Draft genome sequence of Rubidibacter lacunae strain KORDI 51-2(T), a cyanobacterium isolated from seawater of Chuuk lagoon.

A photoautotrophic cyanobacterium, Rubidibacter lacunae was reported in 2008 for the first time. The type strain, KORDI 51-2(T), was isolated from seawater of Chuuk lagoon located in a tropical area. Although it belonged to a clade exclusively comprised of extremely halotolerant strains by phylogenetic analyses, R. lacunae is known to be incapable of growth at high salt concentration over 10%. Here we report the main features of the genome of R. lacunae strain KORDI 51-2(T). The genome of R. lacunae contains a gene cluster for phosphonate utilization encoding three transporters, one regulator and eight C-P lyase subunits.

Introduction

type strain KORDI 51-2T (=KCTC 40015T=UTEX L2944T) is a photoautotrophic cyanobacterium isolated from lagoon seawater of Chuuk, Micronesia [1]. At this time, the genus is comprised of a single isolate. Further, only three environmental 16S rRNA gene sequences in the NCBI showed relatively high sequence similarity of ca. 96% to 16S rRNA gene of the strain. Thus, the genus seems either to be a numerically rare cyanobacterium or, to exploit specific environments such as microbial mats. Actually, the most similar sequences (accession no. of DQ861063 and DQ861117 in GenBank) to were obtained in microbial mats of a coastal hypersaline pool. Nonetheless, the strain KORDI 51-2T is a non-extreme halotolerant member in the Halothece cluster, exclusively composed of extremely halophilic/halotolerant bacteria. Considering this contrasting phenotypic trait, genomic information of KORDI 51-2T could provide a good clue to understand genomic adaptation of cyanobacteria at extreme salt condition. Here we present a summary of the genomic features of strain KORDI 51-2T.

Classification and features

By phylogenetic analysis of 16S ribosomal RNA genes (Figure 1), KORDI 51-2T was clustered into the Halothece cluster. Four Euhalothece strains belonging to the cluster were isolated from a hypersaline pond (strains MPI 96N303 and MPI 96N304) or a solar evaporation pond (strains MPI95AH10 and MPI95AH13) in Mexico [2]. These strains showed sustained growth between 6-16% salinity, and several strains could grow even in NaCl saturated brine, suggesting that they are at least extremely halotolerant cyanobacteria [2]. and other Halothece strains belonging to the cluster were also isolated from various hypersaline environments, such as a solar lake in Egypt, a solar evaporation pond in Spain and hypersaline lagoon in Australia [2,3]. On the contrary, KORDI 51-2T was isolated from natural seawater and able to grow at a salinity between 2 and 7% (Table 1). In addition, KORDI 51-2T contains phycoerythrin, which differentiated it from the other strains belonging to the ‘Halothece’ cluster [1]. The epifluorescence micrograph of the cells and other classification and general features were shown in Figure 2 and Table 1, respectively.

Figure 1

Neighbor-joining tree showing the phylogenetic position of KORDI 51-2T relative to other close cyanobacterial strains. GenBank accession numbers for each strain are shown in parenthesis. The tree uses the Jukes-Cantor corrected distance model to construct a distance matrix. Bootstrap values above 60%, based on 1,000 resamplings, are shown at the branching points. Strains with genome sequence are underlined.

Table 1

Classification and general features of strain KORDI 51-2T according to the MIGS recommendations [4]

MIGS ID	Property	Term	Evidence codea
		Domain Bacteria	TAS [5]
		Phylum Cyanobacteria	TAS [6-8]
		Class Cyanobacteria	TAS [8,9]
	Current classification	Order Unknown
		Family 1.1	TAS [7]
		Genus Rubidibacter	TAS [1]
		Species Rubidibacter lacunae	TAS [1]
		Type strain KORDI 51-2	TAS [1]
	Gram stain	Not reported
	Cell shape	Rods	TAS [1]
	Motility	None	TAS [1]
	Sporulation	None	IDA
	Temperature range	25-35oC	TAS [1]
	Optimum temperature	30oC	TAS [1]
MIGS-6	Habitat	Seawater	TAS [1]
MIGS-6.3	Salinity	2-7% (optimum: 5)	TAS [1]
MIGS-22	Oxygen     Carbon source     Energy source	Aerobic     Autotroph     Phototroph	TAS [1]      TAS [1]      TAS [1]
MIGS-15	Biotic relationship	Free living	TAS [1]
MIGS-14	Pathogenicity	None	NAS
MIGS-4	Geographic location	Chuuk state, Micronesia	TAS [1]
MIGS-5	Sample collection time	July, 2004	IDA
MIGS-4.1	Latitude	7o 13’ N	IDA
MIGS-4.2	Longitude	151o 58’ E	IDA
MIGS-4.3	Depth	40 m	IDA
MIGS-4.4	Altitude	Not applicable	NAS

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). These evidence codes are from the Gene Ontology project [9].

Figure 2

Epifluorescence micrograph of KORDI 51-2T. The picture was taken under green excitation and then converted to gray scale. Bar, 3 μm.

Genome sequencing and annotation

Genome project history

The organism was selected for sequencing on the basis of its phylogenetic position. The genome project was deposited in the Genomes On Line Database [10] and draft genome sequence was deposited in GenBank database (accession number ASSJ00000000). The genome sequencing was carried out in Macrogen Inc. (Seoul, Korea) using GS-FLX Titanium sequencing technology. Table 2 presents the project information and its association with MIGS version 2.0 compliance [4].

Table 2

Genome sequencing project information

MIGS ID	Property	Term
MIGS-31	Finishing quality	Draft
MIGS-28	Libraries used	Shotgun library
MIGS-29	Sequencing platforms	454 GS-FLX Titanium
MIGS-31.2	Sequencing coverage	30×
MIGS-30	Assemblers	Newbler version 2.3
MIGS-32	Gene calling method	Prodigal, GenePRIMP
	Genbank ID	ASSJ00000000
	Genbank Date of Release	October 7, 2013
	GOLD ID	Gi22154
	Project relevance	Cyanobacterial ecology

Growth conditions and DNA isolation

KORDI 51-2T was grown in a 50 ml culture flask filled with 50 ml of modified f/2 medium in which silicate was omitted and ammonium chloride was supplemented (final conc. of 100 μM). The culture flask with inoculum was incubated at 25oC at about 20 μE m-2 s-1 (light:dark=14:10) for 3 weeks. Genomic DNA was isolated using Qiagen Genomic-tip 100/G (Qiagen) according to the manufacturer’s instruction.

Genome sequencing and assembly

The genome was sequenced by pyrosequencing (GS-FLX Titanium). A shotgun library was constructed according to GS FLX Titanium Sequencing Method Manual. The 291,414 pyrosequencing reads obtained has an average length of 442.12 bp and were assembled using the Newbler assembler (version, 2.3; Roche) with default options. The final assembly resulted in 126 contigs longer than or equal to 500 bp with the contigs sum of 4,215,105 bp. After removing 27 short contigs with low coverage in order to minimize possible contamination, the remaining 99 contigs were used for further analyses (Table 3).

Table 3

Genome statistics

Attribute	Value	% of totala
Genome size (bp)	4,153,658
DNA Coding region (bp)	3,323,928	80.02
DNA G+C content (bp)	2,335,216	56.22
No. of contigs	99
Total genesb	3,790
RNA genes	50	1.32
Protein-coding genes	3,740	98.68
Genes with functional prediction	2411	63.61
Genes with enzymes	775	20.45
Genes with transporter classification	343	9.05
Genes assigned to COGs	2,228	58.79
Genes assigned to Pfam	2,511	66.25
Genes assigned to TIGRFam	976	25.75
Genes assigned in paralog clusters	2427	64.04
Genes with signal peptides	137	3.61
Genes with transmembrane helices	810	21.37

a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

b) Also includes 283 pseudogenes.

Genome annotation

The gene prediction and functional annotation of the genome sequence was basically performed within the Integrated Microbial Genomes – Expert Review (IMG-ER) platform [11]. The tRNAScan-SE was used to find tRNA genes [12]. Ribosomal RNA genes and ncRNA were predicted using RNAmmer [13] and Infernal [14] using the Rfam model [15], respectively. Identification of protein coding genes was performed using Prodigal [16], followed by a round of manual curation using the JGI GenePRIMP pipeline [17]. The predicted CDS were searched using the TIGR-fam, Pfam and COG databases implemented in the IMG systems.

Genome properties

The draft genome of KORDI 51-2T, with a total of 4.15 Mbp from 99 contigs, contains 56.22% G+C contents (Figure 3 and Table 3). A total of 3,790 genes were predicted. Of these, 283 pseudogenes. The remaining 3,457 were annotated as protein-coding genes and 50 for RNA genes (3 for rRNA, 41 for tRNA and 6 other nc RNA). The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4.

Figure 3

Graphical circular map of the genome. From outside to the center: color by COG categories and RNAs on forward strand, genes on forward strand, genes on reverse strand, color by COG categories and RNAs on reverse strand, GC content, GC skew.

Table 4

Number of genes associated with the 25 general COG functional categories

Code	Value	% age	Description
J	149	6.08	Translation
A	1	0.04	RNA processing and modification
K	109	4.45	Transcription
L	127	5.18	Replication, recombination and repair
B	1	0.04	Chromatin structure and dynamics
D	24	0.98	Cell cycle control, mitosis and meiosis
Y	0	-	Nuclear structure
V	43	1.75	Defense mechanisms
T	105	4.28	Signal transduction mechanisms
M	166	6.77	Cell wall/membrane biogenesis
N	25	1.02	Cell motility
Z	1	0.04	Cytoskeleton
W	0	-	Extracellular structures
U	62	2.53	Intracellular trafficking and secretion
O	118	4.81	Posttranslational modification, protein turnover, chaperones
C	149	6.08	Energy production and conversion
G	126	5.14	Carbohydrate transport and metabolism
E	172	7.01	Amino acid transport and metabolism
F	58	2.37	Nucleotide transport and metabolism
H	157	6.4	Coenzyme transport and metabolism
I	51	2.08	Lipid transport and metabolism
P	158	6.44	Inorganic ion transport and metabolism
Q	75	3.06	Secondary metabolites biosynthesis, transport and catabolism
R	331	13.5	General function prediction only
S	244	9.95	Function unknown
-	1562	41.21	Not in COGs

Insights from the genome sequence

A genome analysis of KORID 51-2T, revealed that it contains a gene cluster participating in organic phosphonate utilization. Likewise with a marine nitrogen-fixing cyanobacterium, IMS101 [18], the strain KORDI 51-2T has orthologs to phnC-E (transporters) and phnG-M (C-P lyase complex) (Figure 4A). Additionally, an ortholog to phnF (transcriptional regulator) is found in strain KORDI 51-2T, but not in T. erythraeum IMS101. Phylogenetic analysis of PhnJ proteins found in various bacterial strains, showed that PhnJ proteins of cyanobacteria form polyphyletic lineages (Figure 4B), suggesting that the phn gene cluster of cyanobacteria might be acquired by horizontal gene transfer. As KORDI 51-2T can grow in media supplemented with variety of organic phosphonate substrates (2-aminoethylphosphonate, methylphosphonate, phosphonoacetic acid and phosphonoformic acid) as a sole P-source (data not shown), the strain must be able to cleave C-P bonds of organic phosphonate by C-P lyase pathways and utilize them as a P-source.

Figure 4

DNA topology of the phn cluster (A) and phylogenetic analysis of the PhnJ protein (B). A, Genes encoding phosphonate transport (gray), regulation (light gray), and the C-P lyase subunits (dark gray) are shown. Additional two sets of transporters were not shown. B, Phylogenetic relationship of the PhnJ protein from a variety of bacteria determined by maximum-likelihood analysis. Bootstrap values >70 are shown at the nodes. The scale bar represents amino-acid substitution per site.