia sp. strains reflect host range and host plant Soil. 1984;78:23–8. biogeography. Genome Res. 2007;17(1):7–15. 38. Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5(4):433–8. 21. Nouioui I, Beauchemin N, Cantor MN, Chen A, Detter JC, Furnholm T, 39. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina Ghodhbane-Gtari F, Goodwin L, Gtari M, Han C, et al. Draft Genome sequence data. Bioinformatics. 2014;30(15):2114–20. Sequence of Frankia sp. Strain BMG5.12, a Nitrogen-Fixing Actinobacterium 40. Nurk S, Bankevich A, Antipov D, Gurevich AA, Korobeynikov A, Lapidus A, Isolated from Tunisian Soils. Genome Announc. 2013;1(4). Prjibelski AD, Pyshkin A, Sirotkin A, Sirotkin Y, et al. Assembling single-cell 22. Nouioui I, Gtari M, Goker M, Ghodhbane-Gtari F, Tisa LS, Fernandez MP, genomes and mini-metagenomes from chimeric MDA products. J Comput Normand P, Huntemann M, Clum A, Pillay M, et al. Draft Genome Sequence Biol. 2013;20(10):714–37. of Frankia Strain G2, a Nitrogen-Fixing Actinobacterium Isolated from 41. Gnerre S, Maccallum I, Przybylski D, Ribeiro FJ, Burton JN, Walker BJ, Sharpe Casuarina equisetifolia and Able To Nodulate Actinorhizal Plants of the T, Hall G, Shea TP, Sykes S, et al. High-quality draft assemblies of mammalian Order Rhamnales. Genome Announc. 2016;4(3). genomes from massively parallel sequence data. PNAS. 2011;108(4):1513–8. 23. Oshone R, Hurst SGt, Abebe-Akele F, Simpson S, Morris K, Thomas WK, Tisa 42. Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides LS. Permanent Draft Genome Sequences for Two Variants of Frankia sp. NCIMGER. A system for microbial genome annotation expert review and Strain CpI1, the First Frankia Strain Isolated from Root Nodules of curation. Bioinformatics. 2009;25:2271–8. Comptonia peregrina. Genome Announc. 2016;4(1). 43. Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Prome JC, Denarie J. 24. Oshone R, Ngom M, Abebe-Akele F, Simpson S, Morris K, Sy MO, Champion Symbiotic host-specificity of Rhizobium meliloti is determined by a A, Thomas WK, Tisa LS. Permanent Draft Genome Sequence of Frankia sp. sulphated and acylated glucosamine oligosaccharide signal. Nature. 1990; Strain Allo2, a Salt-Tolerant Nitrogen-Fixing Actinobacterium Isolated from 344(6268):781–4. the Root Nodules of Allocasuarina. Genome Announc. 2016;4(3). 44. Chaintreuil C, Arrighi JF, Giraud E, Miche L, Moulin L, Dreyfus B, Munive- 25. Pujic P, Bolotin A, Fournier P, Sorokin A, Lapidus A, Richau KH, Briolay J, Hernandez JA, Villegas-Hernandez Mdel C, Bena G. Evolution of symbiosis in Mebarki F, Normand P, Sellstedt A. Genome Sequence of the Atypical the legume genus Aeschynomene. New Phytol. 2013;200(4):1247–59. Symbiotic Frankia R43 Strain, a Nitrogen-Fixing and Hydrogen-Producing 45. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Actinobacterium. Genome Announc. 2015;3(6). Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a 26. Sen A, Beauchemin N, Bruce D, Chain P, Chen A, Walston Davenport K, genome sequence (MIGS) specification. Nat Biotechnol. 2008;26(5):541–7. Deshpande S, Detter C, Furnholm T, Ghodbhane-Gtari F, et al. Draft genome 46. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: sequence of Frankia sp. strain QA3, a nitrogen-fixing actinobacterium isolated proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci from the root nodule of Alnus nitida. Genome Announc. 2013;1(2):e0010313. U S A. 1990;87(12):4576–9. 27. Swanson E, Oshone R, Simpson S, Morris K, Abebe-Akele F, Thomas WK, Tisa 47. Goodfellow M. Phylum XXVI. Actinobacteria phyl. nov. Bergey’s Manual of LS. Permanent Draft Genome Sequence of Frankia sp. Strain ACN1ag, a Systematic Bacteriology, vol 5; 2012. p. Part A: 33. doi:10.1007/978-0-387- Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Alnus 68233-447. glutinosa. Genome Announc. 2015;3(6). 48. Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a new hierarchic 28. Swanson E, Oshone R, Simpson S, Morris K, Abebe-Akele F, Thomas WK, classification system, Actinobacteria classis nov. Int J Syst Bacteriol. 1997;47: Tisa LS. Permanent Draft Genome Sequence of Frankia sp. Strain AvcI1, 479–91. a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of 49. Sen A, Daubin V, Abrouk D, Gifford I, Berry AM, Normand P. Phylogeny of Alnus viridis subsp. crispa Grown in Canada. Genome Announc. the class Actinobacteria revisited in the light of complete genomes. The 2015;3(6). orders ‘Frankiales’ and Micrococcales should be split into coherent entities: 29. Tisa LS, Beauchemin N, Cantor MN, Furnholm T, Ghodhbane-Gtari F, proposal of Frankiales ord. nov., Geodermatophilales ord. nov., Goodwin L, Copeland A, Gtari M, Huntemann M, Ivanova N, et al. Draft Acidothermales ord. nov. and Nakamurellales ord. nov. Int J Syst Evol Genome Sequence of Frankia sp. Strain DC12, an Atypical, Noninfective, Microbiol. 2014;64(Pt 11):3821–32. Ineffective Isolate from Datisca cannabina. Genome Announc. 2015;3(4). 50. Becking JH. Frankiaceae fam. nov. (Actinomycetales) with one new 30. Wall LG, Beauchemin N, Cantor MN, Chaia E, Chen A, Detter JC, Furnholm T, combination and six new species of the genus Frankia Brunchorst. Int J Syst Ghodhbane-Gtari F, Goodwin L, Gtari M, et al. Draft Genome Sequence of Bacteriol. 1970;20:201–20. Frankia sp. Strain BCU110501, a Nitrogen-Fixing Actinobacterium Isolated 51. Normand P, Orso S, Cournoyer B, Jeannin P, Chapelon C, Dawson J, from Nodules of Discaria trinevis. Genome Announc. 2013;1(4). Evtushenko L, Misra AK. Molecular phylogeny of the genus Frankia and Ktari et al. Standards in Genomic Sciences (2017) 12:51 Page 10 of 10

related genera and emendation of the family Frankiaceae. Int J Syst Bacteriol. 1996;46(1):1–9. 52. Brunchorst J. Über einige Wurzelanschwellungen, besonders diejenigen von Alnus und den Elaeagnaceen. Unters Bot Inst Tübingen. 1886;2:151–77. 53. Skerman VBD, McGowan V, Sneath PHA. Approved lists of bacterial names. Int J Syst Evol Microbiol. 1980;30:225–420.

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