Draft Genome Sequence of the Symbiotic Frankia Sp. Strain KB5 Isolated from Root Nodules of Casuarina equisetifolia.

Frankia sp. strain KB5 was isolated from Casuarina equisetifolia and previous studies have shown both nitrogenase and uptake hydrogenase activities under free-living conditions. Here, we report 5.5-Mbp draft genome sequence with a G+C content of 70.03 %, 4,958 candidate protein-encoding genes, and 2 rRNA operons.

Introduction

Biological nitrogen fixation, which is carried out by prokaryotes, converts atmospheric dinitrogen gas into a reduced biologically useful form. One group of these nitrogen-fixing microbes is the genus Frankia, which is found in association with actinorhizal plants or free-living in the soil 1, 2. Based on molecular studies, there are four distinct Frankia lineages 3-5. Three of the Clusters (I, II, and III) are infective on actinorhizal host plants, while members of Cluster IV are considered “atypical” strains, which are unable to re-infect the plants or form ineffective nodules that unable to fix nitrogen.

Frankia sp. strain KB5 was isolated from the root nodules of Casuarina equisetifolia ssp. incana in Australia 6. This strain is a member of Frankia subcluster Ic, which has its host range limited to Casuarina and Allocasuarina plants. The physiology of Frankia sp. strain KB5 has been well studied especially the close relationship between hydrogenase and nitrogenase activities 7-9. Interestingly, the addition of nickel results in increased uptake hydrogenase activity 7. Frankia sp. strain KB5 genome was chosen to be sequenced for several reasons including an interesting physiology. Furthermore, this strain represents an isolate from Australia, the native biogeographical region of Frankia subcluster Ic 10. This database could provide more information on the evolution of this Frankia subcluster.

Sequencing of the draft genome of Frankia sp. strain KB5 was performed at the Hubbard Center for Genome Studies (University of New Hampshire, Durham, NH) using Illumina technology techniques 11. A standard Illumina shotgun library was constructed and sequenced using the Illumina HiSeq2500 platform, using a pair-end library with an average size of 600 bp obtaining 10,539,470 reads of 250 bp in length. The Illumina sequence data were trimmed by Trimmonatic version 0.32 12, assembled using Spades version 3.5 12 and ALLPaths-LG version r52488 13. The final draft assembly for Frankia sp. strain KB5 consisted of 420 contigs with an N50 contig size of 24.2 kb and 236.8X coverage of the genome. The final assembled genome contained a total sequence length of 5,455,564 bp with a G+C content of 70.03%.

The assembled Frankia sp. strain KB5 genome was annotated via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and resulted in 4,958 candidate protein-encoding genes and 2 rRNA operons. The genome features of Frankia sp. strain KB5 fall with the realm of the other Casuarina genomes including Frankia casuarinae strain (CcI3) 14 the type strain (Table 1).

Table 1

Genome features of Frankia sp. strain KB5 and other Frankia strains isolated from Casuarina root nodules.

Strain	Source	Location1	Size (Mb)	No. of Contig(s)	G+C (%)	No. of CDS	No. of rRNA	No. of tRNA
KB5	This study	Australia	5.46	420	70.0	4,958	6	45
CcI3	[10]	USA	5.43	1	70.1	4,598	6	46
CeD	[20]	Senegal	5.00	120	70.1	4,403	7	45
Allo2	[21]	Uruguay	5.33	110	69.8	4,838	7	46
Thr	[22]	Egypt	5.31	171	70.0	4,805	5	46
BMG5.23	[23]	Tunisia	5.27	167	70.0	4,747	9	47
CcI6	[24]	Egypt	5.39	138	67.6	4,902	9	46
BR	[25]	Brazil	5.23	180	70.0	4,777	5	46

1 The source of the isolate.

The genome also contained a nif and 2 hup operons encoding the nitrogenase and uptake hydrogenase enzymes, respectively. The operons were organized similar to those reported for Frankia cluster I genomes 15. The 2 hup operons have been shown to be expressed differently, with hup 1 being mainly expressed in free-living conditions and hup 2 in symbiotic 16. Bioinformatic analysis of this genomes by the use of the AntiSMASH program 17, 18 revealed the presence of high numbers of secondary metabolic biosynthetic gene clusters, which is consistent with previous results with other Frankia genomes including subcluster Ic 15, 19. Table 2 shows a comparison of the various profiles of different Casuarina isolates for these secondary metabolic biosynthetic gene clusters. Although the majority of these secondary metabolic biosynthetic gene clusters were shared among the Casuarina genomes, the Frankia sp. strain KB5 genome contained several unique clusters that had homologues in other bacteria or were completely novel. For example, cluster KB-24 (location: KBI5_16785-KBI5_16785 genes) involved in terpene biosynthesis had no homology with any of the Frankia genomes, but was homologous to the phenalinolactoneA biosynthetic gene cluster of Streptomyces pactum KLBMP 5084, including the surrounding gene neighborhood. Cluster KB-26 (location: KBI5_17660-KBI5_17705 genes) containing a nonribosomal peptide synthase (NRPS) is only found in the Frankia sp. strain KB5 genome and predicted to produce a core structure (Fig. 1). Further bioinformatics analysis revealed that the Frankia sp. strain KB5 genome contained 715 unique genes that had no homologues in any of the other Casuarina genomes. Although some of these genes like cluster KB-24 genes have predicted functions, the majority of the genes code for hypothetical proteins without a known function. The hypothesis that these genes play a role in the biogeographical distribution of this strain remains to be tested.

Table 2

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

Strain	No. of Biosynthetic gene clusters 1	NRPS 2	PKS 3	Terpene	Siderophore	Bacteriocin	Lantipeptide
KB5	34	4	9	6	1	1	4
CcI3	29	3	5	4	1	3	6
CeD	30	7	7	4	1	1	4
Allo2	32	7	9	4	1	3	5
Thr	33	6	7	4	1	1	6
BMG5.23	31	8	6	4	1	2	4
CcI6	33	8	8	4	1	3	5
BR	29	5	5	4	1	2	5

1 Biosynthetic gene clusters were identified by the use of the AntiSMASH software 17, 18

2 NRPS: Nonribosomal peptide synthase

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

Figure 1

The predicted Frankia chemical structure for secondary metabolic biosynthetic gene cluster KB-24.

In summary, the Frankia sp. strain KB5 genome has revealed an interesting potential natural product profile and serves as a representative of Frankia subcluster Ic from its native environment. Further analysis of this genome and experimental evidence will be needed to support the predicted natural product profile and to provide insight on the evolution of this Frankia subcluster.

Nucleotide sequence accession numbers

This whole-genome shotgun sequence has been deposited at DDBJ/EMBL/GenBank under the accession number MRUJ00000000. The version described in this paper is the first version, MRUJ01000000.