Introduction

The genus Marinobacterium within the family Alteromonadaceae was established in 1997 by González et al. [1]. Currently the genus Marinobacterium contains 16 species with validly published names (Fig. 1). All Marinobacterium strains have been isolated from marine environments [1,2,3,4,5,6,7,8,9,10,11] such as sea water, tidal flat, deep-sea sediment, and coral mucus. Interestingly, their habitats include tropical waters [12, 13], Arctic marine sediment [7], tidal flats [4, 11] as well as deep sea sediment [10], indicating that the genus has well adapted to diverse environments. In the GOLD database [14], genome sequencing of 38 strains from 11 Marinobacterium species are identified to be finished or in progress. In addition, six genome sequences from five species ( M. jannaschii , M. litorale , M. rhizophilum , M. stanieri and M. profundum ) and one unidentified strain are found in the GenBank database. Among them, genomic features of M. rhizophilum CL-YJ9T (=DSM 18822=KCCM 42386 T), isolated from the rhizosphere of a plant Suaeda japonica inhabiting a coastal tidal flat, Korea, will be presented here.

Fig. 1
figure 1

Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between strain CL-YJ9T, members of the genus Marinobacterium and other related genera. Bootstrap percentages >60% (based on 1000 resamplings) are shown below or above the corresponding branches. Solid circles indicate that the corresponding nodes are also recovered in the maximum-likelihood and maximum-parsimony trees. Terasakiella pusillum IFO 13613T (AB006768) was used as an outgroup. Bar, 0.02 nucleotide substitutions per site

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Organism information

Classification and features

By phylogenetic analysis of the 16S rRNA gene sequence (Fig. 1), M. rhizophilum strain CL-YJ9T was positioned within the genus Marinobacterium and formed a distinct branch together with Marinobacterium profundum PAMC 27536 T and Marinobacterium nitratireducens CN44T (Fig. 1). Strain CL-YJ9T was most closely related to Marinobacterium profundum PAMC 27536 T, which appeared as its sister species in the tree. Strain CL-YJ9T grows under strictly aerobic conditions (Table 1). The optimal growth of strain CL-YJ9T 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-YJ9T 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).

Table 1 Classification and general features of M. rhizophilum CL-YJ9T [8, 9]
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Fig. 2
figure 2

Transmission electron microscopy image of Marinobacterium rhizophilum CL-YJ9T

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Genome sequencing information

Genome project history

The strain CL-YJ9T 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.

Table 2 Genome sequencing project information
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Growth conditions and genomic DNA preparation

M. rhizophilum strain CL-YJ9T 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 predicted CDSs, they were used to search the databases, such as National Center for Biotechnology Information non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional analysis and functional annotation were performed within the Integrated Microbial Genomes [32].

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.

Table 3 Genome statistics
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Table 4 Number of genes associated with general COG functional categories
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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-YJ9T 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 . Function of the TRAP transporter is elucidated in both Halomonas elongata DSM 2581 [35] and Silicibacter pomeroyi DSS-3 [36]. Ectoine/5-hydroxyectoine-binding periplasmic protein in M. rhizophilum showed amino acids sequence similarity of 35.1% and 33.8% with those of H. elongata (TeaA) and S. pomeroyi (UehA), respectively. The transported ectoine is used as the sole carbon and nitrogen 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].

Table 5 Enzymes and gene-loci participating in selected pathways identified in the draft genome of M. rhizophilum CL-YJ9T. Gene-loci are from the IMG/MER database
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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 catalase-peroxidae (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 tidal-flat 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.