Genome sequence of the Wenxinia marina type strain (DSM 24838T), a representative of the Roseobacter group isolated from oilfield sediments
© The Author(s) 2014
Published: 15 June 2014
Wenxinia marina Ying et al. 2007 is the type species of the genus Wenxinia, a representative of the Roseobacter group within the alphaproteobacterial family Rhodobacteraceae, isolated from oilfield sediments of the South China Sea. This family was shown to harbor the most abundant bacteria especially from coastal and polar waters, but was also found in microbial mats, sediments and attached to different kind of surfaces.
Here we describe the features of W. marina strain HY34T together with the genome sequence and annotation of strain DSM 24838T and novel aspects of its phenotype. The 4,181,754 bp containing genome sequence encodes 4,047 protein-coding genes and 59 RNA genes. The genome of W. marina DSM 24838T was sequenced as part of the activities of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project funded by the DoE and the Transregional Collaborative Research Centre 51 (TRR51) funded by the German Research Foundation (DFG).
Strain HY34T (= DSM 24838T = CGMCC 1.6105T = JCM 14017T) is the type strain of Wenxinia marina in the monospecific genus Wenxinia [1,2], which belongs to the widely distributed marine Roseobacter group . The strain was isolated from sediments of the Xijiang oilfield located in the South China Sea (China) . The genus Wenxinia was named after Professor Wen-Xin Chen, a Chinese pioneer in soil microbiology. The species epithet marina refers to the Latin adjective marina (‘of or belonging to the sea’) [1,2]. Current PubMed records do not indicate any follow-up research with strain HY34T after the initial description of W. marina .
In this study we analyzed the genome sequence of W. marina DSM 24838T. We present a description of the genome sequencing and annotation and present a summary classification together with a set of features for strain HY34T, including novel aspects of its phenotype.
Classifications and features
16S rRNA gene analysis
A representative genomic 16S rRNA gene sequence of W. marina DSM 24838T was compared with the Greengenes database for determining the weighted relative frequencies of taxa and (truncated) keywords as previously described . The most frequently occurring genera were Ruegeria (41.6%), Paracoccus (31.0%), Oceanicola (14.0%), Silicibacter (5.0%) and Loktanella (3.3%) (60 hits in total). Among all other species, the one yielding the highest score was Oceanicola granulosus (AAOT01000021), which corresponded to an identity of 94.7% and an HSP coverage of 99.6%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was DQ640643 (Greengenes short name ‘Rhodobacteraceae South China Sea oil field sediment isolate HY34 Rhodobacteraceae str. HY34’), which showed an identity of 99.8% and an HSP coverage of 100.0%. The most frequently occurring keywords within the labels of all environmental samples that yielded hits were ‘microbi’ (4.3%), ‘coral’ (3.6%), ‘sea’ (2.6%), ‘diseas’ (2.5%) and ‘china’ (2.4%) (190 hits in total). The most frequently occurring keywords within the labels of those environmental samples which yielded hits of a higher score than the highest scoring species were ‘antecubit, fossa, skin’ (13.9%) and ‘china, field, oil, rhodobacteracea, sea, sediment, south’ (8.3%) (3 hits in total). Some of these keywords fit well to the isolation site of strain HY34T .
Morphology and physiology
Species Wenxinia marina
ovoid or short rods
Yeast extract, peptone
Xijiang oilfield, South China Sea (China)
Sample collection time
The utilization of carbon compounds by W. marina DSM 24838T grown at 28°C was also determined for this study using Generation-III microplates in an OmniLog phenotyping device (BIOLOG Inc., Hayward, CA, USA) . The microplates were inoculated with a cell suspension at a cell density of 95–96% turbidity and dye IF-A. Further additives were vitamin, micronutrient and sea-salt solutions, which had to be added for dealing with such marine bacteria . The plates were sealed with parafilm to avoid a loss of fluid.
The exported measurement data were further analyzed with the opm package for R [9,10], using its facilities for statistically estimating parameters from the respiration curves such as the maximum height, and automatically translating these values into negative, ambiguous, and positive reactions. The reactions were recorded in three biological replicates.
On the Generation-III plates, the strain was positive for pH 6, 1% NaCl, 4% NaCl, 8% NaCl, D-galactose, 3-O-methyl-D-glucose, D-fucose, L-fucose, L-rhamnose, 1% sodium lactate, myoinositol, rifamycin SV, L-aspartic acid, L-glutamic acid, L-histidine, L-serine, D-glucuronic acid, quinic acid, L-lactic acid, citric acid, α-keto-glutaric acid, D-malic acid, L-malic acid, nalidixic acid, and sodium formate.
W. marina HY34T was negative for the following tests: dextrin, D-maltose, D-trehalose, D-cellobiose, β-gentiobiose, sucrose, D-turanose, stachyose, pH 5, D-raffinose, α-D-lactose, D-melibiose, β-methyl-D-galactoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid, D-glucose, D-mannose, D-fructose, inosine, fusidic acid, D-serine, D-sorbitol, D-mannitol, D-arabitol, glycerol, D-glucose-6-phosphate, D-fructose-6-phosphate, D-aspartic acid, D-serine, troleandomycin, minocycline, gelatin, glycyl-L-proline, L-alanine, L-arginine, L-pyroglutamic acid, lincomycin, guanidine hydrochloride, niaproof, pectin, D-galacturonic acid, L-galactonic acid-γ-lactone, D-gluconic acid, glucuronamide, mucic acid, D-saccharic acid, vancomycin, tetrazolium violet, tetrazolium blue, phydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, bromo-succinic acid, lithium chloride, potassium tellurite, tween 40, γ-amino-n-butyric acid, α-hydroxy-butyric acid, β-hydroxy-butyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, acetic acid, aztreonam, butyric acid and sodium bromate.
The phenotype microarray results fit to those reported by Ying and colleagues  in large part. Only the utilization of lactose and D-trehalose could not be confirmed by respiration measurements under the given conditions. Interestingly, W. marina DSM 24838T showed a varying phenotype both in growth measurement  and in the respiration curves among replicates. Ying and colleagues reported eleven substrates yielding “weak” results, which complicates the exact comparison of substrate utilization . In contrast to Ying and colleagues, the OmniLog measurements gave positive reactions for L-histidine and myoinositol. This may be due respiratory measurements being more sensitive than growth measurements .
The principal cellular fatty acids of strain HY34T are C18:1 ω7c (57.1%), C16:0 (16.5%), 11-methyl C18:1 ω7c (5.4%), C18:0 (3.9%), C14:0 (3.7%), C15:1 iso G and C15:1 iso I (3.4%), summed feature 3 C16:1 ω7c and/or C15:0 2-OH (1.9%), C12:0 (1.6%) and C13:0 2-OH (1.2%). The major respiratory lipoquinone was ubiquinone 10, which is a well-known characteristic of the Alphaproteobacteria. Phosphatidylglycerol and phosphatidylcholine were identified as the major polar lipids. In contrast to other representatives of the Roseobacter group such as Marinovum algicola FF3T (DSM 10251T) [12,13], strain HY34T also contains an unidentified glycolipid called L1, which shows similarities to an unidentified phospholipid of Ruegeria atlantica DSM 5828T (all data from ).
Genome sequencing and annotation
Genome project history
Genome sequencing project information
Two genomic libraries: one Illumina PE library (539 bp insert size), one 454 PE library (3kb insert size)
Illumina GA IIx, Illumina MiSeq, 454 GS-FLX Titanium
velvet version 1.1.36, Newbler version 2.3, consed 20.0
Gene calling method
GenBank Date of Release
NCBI project ID
2519899719 (8 scaffold version)
and 2515154190 (41 scaffold version)
Source material identifier
Tree of Life, biodiversity
Growth conditions and DNA isolation
A culture of W. marina DSM 24838T was grown aerobically in DSMZ medium 514  at 30°C. Genomic DNA was isolated using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol provided by the manufacturer but modified by an incubation time of 60 min, incubation on ice overnight on a shaker, the use of an additional 50 µl proteinase K, and the addition of 100 µl protein precipitation buffer. The DNA is available from the Leibniz-Institute DSMZ through the DNA Bank Network .
Genome sequencing and assembly
The genome sequencing under the DFG funded part of the project was perform as previously described for Rubellimicrobium thermophilum , with 3.3 million reads delivered by the first run on an Illumina GAII platform. To increase the sequencing depth, a second Ilumina run was performed, providing another 8.1 million reads. The first draft assembly from 9,139,639 filtered reads (median read length 122 nt) resulted in more than 300 contigs. To gain information on the contig arrangement an additional 454 run was performed. The paired-end pyrosequencing jumping library resulted in 158,608 reads, with an average read length of 450 bp. Both draft assemblies (Illumina and 454 sequences) were fractionated into artificial Sanger reads of 1,000 nt in length plus 75 bp overlap on each site. These artificial reads served as an input for the phred/phrap/consed package . In combination the assembly resulted in 265 contigs in 26 scaffolds.
The genome sequencing under the DoE funded part of the project was performed as previously described for Halomonas zhanjiangensis  also using the Illumina technology . An Illumina Standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform. All general aspects of library construction and sequencing performed at the JGI can be found at . The final assembly for this part of the project resulted in 41 scaffolds covering 4,175,892 bp (ARAY00000000).
The draft sequence from the first (DFG-funded) part was mapped to the permanent draft version ARAY00000000 using minimus2 . By manual editing the number of contigs was reduced to 22 in 8 scaffolds (AONG00000000). The combined sequences provided a 356 × coverage of the genome.
Genes were identified using Prodigal  as part of the JGI genome annotation pipeline. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Identifications of RNA genes were carried out by using HMMER 3.0rc1  (rRNAs) and tRNAscan-SE 1.23 (tRNAs) . Other non-coding genes were predicted using INFERNAL 1.0.2 . Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform  CRISPR elements were detected using CRT  and PILER-CR .
% of Total
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Number of scaffolds
Genes with function prediction (proteins)
Genes in paralog clusters
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with the general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane/envelope biogenesis
Intracellular trafficking and secretion, and vesicular transport
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Insights into the genome
Genome sequencing of W. marina DSM 24838T reveals the presence of one plasmid with a size of about 101 kb. The plasmid contains a characteristic replicase of the RepA-I type , but the typical module structure containing the replicase as well as a parAB partitioning operon was not found. A single parA gene (wenxma_04096) is located adjacent to the replicase and an additional parAB operon (wenxma_04090 to wenxma_04091) is located downstream of repA-I. The plasmid harbors neither a plasmid stability module nor a type-IV secretion system.
The plasmid contains a large RTX-toxin (wenxma_04058) and is dominated by genes that are required for polysaccharide biosynthesis. It includes all four genes of the rhamnose pathway , but the rmlA gene for the glucose-1-phosphate thymidylyltransferase (EC 220.127.116.11; wenxma_04097) is separated from the remaining clustered genes (rmlC, rmlB, rmlD; wenxma_04094 to wenxma_04092). The extrachromosomal replicon may be involved in surface attachment. Comparable RepA-I type plasmids with a similar genetic composition are also present in other Rhodobacterales including several Phaeobacter strains .
Many bacteria encode genome-inserted gene sequences, which are associated with prophages, one of the major reason for horizontal gene transfer and bacterial diversity [34,35]. The genome sequence of W. marina DSM 24838T was found to encode several prophage-associated gene sequences (e.g., wenxma_00641 to wenxma_00646, wenxma_00930 to wenxma_00936, wenxma_01496 to wenxma_01510).
Analysis of the DSM 24838T genome sequence revealed the presence of gene sequences associated to quorum sensing (QS) [36–38]. QS is a bacterial communication system via chemical signal molecules called autoinducers, which are produced and released by QS bacteria to coordinate behaviors with respect to their population density . Interestingly and surprisingly, QS induces also individual morphologies and cell division modes, which was recently shown for D. shibae DFL-12, another representative of the Roseobacter group [39,40]. Regarding to QS the genome of DSM 24838T codes for, e.g., two N-acyl-L-homoserine-lactone synthetases (LuxI homologues, wenxma_01086 and wenxma_03269) and two genes possibly encoding QS-involved response and transcriptional regulators (LuxR homologues, wenxma_01085 and wenxma_03267).
With regard to morphological traits, several genes associated with the putative production, biosynthesis and export of exopopolysaccharides (wenxma_00281, wenxma_02363 and wenxma_02364, wenxma_03720 and wenxma_03721) and capsule polysaccharides (wenxma_00822, wenxma_02023 to wenxma_02025, wenxma_02704 and wenxma_02705, wenxma_04069) were detected.
Interestingly, the genome of DSM 24838T was found to encode several gene sequences putatively involved in pili formation (e.g., wenxma_01776 to wenxma_01787, wenxma_03426 to wenxma_03435) and chemotaxis (e.g., wenxma_3823 to wenxma_03830), although the strain was described as non-motile . Hence, it could be that the formed pili play a role for adhesion or switching-type motility on solid surfaces.
Further, according to its genome strain DSM 24838T accumulates polyhydroxyalkanoates as storage compounds (wenxma_02601 to wenxma_02604), which is in accordance with the findings of Ying and colleagues for strain HY34T .
The authors would like to gratefully acknowledge the assistance of Iljana Schröder for growing W. marina cultures and Meike Döppner for DNA extraction and quality control (both at the Leibniz-Institute DSMZ). This work was performed under the auspices of the German Research Foundation (DFG) Transregio-SFB51 Roseobacter grant and the US Department of Energy’s Office of Science, Biological and Environmental Research Program and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344.
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