Draft genome sequence of Marinobacterium rhizophilum CL-YJ9T (DSM 18822T), isolated from the rhizosphere of the coastal tidal-flat plant Suaeda japonica

The genus Marinobacterium belongs to the family Alteromonadaceae within the class Gammaproteobacteria and was reported in 1997. Currently the genus Marinobacterium contains 16 species. Marinobacterium rhizophilum CL-YJ9T was isolated from sediment associated with the roots of a plant growing in a tidal flat of Youngjong Island, Korea. The genome of the strain CL-YJ9T was sequenced through the Genomic Encyclopedia of Type Strains, Phase I: KMG project. Here we report the main features of the draft genome of the strain. The 5,364,574 bp long draft genome consists of 58 scaffolds with 4762 protein-coding and 91 RNA genes. Based on the genomic analyses, the strain seems to adapt to osmotic changes by intracellular production as well as extracellular uptake of compatible solutes, such as ectoine and betaine. In addition, the strain has a number of genes to defense against oxygen stresses such as reactive oxygen species and hypoxia.


Classification and features
By phylogenetic analysis of the 16S rRNA gene sequence (Fig. 1), M. rhizophilum strain CL-YJ9 T was positioned within the genus Marinobacterium and formed a distinct branch together with Marinobacterium profundum PAMC 27536 T and Marinobacterium nitratireducens CN44 T (Fig. 1). Strain CL-YJ9 T was most closely related to Marinobacterium profundum PAMC 27536 T , which appeared as its sister species in the tree. Strain CL-YJ9 T grows under strictly aerobic conditions ( Table 1). The optimal growth of strain CL-YJ9 T occurs at pH 7.0, with a growth range of pH 6.0-9.0. Growth occurs in the presence of 1.0-5.0% (w/v) NaCl (optimum 3.0%) and at 5-30°C (optimum 25°C) ( Table 1). Cells of strain CL-YJ9 T are rod-shaped, on average approximately 0.3-0.4 μm wide and 0.6-0.8 μm long and motile by means of monopolar flagella (Fig. 2).

Genome project history
The strain CL-YJ9 T was chosen for genome sequencing by the phylogeny-based selection [15,16] as a part of the Genomic Encyclopedia of Type Strains, Phase I: the KMG project [17]. The KMG project, the first of the production phases of the GEBA: sequencing a myriad of type strains initiative [18,19] and a Genomic Standards Consortium project [20] was set up to increase the sequencing coverage of key reference microbial genomes and to generate a large genomic basis for the discovery of genes encoding novel enzymes [21]. The genome sequencing, finishing and annotation were performed by the DOE-JGI using state of the art sequencing technology [22]. A summary of the project information is presented in Table 2.
Growth conditions and genomic DNA preparation M. rhizophilum strain CL-YJ9 T was grown in DSMZ medium 514 (http://www.dsmz.de) at 28°C and aerobe conditions. Genomic DNA was isolated using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol provided by the manufacturer but additionally applying 50 μl proteinase K and using a 60 min incubation time. DNA is available through the DNA Bank Network [23].

Genome sequencing and assembly
Using the purified genomic DNA, the draft genome of M. rhizophilum CL-YJ9 T was generated at the DOE-JGI using the Illumina technology [24]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 7,253,734 reads totaling 1088.1 Mbp. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library-preparation artifacts [25]. The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet (version 1.1.04) [26], (2) 1-3 Kbp simulated paired end reads were created from Velvet contigs using wgsim (https:// github.com/lh3/wgsim), (3) Illumina reads were assembled with simulated read pairs using Allpaths-LG (version r41043) [27]. Parameters for assembly steps were exactly same as in Choi et al. [28]. The final draft assembly contained 68 contigs in 58 scaffolds. The total size of the genome is 5.4 Mbp and the final assembly is based on 638.1 Mbp of Illumina data, which provides an average 119.1X coverage of the genome.

Genome annotation
As described in Choi et al. [28], identification of genes was performed using Prodigal [29] as part of the DOE-JGI Annotation pipeline [30,31]. After translation of the

Genome properties
The genome is 5,364,574 bp long and comprises 58 scaffolds ranging 1097 to 401,958 bp, with an overall G + C content of 58.5% (Table 3). Of the 4853 genes predicted, 4762 were protein coding genes, and 91 were RNA genes. A total of 3878 genes (79.9%) 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
To cope with osmotically varying conditions in tidal flat (e.g., exposure to heavy rainfalls or desiccation during low tides), M. rhizophilum CL-YJ9 T seems to display diverse mechanisms of adaption. For instance, the strain can synthesize compatible solutes such as betaine, ectoine and 5-hydroxyectoine. The strain has two kind of genes (choline dehydrogenases and betaine aldehyde dehydrogenase; Table 5) participating in glycine-betaine biosynthesis from choline, which is found in Gram-negative bacteria [33]. The strain also has essential genes participating in the ectoine biosynthesis and the 5-hydroxyectoine biosynthesis (five enzymes for the steps from aspartate to ectoine as well as ectoine hydroxylase, respectively; Table 5) [34]. In addition, the strain seems to uptake osmolytes by transport from the external environment. In the genomic analysis, the glycine betaine/L-proline ABC transporter system known as proU, which is an operon that encodes a high-affinity ABC transporter system consisting of three proteins (ProV, ProW and ProX; F451DRAFT_00884, F451DRAFT_00885, F451DRAFT_00886, respectively) is found in the strain. Further, the homologue of the TRAP transporter (F451DRAFT_00922) involved in transport of external ectoine and hydroxyectoine is found in M. rhizophilum.

Not in COGs
The total is based on total number of protein coding genes in the annotated genome 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 source in S. pomeroyi, but H. elongata can use it as a compatible solute. Considering that ectoine can be de novo produced in M. rhizophilum as well as actively transported from the environment, the role of the TRAP transporter in M. rhizophilum could be thought to recover endogenously synthesized ectoine that has leaked through the membrane as known in H. elongata [35].
In the rhizosphere of tidal flat, oxygen tension varies in a wide range due to temperature change, repetitive exposure to atmosphere and seawater during tidal cycle and oxygen release from the roots of plants. Further, M. rhizophilum has a molybdopterin biosynthesis pathway ( Table 5) and molybdoenzymes that use molydopterin as cofactor or prosthetic group such as formate dehydrogenase (F451DRAFT_01667, F451DRAFT_01668, F451DRAFT_01669, F451DRAFT_01665) and arsenate reductase (F451DRAFT_01068). ROS can be generated during the molybdopterin metabolism. Thus, defense mechanisms to ROS are required. Alteromonas sp. SN2, isolated from marine tidal flat, increased the number of oxidative stress tolerance genes to deal with ROS [37]. Similarly, many genes encoding ROS defense mechanisms are present in M. rhizophilum, including catalaseperoxidae (F451DRAFT_01727, F451DRAFT_04596), superoxide dismutase (F451DRAFT_03202), alkyl hydroperoxide reductase (F451DRAFT_02876, F451DRAFT_01413, F451DRAFT_00847), glutathione peroxidase (F451DRAFT_01603) and glutaredoxin (F451DRAFT_00578, F451DRAFT_01573, F451DRAFT _04005) as direct ROS scavengers. This line of data indicates a lifestyle of M. rhizophilum closely associated with the rhizosphere where substantial amounts of oxygen might be released from the roots of a well-adapted tidalflat plant, Suaeda japonica. On the contrary, truncated bacterial hemoglobins (F451DRAFT_00578, F451DRAFT _01573, F451DRAFT_04005) involved in protection from oxidative stress and enhanced respiration under hypoxic conditions are present, indicating M. rhizophilum is adapted to the hypoxic rhizosphere in tidal-flat sediments, too.
The presence of motility by means of monopolar flagella was reported in a previous report [4]. Consistently, a number of genes encoding flagellar basal body proteins, flagellar hook-associated proteins and flagellar biosynthesis proteins are found in the genomic analyses, suggesting that M. rhizophilum could explore more favorable microenvironments using flagella in the rhizosphere. In contrast to a recent study that genes encoding steroid catabolism were identified in Marinobacterium stanieri S30 [38], most of these genes were not identified in the M. rhizophilum.

Conclusions
The genome of a representative of the genus Marinobacterium from the Proteobacteria phylum is reported here for the first time. In addition to detailed information on genome sequencing and annotation, genetic adaptation in environmental conditions closely associated with rhizosphere of a tidal flat plant such as salinity change and oxygen stress could be understood on the basis of genomic analyses.