Open Access

Permanent draft genome sequence of Vibrio tubiashii strain NCIMB 1337 (ATCC19106)

  • Ben Temperton,
  • , Simon Thomas,
  • , Karen Tait
  • , Helen Parry
  • , Matt Emery
  • , Mike Allen
  • , John Quinn
  • , John MacGrath
  • and Jack Gilbert, ,

DOI: 10.4056/sigs.1654066

Received: 29 April 2011

Published: 29 April 2011

Abstract

Vibrio tubiashii NCIMB 1337 is a major and increasingly prevalent pathogen of bivalve mollusks, and shares a close phylogenetic relationship with both V. orientalis and V. coralliilyticus. It is a Gram-negative, curved rod-shaped bacterium, originally isolated from a moribund juvenile oyster, and is both oxidase and catalase positive. It is capable of growth under both aerobic and anaerobic conditions. Here we describe the features of this organism, together with the draft genome and annotation. The genome is 5,353,266 bp long, consisting of two chromosomes, and contains 4,864 protein-coding and 86 RNA genes.

Introduction

The genus Vibrio is both numerous and ubiquitous within marine environments, with Vibrio species harbored within many diverse marine organisms, such as mollusks, shrimps, fishes, cephalopods and corals [1]. Comparative genome analysis has revealed a huge genetic diversity within this genus, which is driven by mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g. the acquisition of bacteriophages, pathogenicity islands, and super-integrons), the combination of which presumably stimulates genetic and functional diversity and allows this group to colonize a wide variety of ecological niches and hosts [1,2].

Vibrio tubiashii was first described as three strains of Vibrio anguillarum by Tubiash et al [3] in 1965. The organisms were isolated from bivalve mollusks during an outbreak of bacillary necrosis in Milford, Connecticut, and deposited in the American Type Culture Collection as ATCC 19105, 19106 and 19109. These three strains were further elucidated and formally named as V. tubiashii by Hada et al [4] in 1984. Subsequently, several virulence factors have been identified [5,6] and the organism is increasingly implicated in major disease outbreaks in bivalve mollusks [1].

V. tubiashii is closely related to the proposed coral pathogen V. coralliilyticus, as well as V. orientalis, a bacterium associated with penaeid shrimps [7]. Indeed, V. coralliilyticus was initially designated as a V. tubiashii strain [8,9] due to their close similarity.

Classification and features

Vibrio tubiashii 1337 belongs to the Gammaproteobacteria and are contained within the family, Vibrionaceae [Table 1]. Cells of Vibrio tubiashii are Gram-negative curved-rods of approximately 0.5 by 1.5 µm, which are motile in liquid media by means of a single sheathed, polar flagellum [3,4] These cells are facultative anaerobes, [3,4,22]. It is catalase and oxidase positive, capable of splitting indole from tryptophan, and can use glucose, xylose, mannitol, rhamnose, sucrose, arabinose and acetate as sole carbon sources, and has β-galactosidase activity, despite an apparent inability to ferment lactose. V. tubiashii is capable of dissimilatory nitrate and nitrite reduction under anaerobic conditions, can use organic phosphorus during phosphate limitation, and can utilize 2-aminoethylphosphonate as a sole phosphorus source.

Table 1

Classification and general features of V. tubiashii according to the MIGS recommendations

     MIGS ID

    Property

     Term

     Evidence code

     Domain Bacteria

     TAS [10]

     Phylum Proteobacteria

     TAS [11]

     Class Gammaproteobacteria

     TAS [12,13]

    Current classification

     Order Vibrionales

     TAS [14]

     Family Vibrionaceae

     TAS [15,16]

     Genus Vibrio

     TAS [15,17-19]

     Species Vibrio tubiashii NCIMB 1337

     TAS [4]

    Gram stain

     negative

     IDA

    Cell shape

     Curved rods (vibroid)

     IDA

    Motility

     motile via single polar flagellum

     IDA

    Sporulation

     Non-sporulating

     IDA

    Temperature range

     Mesophile 12-30oC

     IDA

    Optimum temperature

     25oC

     IDA

     MIGS 6.3

    Salinity

     Slightly halophylic, optimum 1-3% NaCl

     IDA

     MIGS-22

    Oxygen requirement

     Aerobic/ facultative anaerobic

     IDA

    Carbon source

     Highly diverse

     IDA

    Energy source

     Highly diverse

     IDA

     MIGS-6

    Habitat

     Marine invertebrates

     TAS [20]

     MIGS-16

    Biotic relationship

     Parasitic

     TAS [3]

     MIGS-14

    Biosafety level

     2

     TAS [4]

    Isolation

     Moribund juvenile oyster (Crassostrea virginica)

     TAS [3,4]

     MIGS-4

    Geographical location

     Milford, Connecticut, USA

     TAS [3]

     MIGS-5

    Sample collection time

     01/02/1965

     TAS [3]

     MIGS 4.1

    latitude

     41.22 N

     TAS [3]

     MIGS 4.2

    longitude

     -73.06 W

     TAS [3]

     MIGS 4.3

    Depth

     Not reported

     MIGS 4.4

    Altitude

     Marine

     TAS [3]

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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 [21]. If the evidence code is IDA, then the property was directly observed, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.

V. tubiashii has an absolute requirement for sodium and chloride ions, and is incapable of growth on media containing less than 0.5% W/V NaCl. The temperature optimum for growth is 25oC, but growth does occur in the range of 12-30oC. The organism is killed at 37oC. V. tubiashii has a biphasic pH response and grows optimally at both pH 8.0 and 6.5, but displays weakened growth at pH 7.0 and 7.5. The bacterium shows rapid growth on marine broth and produces buff colored, opaque, irregular, slightly convex colonies on marine agar, and yellow colonies, characteristic of the Vibrionaceae, on Thiosulfate-Citrate-Bile-Sucrose Agar (TCBS).

Growth conditions and DNA isolation

Vibrio tubiashii NCIMB 1337 (ATCC19106) was grown in marine broth (seawater + 1 gl-1 yeast extract and 0.5 gl-1 tryptone) at 25oC for 24 hours. DNA was extracted using the Qiagen DNAeasy blood and tissue kit, without modification of the manufacturer’s protocol.

Genome sequencing and annotation

Genome sequencing

The genome was sequenced using the Illumina sequencing platform. All general aspects of library construction and sequencing performed at the NERC Biomolecular analysis facility can be found on the NBAF website [23]. SOLEXA Illumina reads were assembled using VELVET Large Newbler contigs that were broken into 4,074 overlapping fragments of 1,000 bp and entered into the assembly as pseudo-reads. The sequences were assigned quality scores based on consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. The error rate of the completed genome sequence is less than 1 in 100,000. Overall sequencing provided 131 × coverage of the genome.

Genome annotation

Genes were identified using the RAST server The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. The tRNAScanSE tool [24] was used to find tRNA genes, whereas ribosomal RNAs were found by using BLASTn against the ribosomal RNA databases. The RNA components of the protein secretion complex and the RNaseP were identified by searching the genome for the corresponding Rfam profiles using INFERNAL [25]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform developed by the Joint Genome Institute, Walnut Creek, CA, USA [26,27].

Genome project information

This organism was selected for sequencing on the basis of its increasing impact as a bivalve pathogen, and was funded by i-G Peninsula. The genome project is deposited in the IMG database and the complete genome sequence in GenBank (CP001643). Sequencing, finishing and annotation were performed by the GenePool Team at NERC Biomolecular Analysis Facility (NBAF) Edinburgh. A summary of the project information is shown in Table 2.

Table 2

Project information

    MIGS ID

     Property

      Term

    MIGS-31

     Finishing quality

      Draft

    MIGS-28

     Libraries used

      Illumina

    MIGS-29

     Sequencing platforms

      Illumina SOLEXA GAIIx

    MIGS-31.2

     Fold coverage

      131×

    MIGS-30

     Assemblers

      Velvet

    MIGS-32

     Gene calling method

      RAST

     Genome Database release

      181

     Genbank ID

      866909

     Genbank Date of Release

      December 12, 2010

     GOLD ID

      Gi07317

Genomic properties

The genome was assembled into 335 contigs and includes two circular chromosomes combining to give a total size of 5,353,266 bp (44.84% GC content). A total of 4,950 genes were predicted, 4,864 of which are protein-coding genes. 74.22% of protein coding genes were assigned to a putative function with the remaining annotated as hypothetical proteins. 658 protein coding genes belong to paralogous families in this genome corresponding to a gene content redundancy of 13.29%. The properties and the statistics of the genome are summarized in Tables 3-5.

Table 3

Summary of genome*

    Label

    Size (Mb)

    Chromosome 1

    3.4

    Chromosome 2

    1.9

* Two chromosomes with no plasmids. Approximate chromosome size estimated by Pulse field gel electrophoresis

Table 5

Number of genes associated with the 25 general COG functional categories

Code

    Value

   %age

     Description

J

    200

   4.86

     Translation

A

    1

   0.02

     RNA processing and modification

K

    369

   8.96

     Transcription

L

    154

   3.74

     Replication, recombination and repair

B

    1

   0.02

     Chromatin structure and dynamics

D

    37

   0.9

     Cell cycle control, mitosis and chromosome partitioning

Y

     Nuclear structure

V

    75

   1.82

     Defense mechanisms

T

    432

   8.31

     Signal transduction mechanisms

M

    227

   5.51

     Cell wall/membrane biogenesis

N

    148

   3.59

     Cell motility

U

    146

   3.55

     Intracellular trafficking and secretion

O

    173

   4.2

     Posttranslational modification, protein turnover, chaperones

C

    203

   4.93

     Energy production and conversion

G

    248

   6.02

     Carbohydrate transport and metabolism

E

    348

   8.45

     Amino acid transport and metabolism

F

    105

   2.55

     Nucleotide transport and metabolism

H

    159

   3.86

     Coenzyme transport and metabolism

I

    119

   2.89

     Lipid transport and metabolism

P

    188

   4.57

     Inorganic ion transport and metabolism

Q

    77

   1.77

     Secondary metabolites biosynthesis, transport and catabolism

R

    445

   10.81

     General function prediction only

S

    356

   8.65

     Function unknown

-

    1276

   25.78

     Not in COGs

a) The total is based on the total number of protein coding genes in the annotated genome.

Table 4

Nucleotide content and gene count levels of the genome

     Attribute

    Value

    % of totala

     Size (bp)

    5,353,266

    100%

     G+C content (bp)

    2,400,750

    44.87%

     Coding region (bp)

    4,627,782

    86.45%

     Total genesb

    4950

    100%

     RNA genes

    86

    1.74%

     Protein-coding genes

    4864

    98.26%

     Genes in paralog clusters

    658

    13.29%

     Genes assigned to COGs

    3674

    74.22%

     Genes with signal peptides

    1655

    33.43%

     Genes with transmembrane helices

    1167

    23.58%

     Paralogous groups

    658

    13.29%

a)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.

b)Also includes 54 pseudogenes and 5 other genes.

Genomic comparison

Based on COG I.D the Vibrio tubiashii genome shows most similarity to the genome of V coralliilyticus (R2 = 0.96) and to V. orientalis (R2 = 0.94), while showing less similarity to V. shilonii (R2= 0.86) [Table 6]. This is in contrast to the 16S-based analysis shown in Figure 1. However, it should be noted that 16S rRNA analysis often poorly discriminates vibrios due to low sequence heterogeneity in the 16S gene [28].

Table 6

Comparison of the genome of Vibrio tubiashii NCIMB 1337 with other sequenced Vibrios

Genome Name

   Vibrio coralliilyticus ATCC BAA-450

    Vibrio orientalis CIP 102891

   Vibrio shilonii AK1

   Vibrio tubiashii NCIMB 1337

Genes

   5,144

4,297

5,438

4,950

RNA

   122

128

78

86

w/ Func Pred

   3,687

3185

3,517

4,062

w/ Func Pred %

   71.68%

74.12%

64.67%

82.06%

Enzymes

   1,143

1,058

1,258

1,116

Enzymes %

   22.22%

24.62%

23.13%

22.55%

KEGG

   1397

1,257

1,511

1,354

KEGG %

   27.16%

29.25%

27.79%

27.35%

COG

   3815

3,302

4,093

3,674

COG %

   74.16%

76.84%

75.27%

74.22%

Pfam

   4127

3,520

4,379

3,976

Pfam %

   80.23%

81.92%

80.53%

80.32%

TIGRfam

   1,643

1,515

1,708

1,651

TIGRfam %

   31.94%

35.26%

31.41%

33.35%

Signal peptide

   1,733

1,408

1,214

1,655

Signal peptide %

   33.69%

32.77%

22.32%

33.43%

TransMb

   1,227

1,018

1,326

1,167

TransMb Perc

   23.85%

23.69%

24.38%

23.58%

Pfam Clusters

   2,183

2,091

2,163

2,186

COG Clusters

   2,030

1,943

2,087

2,041

TIGRfam Clusters

   1,310

1,246

1,300

1,323

GC Perc

   0.46

0.45

0.44

0.45

Bases

   5,680,628

4698244

5,701,826

5,353,266

Figure 1

Phylogenetic tree highlighting the position of V. tubiashii NCIMB 1337 relative to other Vibrio strains. The tree was inferred from 1,159 aligned characters of the 16S rRNA gene sequence under the neighborhood joining criterion. Numbers above the branches are support values from 1,000 bootstrap replicates if greater than 60%.

Regulatory systems

The Vibrio tubiashii NCIMB 1337 genome contains multiple quorum sensing systems, most notably a luxM/N system which has two adjacent copies of the luxN gene. In addition, there is a luxS/PQ system, with the lux P and Q gene appearing consecutively. There is also a cqsA/S system. It is probable that these three systems converge on the phospho-relay transfer system encoded by the luxO/luxU/hapR genes. There are two additional lux genes (LuxT and LuxZ). The genome also contains the rpoN gene encoding for the sigma-54 factor, which may indicate the presence of the two-component phosphorylation-dephosphorylation cascade described in V. harveyi [29] (note: Vibrio harveyi is also known as Lucibacterium harveyi and Beneckea harveyi.)

Antibiotic resistance

There are six separate genes encoding for putative β-lactamases within the genome, but only two have homology at the protein levels with any know Vibrio β-lactamases. There is also a multi-antibiotic resistance protein MarC, associated with an operon containing a variety of multidrug resistance proteins. This operon is controlled by a MerR type transcriptional regulator, which is often associated with antibiotic resistance [30], and may account for the kanamycin resistance observed in this strain by the authors.

Declarations

Acknowledgements

We wish to thank i-G Peninsula (Prospect Place, the Hoe, Plymouth, Devon, UK) for providing funding for this project, and NBAF Edinburgh for performing the sequencing.


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