Open Access

Partial genome sequence of the haloalkaliphilic soda lake bacterium Thioalkalivibrio thiocyanoxidans ARh 2T

Standards in Genomic Sciences201510:85

https://doi.org/10.1186/s40793-015-0078-x

Received: 27 March 2015

Accepted: 13 October 2015

Published: 26 October 2015

Abstract

Thioalkalivibrio thiocyanoxidans strain ARh 2T is a sulfur-oxidizing bacterium isolated from haloalkaline soda lakes. It is a motile, Gram-negative member of the Gammaproteobacteria. Remarkable properties include the ability to grow on thiocyanate as the sole energy, sulfur and nitrogen source, and the capability of growth at salinities of up to 4.3 M total Na+. This draft genome sequence consists of 61 scaffolds comprising 2,765,337 bp, and contains 2616 protein-coding and 61 RNA-coding genes. This organism was sequenced as part of the Community Science Program of the DOE Joint Genome Institute.

Keywords

Haloalkaliphilic Soda lakes Sulfur-oxidizing bacteria Thiocyanate

Introduction

Soda lakes are found in many arid zones across the world, such as the Kulunda Steppe in Russia, North-Eastern China, the Rift Valley in Africa, and in arid parts of North America, i.e. California and Nevada. The defining characteristics of these lakes are the abundance of carbonate/bicarbonate anions rather than chloride and their moderate to high salinities. This makes soda lakes a unique habitat with stable, alkaline pH values above nine and up to 11 [1]. Despite the high salinity and alkalinity, soda lakes harbor a rich microbial diversity that is responsible for highly active elemental cycles. Aside from the carbon cycle, the sulfur cycle is of great importance in these lakes [2], yet little is known about their precise biogeochemistry and dynamics [3]. A better understanding of these processes will lead to improved insights into the ecology and biogeochemical cycling in soda lakes. Additionally, sulfur-cycling extremophilic prokaryotes have important applications in bioremediation [4] and more detailed knowledge of their physiology may improve industrial waste processing. For these reasons, we have sequenced more than 70 strains belonging to the genus Thioalkalivibrio , a dominant cultivated group of chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria in soda lakes worldwide. Here we present the partial genome sequence of Thioalkalivibrio thiocyanoxidans ARh 2T.

Organism information

Classification and features

T. thiocyanoxidans ARh 2T forms motile vibrio-like cells of approximately 0.5–0.6 by 0.8–1.4 μm (basic properties are summarized in Table 1). The cells grown with thiocyanate as electron source have a remarkably extended periplasm (Fig. 1). It is a Gram-negative bacterium belonging to the Gammaproteobacteria (Fig. 2). The species description is based on four strains (ARh 2, ARh 3, ARh 4 and ARh 5) that were isolated from sediment samples of South-Western Siberian, Kenyan and Egyptian soda lakes. Strain ARh 2 is a type strain of the T. thiocyanoxidans species. As a chemolithoautotroph, ARh 2T derives energy from the oxidation of inorganic sulfur compounds, such as sulfide, thiosulfate, thiocyanate, elemental sulfur and polysulfides. The most interesting properties are its ability to grow on thiocyanate as the sole source of energy, sulfur and nitrogen and its ability to grow in saturated soda brines brines with thiosulfate as energy source [5].
Table 1

Classification and general features of Thioalkalivibrio thiocyanoxidans ARh 2T [12]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [13]

  

Phylum Proteobacteria

TAS [14, 15]

  

Class Gammaproteobacteria

TAS [15, 16]

  

Order Chromatiales

TAS [15, 17]

  

Family Ectothiorhodospiraceae

TAS [18]

  

Genus Thioalkalivibrio

TAS [19]

  

Species Thioalkalivibrio thiocyanoxidans

TAS [5]

  

Type strain: ARh 2T (DSM 13532)

 
 

Gram stain

Negative

TAS [5, 19]

 

Cell shape

Vibrios

TAS [5]

 

Motility

Motile

TAS [5]

 

Sporulation

Non-sporulating

NAS

 

Temperature range

Mesophilic

TAS [5]

 

Optimum temperature

35–37 °C

TAS [5]

 

pH range; Optimum

8.5–10.5

TAS [5]

 

Carbon source

Inorganic carbon

TAS [5]

MIGS-6

Habitat

Soda lakes

TAS [5]

MIGS-6.3

Salinity

0.3–4.3 M Na+

TAS [5]

MIGS-22

Oxygen requirement

Aerobe

TAS [5]

MIGS-15

Biotic relationship

Free-living

NAS

MIGS-14

Pathogenicity

Non-pathogenic

NAS

MIGS-4

Geographic location

Kenya

TAS [5]

MIGS-5

Sample collection

1999

TAS [5]

MIGS-4.1

Latitude

Not reported

 

MIGS-4.2

Longitude

Not reported

 

MIGS-4.4

Altitude

Not reported

 

aEvidence 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]

Fig. 1

Thin section electron microscopy photograph of cells of strain ARh 2T grown with thiocyanate in batch culture at pH 9.8 and 0.6 M total Na+. OM - outer cell membrane; CM - cytoplasmic membrane; P - periplasm; C - cytoplasm

Fig. 2

Phylogenetic tree based on 16S rRNA sequences comprising the Thioalkalivibrio type strains and several other members of the Ectothiorhodospiraceae family. Black dots mark nodes with a bootstrap value between 90 and 100 %. 16S rRNA sequences of members of the Alphaproteobacteria were used as the outgroup, but pruned from the tree. The tree was constructed using ARB [21] and bootstrap values calculated using MEGA6 [22]

Genome sequencing information

Genome project history

Thioalkalivibrio thiocyanoxidans ARh 2T was sequenced as part of a project aimed at sequencing a large number of Thioalkalivibrio isolates. The goal of this project is to enable the study of the genomic diversity of the dominant genus of sulfur-oxidizing bacteria in soda lakes. T. thiocyanoxidans ARh 2T was selected for its ability to grow in salt-saturated brines (4.3 M Na+) and for its ability to grow on thiocyanate as the sole energy, sulfur and nitrogen source. The permanent draft genome we present here consists of approximately 2.8 million basepairs divided over 61 scaffolds. Sequencing was performed at the Joint Genome Institute under project 1008667. The genome sequence was released in Genbank on December 25, 2014. An overview of the project is given in Table 2.
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Improved high-quality draft

MIGS-28

Libraries used

Illumina standard fragment, 270 bp

MIGS 29

Sequencing platforms

Illumina HiSeq 2000

MIGS 31.2

Fold coverage

1819

MIGS 30

Assemblers

Velvet 1.1.04 [7], ALLPATHS R39750 [8]

MIGS 32

Gene calling method

Prodigal [9], GenePRIMP [10]

 

Locus Tag

G372

 

Genbank ID

ARQK00000000

 

GenBank Date of Release

2014-12-25

 

GOLD ID

Gp0025980

 

BIOPROJECT

PRJNA185302

 

IMG submission ID

12214

MIGS 13

Source Material Identifier

DSM 13532

 

Project relevance

Biotechnology

Growth conditions and genomic DNA extraction

T. thiocyanoxidans ARh 2T (DSM 13532) was cultured in a standard buffer containing sodium carbonate and bicarbonate at pH 10. The total salt concentration was 0.6 M Na+ [6]. The energy source was thiosulfate, at a concentration of 40 mM. After harvesting, the cells were stored at −80 °C for further processing. Genomic DNA was extracted using a chloroform-phenol-isoamylalcohol mixture and precipitated with ethanol. After vacuum drying, the pellet was dissolved in water and the quantity and quality of the DNA determined using the JGI-provided Mass Standard Kit.

Genome sequencing and assembly

This strain was sequenced as part of the Community Science Program of the US Department of Energy Joint Genome Institute. The Illumina HiSeq 2000 platform was used for sequencing, with a depth of 1819X. More details regarding the library construction and sequencing are available at the JGI website. Reads were filtered using DUK and assembled using Velvet 1.1.04 [7]. Pseudoreads (1–3 Kb) were generated from the Velvet output using wgsim and reassembled using ALLPATHS-LG r42328 [8]. The final assembly consists of 61 scaffolds.

Genome annotation

Genes were predicted using Prodigal [9], followed by a round of manual curation using GenePRIMP [10] to detect pseudogenes. The resulting predicted genes were translated and annotated using the NCBI NR database in combination with the UniProt, TIGRFam, Pfam, KEGG, COG and InterPro databases and tRNAScanSE [11] for tRNA prediction. Ribosomal RNAs were detected using models built from SILVA. Further annotation was performed using the Integrated Microbial Genomes platform. All annotation data is freely available there, with IMG submission ID 12214.

Genome properties

The final draft of the genome comprises 2.8 million base pairs in 61 scaffolds, with a G + C percentage of 66.18 %. The gene calling and annotation pipeline detected 2677 genes, of which 2616 code for proteins. Basic statistics concerning the genome sequence are shown in Table 3. In total, 70 % of the genes could be assigned functional categories based on COGs (see Table 4).
Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

2,765,337

100.00

DNA coding (bp)

2,496,809

90.29

DNA G + C (bp)

1,829,984

66.18

DNA scaffolds

61

100.00

Total genes

2677

100.00

Protein coding genes

2616

97.72

RNA genes

61

2.28

Pseudo genes

Not determined

Not determined

Genes in internal clusters

Not determined

Not determined

Genes with function prediction

2230

83.30

Genes assigned to COGs

1885

70.41

Genes with Pfam domains

1799

78.94

Genes with signal peptides

217

8.11

Genes with transmembrane helices

655

24.47

CRISPR repeats

1

100.00

Table 4

Number of genes associated with the 25 general COG functional categories

Code

Value

% age

Description

J

148

7.09

Translation, ribosomal structure and biogenesis

A

1

0.05

RNA processing and modification

K

70

3.36

Transcription

L

98

4.70

Replication, recombination and repair

B

2

0.10

Chromatin structure and dynamics

D

32

1.53

Cell cycle control, Cell division, chromosome partitioning

V

29

1.39

Defense mechanisms

T

105

5.03

Signal transduction mechanisms

M

153

7.33

Cell wall/membrane biogenesis

N

73

3.50

Cell motility

U

72

3.45

Intracellular trafficking and secretion

O

109

5.23

Posttranslational modification, protein turnover, chaperones

C

148

7.09

Energy production and conversion

G

82

3.93

Carbohydrate transport and metabolism

E

145

6.95

Amino acid transport and metabolism

F

60

2.88

Nucleotide transport and metabolism

H

131

6.28

Coenzyme transport and metabolism

I

86

3.02

Lipid transport and metabolism

P

105

5.03

Inorganic ion transport and metabolism

Q

37

1.77

Secondary metabolites biosynthesis, transport and catabolism

R

228

10.93

General function prediction only

S

195

9.35

Function unknown

-

792

29.59

Not in COGs

The total is based on the total number of protein coding genes in the genome

Conclusions

Sequencing of the genome of Thioalkalivibrio thiocyanoxidans ARh 2T is an important step towards a more comprehensive understanding of the mechanism by which this organism can adapt to extremely high salinity. In addition, it will provide important information on the role of this organism in the carbon and sulfur cycles of natural and engineered environments, in particular in the degradation of thiocyanate.

Declarations

Acknowledgements

The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231. Tom Berben and Gerard Muyzer are supported by ERC Advanced Grant PARASOL (No. 322551). Dimitry Sorokin is supported by RBFR Grant 13-04-00049.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Microbial Systems Ecology, Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam
(2)
Winogradsky Institute of Microbiology, RAS
(3)
Department of Biotechnology, Delft University of Technology
(4)
Joint Genome Institute

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Copyright

© Berben et al. 2015