High-quality draft genome sequence of Gracilimonas tropica CL-CB462T (DSM 19535T), isolated from a Synechococcus culture

Gracilimonas tropica Choi et al. 2009 is a member of order Sphingobacteriales, class Sphingobacteriia. Three species of the genus Gracilimonas have been isolated from marine seawater or a salt mine and showed extremely halotolerant and mesophilic features, although close relatives are extremely halophilic or thermophilic. The type strain of the type species of Gracilimonas, G. tropica DSM19535T, was isolated from a Synechococcus culture which was established from the tropical sea-surface water of the Pacific Ocean. The genome of the strain DSM19535T was sequenced through the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project. Here, we describe the genomic features of the strain. The 3,831,242 bp long draft genome consists of 48 contigs with 3373 protein-coding and 53 RNA genes. The strain seems to adapt to phosphate limitation and requires amino acids from external environment. In addition, genomic analyses and pasteurization experiment suggested that G. tropica DSM19535T did not form spore.


Introduction
The genus Gracilimonas was first established in 2009 [1], and at the time of writing this paper there are three species that comprise this genus, G. tropica [1], G. mengyeensis [2], and G. rosea [3]. They are Gram-negative, catalase-and oxidase-positive, aerobic and facultatively anaerobic and have rod-shaped cells (Fig. 1) [1][2][3]. In addition, they form endospores except G. mengyeensis [3]. Gracilimonas tropica CL-CB462 T (=KCCM 90063 T = DSM 19535 T ), the type strain of the type species of the genus Gracilimonas, was isolated from a Synechococcus culture which was established from the tropical seasurface water of the Pacific Ocean [1]. Interestingly, the genus Gracilimonas formed a robust clade together with extremely halophilic or thermophilic bacteria (Salinibacter ruber and Rhodothermus marinus, respectively). On the contrary, Gracilimonas species show only extremely halotolerant and mesophilic features. Considering the phenotypic diversity within the clade, their comparative genomic analyses could provide a good clue to understand bacterial adaptation to extreme environments based on genomic context. Here we present a summary of the genomic features of G. tropica DSM 19535 T , which is the first genome-sequenced type strain from the genus Gracilimonas.

Classification and features
Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed G. tropica DSM19535 T is classified into the genus Gracilimonas (Fig. 2). The type strains which were most closely related to strain DSM19535 T were Gracilimonas mengyeensis YIM J14 T with 16S rRNA sequence similarity of 96.9 %, and Gracilimonas rosea CL-KR2 T with a similarity of 96.1 %. Strain DSM19535 T is tolerant of high salinity (up to 20 %) with a growth occurring over the range of salinity of 1-20 % (w/v) sea salts (optimum 3-6 %) ( Table 1). Growth occurs under either aerobic or facultatively anaerobic conditions. The optimum pH is 7.0-8.0 with a growth range of pH 6-10 ( Table 1). The strain was auxotroph for isoleucine and methionine (Table 1). Despite the phylum Bacteroidetes is known to be as a non-spore forming group [4], the strain was reported to form endospores, together with G. rosea [3]. However, strain DSM19535 T could not be asserted to form spore by the genomic analysis (see 'Insights from the genome sequence').
By phylogenetic analyses (Fig. 2), the genus Gracilimonas formed a sister clade with the genus Balneola which shows mesophilic features [5,6]. At an outer branch, the clade with Gracilimonas and Balneola formed a robust clade with the moderate halophilic Fodinibius salinus (Fig. 2). Moreover, at a deeper branch, the clade formed a robust association with a clade that includes the thermophilic genus Rhodothermus [7] and the genus of extremely halophilic Salinibacter [8], despite the relatively low (ca. 80 %) similarities between the two clades. Thus, the phylogentically robust clade contains both extremophiles and non-extremophiles.
Auxotrophy for amino acids was examined using a minimal medium (glucose, 2 g; pyruvate, 0.3 g; K 2 HPO 4 , 3 g; NaH 2 PO 4 , 1 g; NH 4 Cl, 1 g; MgSO 4 •7H 2 O, 0.3 g; 1 ml of Holden's trace elements [9]; 1 ml of Balch's vitamin solution [10]; 1 L of artificial seawater [11]) supplemented with 0.3 mM or 3 mM of all amino acids except a focal amino acid. The strain could not grow in minimal medium without supplementation of L-isoleucine and Lmethionine. But, the strain did not require other amino acids (L-alanine, L-arginine, L-asparagine, L-aspartate, Lcysteine, L-glutamate, L-glutamine, glycine, L-histidine, Llysine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and selenocysteine) for growth. , 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 [42] Genome sequencing information

Genome project history
A culture of DSM 19535 T (strain CL-CB462 T ) was selected for sequencing on the basis of its phylogenetic position [12,13], and is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project [14], a follow-up of the Genomic Encyclopedia of Bacteria and Archaea pilot project [15], which aims in increasing the sequencing coverage of key reference microbial genomes and to generate a large genomic basis for the discovery of genes encoding novel enzymes [16]. The one thousand microbial genomes-I is the first of the production phases of the Genomic Encyclopedia of Bacteria and Archaea: sequencing a myriad of type strains initiative [17] and a Genomic Standards Consortium project [18]. The genome project is deposited in the Genomes On Line Database [19] and the genome sequence is available from GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology [20].
A summary of the project information is presented in Table 2.
Growth conditions and genomic DNA preparation G. tropica DSM 19535 T , was grown in DSMZ medium 514 (Bacto Marine Broth) [21] at 28°C. Genomic DNA was isolated from about 0.5 g of cell paste using Jetflex Purification Kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with an additional protease K (50 μl; 21 mg/ml) digest for 60 min. at 58°C followed by addition of 200 μl Protein Precipitation Buffer after protein precipitation and overnight incubation on ice [22]. DNA was quality controlled according to JGI guidelines and is available through the DNA Bank Network [23].

Genome annotation
Genes were identified using Prodigal [29] as part of the DOE-JGI Annotation pipeline [30,31] followed by a round of manual curation using the JGI GenePRIMP pipeline [32]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information non-redundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes [33].

Genome properties
The genome of the strain is 3,831,242 bp long and comprises 48 contigs ranging 1177 to 783,752 bp, with an overall GC content of 42.9 % (Table 3). Of the 3426 genes predicted, 3373 were protein coding genes, and 53 were RNA genes. A total of 2413 genes (70.4 %) were assigned a putative function while the remaining ones were annotated as hypothetical or unknown proteins. The distribution of genes into COG functional categories is presented in Table 4. The properties and the statistics of the genome are summarized in Tables 3 and 4.

Insights from the genome sequence
Based on genomic analysis of the metabolic features, G. tropica DSM19535 T is predicted to be an auxotroph for L-lysine, L-phenylalanine, L-tyrosine, L-arginine, L-aspartic acid, L-isoleucine, L-proline, and L-methionine.
In the auxotroph test, however, the strain was found to be auxotroph only for L-isoleucine and L-methionine (Table 1). This discrepancy might be due to missing annotations of essential genes by incomplete sequencing or presence of unknown genes related with transport and/or assimilation. In addition, despite selenocysteine was one of essential amino acids required for growth by the genomic analysis, the strain could grow in a medium without selenocysteine. Genome analysis also revealed that strain DSM19535 T has a copper-containing nitrite reductase gene (nirK) homolog, suggesting that the strain may transform nitrite to nitric oxide (NO) under low oxygen or anoxic conditions. In addition, the strain contains DnrN (nitric oxide-dependent regulator) gene and this may protect cells from nitrosative stress [34].
However, the nitrate, nitric oxide and nitrous oxide reductases involved in denitrification were not found. The strain has an ATP-dependent glutamine synthetase and a NADPH-dependent glutamate-oxoglutarate amidotransferase, and thus can assimilate ammonia into glutamate and glutamine. In the strain, ammonium may be transported by an ammonium transport protein.
Genes participating in phosphate metabolism were also identified in the genome of the strain DSM19535 T . Inorganic pyrophosphatase catalyzing the conversion of pyrophosphate to phosphate ion, and polyphosphate kinase catalyzing the formation of polyphosphate from ATP were found in the genome. The strain has several genes of Pho regulon (phoH, phoU, phoR and phoB) mediating an adaptive response to inorganic phosphate limitation but not high affinity phosphate binding protein and transporter (pstS and pstACB). In addition, the strain may hydrolyze phosphate groups from many types of organic molecules using alkaline phosphatase.
In the previous study, G. tropica DSM19535 T was reported to be able to form spores [1]. The sporeformation is very unusual in the phylum Bacteroidetes [4]. Despite four and five proteins were annotated as stage II sporulation protein E (SpoIIE) and sporulation related domain, respectively, by search against the Pfam database, more than a hundred sporulation-related genes identified in Bacillus subtilis 168 T were absent from the genome of strain DSM19535 T . Further, the genes found in G. tropica were also found in genomes of phylogenetically close but non-sporulating genera, Balneola vulgaris DSM 17,893 and Salisaeta longa DSM2114. Therefore, 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 further tests to examine spore-formation were conducted in this study. Consistent with the previous study, sporelike spherical cells were found after malachite green staining. However, after pasteurization at 60°C for 10 and 20 min and 80, 90 and 100°C for 10 min, re-growth of cells was never observed, suggesting that the coccoid cells may not be endospore. Actually, non-spore but spore-like spherical cells were also found in aging cultures of a variety of non-sporulating bacteria including Salinispira pacifica belonging to the phylum Spirochaetae, Prolinoborus fasciculus belonging to the class Betaproteobacteria and Anaerophaga thermohalophila belonging to the phylum Bacteroidetes [35][36][37]. The genomic analyses and pasteurization experiment convincingly suggested that the spore-like coccoid cells of G. tropica DSM19535 T are not endospores.

Conclusion
The genome of a member belonging to the genus Gracilimonas in the phylum Bacteroidetes is reported here. In addition to detailed information of genome sequencing and annotation, genetic characteristics related with nitrogen and phosphorus utilization could be understood  The total is based on total number of protein coding genes in the annotated genome