Open Access

Complete genome sequence of Thalassolituus oleivorans R6-15, an obligate hydrocarbonoclastic marine bacterium from the Arctic Ocean

  • Chunming Dong, ,
  • , Xin Chen, , ,
  • , Yanrong Xie, , ,
  • , Qiliang Lai, ,
  • and Zongze Shao, ,
Corresponding author

DOI: 10.4056/sigs.5229330

Received: 01 March 2014

Accepted: 01 March 2014

Published: 15 June 2014

Abstract

Strain R6-15 belongs to the genus Thalassolituus, in the family Oceanospirillaceae of Gammaproteobacteria. Representatives of this genus are known to be the obligate hydrocarbonoclastic marine bacteria. Thalassolituus oleivorans R6-15 is of special interest due to its dominance in the crude oil-degrading consortia enriched from the surface seawater of the Arctic Ocean. Here we describe the complete genome sequence and annotation of this strain, together with its phenotypic characteristics. The genome with size of 3,764,053 bp comprises one chromosome without any plasmids, and contains 3,372 protein-coding and 61 RNA genes, including 12 rRNA genes.

Keywords:

Thalassolituusgenomealkane-degradingsurface seawaterArctic Ocean

Introduction

Thalassolituus spp. belong to the Oceanospirillaceae of Gammaproteobacteria. The genus was first described by Yakimov et.al. (2004), and is currently composed of two type species, T. oleivorans and T. marinus [1,2]. Bacteria of this genus are known as obligate hydrocarbonoclastic marine bacteria [3]. Previous reports showed that Thalassolituus-related species were among the most dominant members of the petroleum hydrocarbon-enriched consortia at low temperature [4-7]. In addition to consortia enriched with oil, Thalassolituus spp. can be detected in variety of cold environments as well [8-10].

Strain R6-15 was isolated from the surface seawater of the Arctic Ocean after enriched with crude oil during the fourth Chinese National Arctic Research Expedition of the “Xulong” icebreaker in the summer of 2010. The 16S rRNA gene sequence shared 99.86% and 96.39% similarities with T. oleivorans MIL-1T and T. marinus IMCC1826T, respectively. Pyrosequencing results (16S rRNA gene V3 region) of fifteen oil-degrading consortia across the Arctic Ocean showed that the dominant member in most of the consortia shared identical sequence of this strain, comprising 8.4-99.6% of the total reads (not published).

Here, we described the complete genome sequence and annotation of strain T. oleivorans R6-15, and its phenotypic characteristics. Moreover, a brief comparison was made between strain R6-15 and the two type strains of the validly named species of this genus, in both phenotypic and genomic aspects.

Classification and features

T. oleivorans R6-15 is closely related with T. oleivorans MIL-1T (Figure 1, Table 1). The strain is aerobic, Gram-negative and motile by a single polar flagellum, exhibiting a characteristic morphology of a curved rod-shape cell (Figure 2). Strain R6-15 is able to utilize a restricted spectrum of carbon substrates for growth, including sodium acetate, Tween-40, Tween-80 and C12-C36 aliphatic hydrocarbons. Its growth temperature ranges from 4 to 32°C with optimum of 25°C.

Figure 1

Phylogenetic tree highlighting the position of T. oleivorans strain R6-15 relative to other type and non-type strains with finished or non-contiguous finished genome sequences within the family Oceanospirillaceae. Accession numbers of 16S rRNA gene sequences are indicated in brackets. Sequences were aligned using DNAMAN version 6.0, and a neighbor-joining tree obtained using the maximum-likelihood method within the MEGA version 5.0 [11]. Numbers adjacent to the branches represent percentage bootstrap values based on 1,000 replicates.

Table 1

Classification and general features of T. oleivorans R6-15 according to the MIGS recommendations [12].

MIGS ID

       Property

      Term

        Evidence codea

      Domain Bacteria

        TAS [13]

      Phylum Proteobacteria

        TAS [14]

      Class Gammaproteobacteria

        TAS [15-17]

       Current classification

      Order Oceanospirillales

        TAS [16,18]

      Family Oceanospirillaceae

        TAS [16,19]

      Genus Thalassolituus

        TAS [1]

      Species Thalassolituus oleivorans

        IDA

       Gram stain

      Negative

        IDA

       Cell shape

      Curved rods

        IDA

       Motility

      Motile

        IDA

       Sporulation

      Non-sporulating

        IDA

       Temperature range

      4-32°C

        IDA

       Optimum temperature

      25°C

        IDA

       Carbon source

      Sodium acetate, Tween-40, Tween-80,      alkanes (C12-C36)

        IDA

       Energy source

      Chemoorganotrophic

        IDA

       Terminal electron receptor

      Oxygen

        IDA

MIGS-6

       Habitat

      Surface seawater

        IDA

MIGS-6.3

       Salinity

      0.5-5% NaCl (w/v)

        IDA

MIGS-22

       Oxygen

      Aerobic

        IDA

MIGS-15

       Biotic relationship

      Free-living

        IDA

MIGS-14

       Pathogenicity

      Unknown

        NAS

MIGS-4

       Geographic location

      Chukchi Sea, Arctic Ocean

        IDA

MIGS-5

       Sample collection time

      July 2010

        IDA

MIGS-4.1

       Latitude

      69°30.00′

        IDA

MIGS-4.2

       Longitude

      -168°59.00′

        IDA

MIGS-4.3

       Depth

      Surface seawater

        IDA

MIGS-4.4

       Altitude

      Sea level

        IDA

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 [20]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.

Figure 2

Transmission electron micrograph of T. oleivorans R6-15, using a JEM-1230 (JEOL) at an operating voltage of 120 kV. The scale bar represents 0.5 µm.

When compared to other Thalassolituus species, strain R6-15 differed from type strain MIL-1T [1] in catalase, urease and acid phosphatase, and in the utilization of n-alkane, pyruvic acid methyl ester, D-mannitol and D-sorbitol (Table 2). Differences were also observed with type strain IMCC1826T [2] in growth temperature range, catalase, nitrate reductase, urease and leucine arylamidase and the utilization of n-alkane, pyruvic acid methyl ester, β-Hydroxybutyric acid and D,L-Lactic acid (Table 2).

Table 2

Differential phenotypic characteristics between T. oleivorans R6-15 and other Thalassolituus species.

Characteristic

       1

       2

       3

Cell diameter (µm)

       0.25-0.4 x 1.2-2.0

       0.32-0.77x1.2-3.1

       0.4-0.5 x1.2-2.5

Salinity/Optimum (w/v)

       0.5-5%/ 3%

       0.5-5.7%/ 2.3%

       0.5-5.0%/ 2.5%

Temperature range (°C)

       4-32

       4-30

       15-42

Number of polar flagella

       1

       1-4

       1

Production of

Catalase

       -

       +

       +

Nitrate reductase

       -

       -

       +

Urease

       w

       -

       +

Acid phosphatase

       +

       -

       +

Leucine arylamidase

       +

       +

       -

Carbon source

Sodium acetate

       +

       +

       na

n-alkane

       C12-C36

       C7-C20

       C14 and C16

Pyruvic acid methyl ester

       w

       -

       +

β-Hydroxybutyric acid

       -

       -

       +

D,L-Lactic acid

       -

       -

       +

D-Mannitol

       -

       +

       -

D-Sorbitol

       -

       +

       -

Geographic location

       Chukchi Sea, Arctic Ocean

       Harbor of Milazzo, Italy

       Deokjeok island, Korea

Habitat

       surface seawater

       seawater/sediment

       surface seawater

G+C content (mol%)

       46.6

       46.6

       54.6

Strains: 1, T. oleivorans R6-15; 2, T. oleivorans MIL-1T; 3, T. marinus IMCC1826T. +: positive result, -: negative result, w: weak positive result, na: data not available.

Genome sequencing information

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position and dominance position in the crude oil-degrading consortia enriched from the surface seawater of the Arctic Ocean. The complete genome sequence was deposited in Genbank under accession number CP006829. Sequencing, finishing and annotation of the T. oleivorans R6-15 genome were performed by the Chinese National Human Genome Center (Shanghai). Table 3 presents the project information and its association with MIGS version 2.0 compliance [21].

Table 3

Project information

MIGS ID

      Property

       Term

MIGS-31

      Finishing quality

       Finished

MIGS-28

      Libraries used

       one 454 pyrosequence standard library

MIGS-29

      Sequencing platforms

       454 GS FLX Titanium

MIGS-31.2

      Fold coverage

       21.1 ×

MIGS-30

      Assemblers

       Newbler version 2.7

MIGS-32

      Gene calling method

       NCBI PGAP pipeline

      GenBank ID

       CP006829

      GenBank Date of Release

       On publication

      GOLD ID

       Gi20060

      Project relevance

       Crude oil-degradation, biogeography

Growth conditions and DNA isolation

Strain R6-15 was grown aerobically in ONR7a medium [22] with sodium acetate as the sole carbon and energy source. The genomic DNA was extracted from the cell, concentrated and purified using the AxyPrep bacterial genomic DNA miniprep Kit (Axygen), as detailed in the manual for the instrument.

Genome sequencing and assembly

The genome was sequenced by using a massively parallel pyrosequencing technology (454 GS FLX) [23]. A total of 140,550 reads counting up to 78,223,504 bases were obtained, covered 21.1-folds of genome. The Newbler V2.7 [24] software package was used for sequence assembly and quality assessment. After assembling, 64 contigs ranging from 500 bp to 304,980 bp were obtained, and the relationship of the contigs was determined by multiplex PCR [25]. Gaps were then filled in by sequencing the PCR products using ABI 3730xl capillary sequencers. A total of 284 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Finally, the sequences were assembled using Phred, Phrap and Consed software packages [26], and low quality regions of the genome were re-sequenced. The final sequence accuracy was approximately 99.999%.

Genome annotation

The protein-coding genes, structural RNAs (5S, 16S, 23S), tRNAs and small non-coding RNAs were predicted and achieved by using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server online [27]. The functional annotation of predicted ORFs was performed using RPS-BLAST [28] against the cluster of orthologous groups (COG) database [29] and Pfam database [30]. TMHMM program was used for gene prediction with transmembrane helices [31] and signalP program was used for prediction of genes with peptide signals [32].

Genome properties

The properties and the statistics of the genome are summarized in Table 4. The genome includes one circular chromosome of 3,764,053 bp (46.6% GC content). In total, 3,489 genes were predicted, 3,372 of which are protein-coding genes, and 61 RNAs; 56 pseudogenes were also identified. The majority of the protein-coding genes (67.07%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 5 and Figure 3.

Table 4

Genome statistics

Attribute

       Value

      % of Totala

Genome size (bp)

       3,764,053

      100.0

DNA coding region (bp)

       3,315,444

      88.08

DNA G+C content (bp)

       1,753,947

      46.60

Number of replicons

       1

Extrachromosomal elements

       0

Total genes

       3,489

      100.00

RNA genes

       61

      1.75

tRNA genes

       48

      1.38

rRNA operons

       4

ncRNA genes

       1

      0.03

Protein-coding genes

       3,372

      96.65

Pseudo genes

       56

      1.61

Genes with function prediction

       2,340

      67.07

Genes in paralog clusters

       1,051

      30.12

Genes assigned to COGs

       2,249

      64.46

Genes assigned Pfam domains

       2,576

      73.83

Genes with signal peptides

       338

      9.69

Genes with transmembrane helices

       775

      22.21

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

Table 5

Number of genes associated with the 25 general COG functional categories

Code

     Value

      %age

       Description

J

     182

      7.11

       Translation, ribosomal structure and biogenesis

A

     1

      0.04

       RNA processing and modification

K

     161

      6.29

       Transcription

L

     132

      5.16

       Replication, recombination and repair

B

     1

      0.04

       Chromatin structure and dynamics

D

     32

      1.25

       Cell cycle control, cell division, chromosome partitioning

Y

     0

      0.00

       Nuclear structure

V

     28

      1.09

       Defense mechanisms

T

     152

      5.94

       Signal transduction mechanisms

M

     150

      5.86

       Cell wall/membrane/envelope biogenesis

N

     85

      3.32

       Cell motility

Z

     1

      0.04

       Cytoskeleton

W

     0

      0.00

       Extracellular structures

U

     83

      3.24

       Intracellular trafficking, secretion, and vesicular transport

O

     127

      4.96

       Posttranslational modification, protein turnover, chaperones

C

     143

      5.59

       Energy production and conversion

G

     76

      2.97

       Carbohydrate transport and metabolism

E

     187

      7.30

       Amino acid transport and metabolism

F

     67

      2.62

       Nucleotide transport and metabolism

H

     115

      4.49

       Coenzyme transport and metabolism

I

     106

      4.14

       Lipid transport and metabolism

P

     138

      5.39

       Inorganic ion transport and metabolism

Q

     57

      2.23

       Secondary metabolites biosynthesis, transport and catabolism

R

     329

      12.85

       General function prediction only

S

     207

      8.09

       Function unknown

-

     1240

      35.54

       Not in COGs

Figure 3

Graphical map of the chromosome. From outside to the center: Genes on forward strand (color by COG categories), genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red), GC content, GC skew.

Comparisons with other Thalassolituus species genomes

Until now, only the genome sequence of the type strain T. oleivorans MIL-1T was available within the genus of Thalassolituus [9]. Here, we compared the genome of strain R6-15 with strain MIL-1T (Table 6). The genome of strain R6-15 is nearly 156 kb smaller in size than strain MIL-1T. The G+C content of strain R6-15 (46.6%) is similar with type strain MIL-1T (46.6%). The gene content of strain R6-15 is smaller than strain MIL-1T (3,489 vs 3,732).

Table 6

Comparison of genomes between T. oleivorans R6-15 and T. oleivorans MIL-1T

Genome Name

      Genome      size (bp)

      Gene      count

      Protein      coding

      Protein with      function

      Without      function

      Plasmid      number

      rRNA      operons

T. oleivorans R6-15

      3,764,053

      3,489

      3,372

      2,340

      1,032

      0

      4

T. oleivorans MIL-1T

      3,920,328

      3,732

      3,603

      2,038

      1,565

      0

      4

Strain R6-15 shares 2,995 orthologous genes with type strain MIL-1T. The average percentage of nucleotide sequence identity is 96.92% between strain R6-15 and MIL-1T. In addition, DNA-DNA hybridization (DDH) estimate value between strain R6-15 and MIL-1T were calculated using the genome-to-genome distance calculator (GGDC2.0) [33,34]. The DDH estimate value between them was 84.5% ± 2.57, which were above the standard criteria (70%) [35]. Therefore, these results confirmed that strain R6-15 belonged to the species of Thalassolituus oleivorans.

Conclusion

Strain R6-15 is the first strain with the complete genome sequence of the genus Thalassolituus isolated from the Arctic Ocean. These genomic data will provide insights into the mechanisms of how this bacterium can thrive on the crude oil in the polar marine environments.

Declarations

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (41206158), the China Polar Environment Investigation and Estimate Project (2012-2015), and the Young Marine Science Foundation of SOA (2012142) .


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