Permanent Draft Genome sequence for Frankia sp. strain CcI49, a Nitrogen-Fixing Bacterium Isolated from Casuarina cunninghamiana that Infects Elaeagnaceae.

Frankia sp. strain CcI49 was isolated from Casuarina cunninghamiana nodules. However the strain was unable to re-infect Casuarina, but was able to infect other actinorhizal plants including Elaeagnaceae. Here, we report the 9.8-Mbp draft genome sequence of Frankia sp. strain CcI49 with a G+C content of 70.5 % and 7,441 candidate protein-encoding genes. Analysis of the genome revealed the presence of a bph operon involved in the degradation of biphenyls and polychlorinated biphenyls.

Soil dwelling actinobacteria of the genus Frankia form an endophytic symbiosis with actinorhizal plants, which are comprised of over 200 species from 8 angiosperm families 1, 2. Actinorhizal plants in symbiosis with Frankia play important ecological roles as pioneer species and are used in agroforestry, land reclamation, crop protection, and soil stabilization projects 3. Molecular phylogenetic approaches have identified four major clusters of Frankia that also follow host plant specificity groups 4-7. Members of cluster 1 are divided into sub-cluster 1a that are infective on Alnus and Myricaceae and sub-cluster 1b strains which are infective on Allocasuarina, Casuarina and Myricacaeae. Cluster 2 represents strains infective on Coriariaceae, Datiscaceae, Dryadoideae and Ceanothus, while cluster 3 comprises strains that are infective on Colletieae, Elaeagnaceae, Gymnostoma and Myricaceae. Finally, cluster 4 groups Frankia strains isolated from actinorhizal nodules that are unable to undertake the nitrogen-fixation process (Fix-) and/or re-infect their host plant causing nodulation (Nod-) and are classified as “atypical Frankia”. Genomes for representatives from each cluster have been sequenced 8. The availability of these Frankia genome databases has opened up the use of “omics” approaches. Analysis of Frankia genomes has revealed new potential in respect to metabolic diversity, natural product biosynthesis, and stress tolerance, which may aid the cosmopolitan nature of the actinorhizal symbiosis.

Several Frankia strains isolated from Casuarina nodules are unable to re-infect it, but are able to infect other actinorhizal plant genera like Elaeagnus 9, 10. Although isolated from Casuarina nodules, these Frankia strains are classified as members of cluster 3 based on molecular phylogeny and genomes for two members of this group have been sequenced 11, 12. Frankia sp. strain CcI49 was isolated from root nodules of Casuarina cunninghamiana grown on the edge of a cultivated field on side of the highway in Ismailia-Port Said, Egypt. The fresh nodules were washed, dissected into individual lobes, and surface-sterilized as described previously 13. Each lobe was checked for sterility in sterile nutrient-rich medium. Nodules that were free from contamination were selected, dissected and homogenized, the homogenates were transferred to 100-ml screw caped bottle containing modified BAP medium for outgrowth. Hyphal outgrowth was homogenized and plated onto solid medium. After 3-4 weeks, colonies picked from the plates were homogenized and incubated in liquid medium. Surprisingly, Frankia sp. strain CcI49 produced reddish colonies, while other Frankia isolates from Casuarina do not. Frankia sp. strain CcI49 produced sporangia and spores that were smaller and narrower than normal Frankia sporangia and spores (Figure 1). Spores from Frankia sp. strain CcI49 had a high germination rate similar to Frankia strain CeI5 14, 15. We tested the ability of Frankia sp. strain CcI49 to re-infect actinorhizal plants. Four different actinorhizal plant species were tested to assay the plant host range and ten plants of each species were inoculated. Frankia sp. strain CcI49 was unable to infect C. cunninghamiana and Alnus glutinosa, but formed nodules on Elaeagnus angustifolia and Hippophäe rhamnoides. All ten of the E, angustifolia and H. rhamnoides plants tested formed nodules. Thus, Frankia sp. strain CcI49 had a plant-host-specificity pattern similar to Frankia sp. strains G2 and R43 9, 10 from cluster 3 also isolated from Casuarina root nodules. Frankia sp. strain CcI49 genome was chosen to be sequenced for several reasons including an interesting physiology including the production of a reddish pigment, development of smaller sporangia and spores than are typical found with Frankia, and providing more information on this Frankia subcluster.

Figure 1

Photomicrograph of Franakia sp. strain CcI49 grown in liquid culture. The elongated arrow shows the presence of a long, narrow sessile sporangium containing differentiated mature spores at distal end (short arrow). Size bar represents 32 µm.

Sequencing of the draft genome of Frankia sp. strain CcI49 was performed at the Hubbard Center for Genome Studies (University of New Hampshire, Durham, NH) using Illumina technology techniques 16. A standard Illumina shotgun library was constructed and sequenced using the Illumina HiSeq2500 platform, which generated 7,939,466 reads (260-bp insert size) totaling 1,921 MBp. The Illumina sequence data were trimmed by Trimmonatic version 0.32 17, assembled using Spades version 3.5 18, and ALLPaths-LG version r52488 19. The final draft assembly for Frankia sp. strain CcI49 consisted of 78 contigs with an N50 contig size of 282.1 kb and 167X coverage of the genome. The final assembled genome contained a total sequence length of 9,758,130 bp with a G+C content of 70.5%.

The assembled Frankia sp. strain CcI49 genome was annotated via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and resulted in 7,411 candidate protein-encoding genes, 46 tRNA and 2 rRNA regions. The genome features of Frankia sp. strain CcI49 fall outside the realm of the other cluster 1b genomes, but similar to other cluster 3 isolates from Casuarina (Table 1). Phylogenetic analysis of the 23S rDNA shows that Frankia sp. strain CcI49 groups with the cluster 3 strains (Figure S1). The genome size and corresponding number of CDSs were larger than the typical cluster 1b, but fit within those values reported for cluster 3 genomes 8. The genome also contained a nif, 2 hup, and 1 shc operons encoding the nitrogenase and uptake hydrogenase enzymes and the hopanoid biosynthetic pathway, respectively. The operons were organized similar to those reported for Frankia cluster 3 genomes 8.

Table 1

Genome features of Frankia sp. strain CcI49 and other Frankia strains isolated from Casuarina root nodules.

Strain	Source	Location1	Size (Mb)	No. of Contigs	Frankia cluster	No. of CDS	Host Plants2
CcI49	This study	Egypt	9.76	78	3	7,441	Elaeagnaceae
R43	[12]	USA	10.45	46	3	7,644	Elaeagnaceae
G2	[11]	Guadeloupe	9.54	90	3	7,790	Elaeagnaceae
KB5	[23]	Australia	5.46	420	1b	4,958	Casuarinaceae
CcI3	[24]	USA	5.43	1	1b	4,598	Casuarinaceae
CeD	[25]	Senegal	5.00	120	1b	4,403	Casuarinaceae
Allo2	[26]	Uruguay	5.33	110	1b	4,838	Casuarinaceae
Thr	[27]	Egypt	5.31	171	1b	4,805	Casuarinaceae
BMG5.23	[28]	Tunisia	5.27	167	1b	4,747	Casuarinaceae
CcI6	[29]	Egypt	5.39	138	1b	4,902	Casuarinaceae
BR	[30]	Brazil	5.23	180	1b	4,777	Casuarinaceae

1 The source of the isolate

2 Re-infection plant host range

Analysis of the Frankia sp. strain CcI49 revealed the presence of the bph operon coding for a potential metabolic pathway involved in the degradation of biphenyl and polychlorinated biphenyls (Figure 2). The bph operon is also present in Frankia sp. strains EuI1c and EUN1f genomes 20 and was also found in the genomes of Frankia sp. strains G2 and R43 11, 12, cluster 3 strains isolated from Casuarina root nodules. Both Frankia sp. strains CcI49 and EUN1f contained the entire bph operon, while two genes (bphA3 and bphH) are missing in Frankia sp. EuI1c (Figure 2). The presence of the complete bph operon suggests that Frankia sp. strain CcI49 may be capable of degrading these recalcitrant xenobiotics.

Figure 2

The bph operon is present in Frankia strains CcI49, EuI1c, G2, R43, and EUN1f. bph genes encode the following : BphA, A2 and A3, Biphenyl 2,3-dioxygenase; BphB, cis -2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase; BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase; BphD, 2-hydroxy-6-phenyl-6-oxohexa-2,4-dieneoate (HOPDA) hydrolase; BphE, 2-hydroxypenta-2,4-dienoate hydratase; BphF, acylating acetaldehyde dehydrogenase; and BphG, 4-hydroxy-2-oxovalerate aldolase.

Bioinformatic analysis of this genomes by the use of the AntiSMASH program 21 revealed the presence of high numbers of secondary metabolic biosynthetic gene clusters, which is consistent with previous results with other Frankia genomes including cluster 3 8, 22. Table 2 shows a comparison of the various profiles of different Frankia strains isolated from Casuarina for these secondary metabolic biosynthetic gene clusters. The profile of Frankia sp. strain CcI49 differed from those shown by Frankia strains that are able re-infect Casuarina and was similar to the pattern exhibited by the other two cluster 3 strains (R43 and G2) isolated from Casuarina nodules. These cluster 3 genomes contained more polyketide synthase (PKS) biosynthetic clusters than the cluster 1b genomes. The Frankia sp. strain CcI49 genome contained several unique clusters that had homologues in other bacteria or were completely novel.

Table 2

Biosynthetic gene clusters for natural products found in the genomes from Casuarina Frankia strains.

Strain	Frankia Cluster	No. of Biosynthetic gene clusters 1	NRPS 2	PKS 3	Terpene	Siderophore	Bacteriocin	Lantipeptide
CcI49	3	42	6	17	3	1	2	5
R43	3	38	4	14	3	1	2	4
G2	3	35	8	13	3	1	2	2
KB5	1b	34	4	9	6	1	1	4
CcI3	1b	29	3	5	4	1	3	6
CeD	1b	30	7	7	4	1	1	4
Allo2	1b	32	7	9	4	1	3	5
Thr	1b	33	6	7	4	1	1	6
BMG5.23	1b	31	8	6	4	1	2	4
CcI6	1b	33	8	8	4	1	3	5
BR	1b	29	5	5	4	1	2	5

1 Biosynthetic gene clusters were identified by the use of the AntiSMASH software 21

2 NRPS: Nonribosomal peptide synthase

3 PKS: polyketide synthase including Type I, II, III, Trans-AT, and other types

In summary, the Frankia sp. strain CcI49 genome has revealed an interesting potential metabolic pathways and natural product profile, and serves as a representative of Frankia cluster 3. Further analysis of this genome and experimental evidence will be needed to support the predicted natural product profile and metabolic potential of Frankia sp. strain CcI49.

Nucleotide sequence accession numbers

This whole-genome shotgun sequence has been deposited at DDBJ/EMBL/GenBank under the accession number MOWP00000000.1. The version described in this paper is the first version, MOWP01000000.

Figure S1.

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