Open Access

Complete genome sequence of Thermanaerovibrio acidaminovorans type strain (Su883T)

  • Mansi Chovatia1,
  • Johannes Sikorski2,
  • Maren Schröder2,
  • Alla Lapidus1,
  • Matt Nolan1,
  • Hope Tice1,
  • Tijana Glavina Del Rio1,
  • Alex Copeland1,
  • Jan-Fang Cheng1,
  • Susan Lucas2,
  • Feng Chen2,
  • David Bruce1, 3,
  • Lynne Goodwin1, 3,
  • Sam Pitluck1,
  • Natalia Ivanova1,
  • Konstantinos Mavromatis1,
  • Galina Ovchinnikova1,
  • Amrita Pati1,
  • Amy Chen4,
  • Krishna Palaniappan4,
  • Miriam Land1, 5,
  • Loren Hauser1, 5,
  • Yun-Juan Chang1, 5,
  • Cynthia D. Jeffries1, 5,
  • Patrick Chain1, 6,
  • Elizabeth Saunders3,
  • John C. Detter1, 3,
  • Thomas Brettin1, 3,
  • Manfred Rohde7,
  • Markus Göker2,
  • Stefan Spring2,
  • Jim Bristow1,
  • Victor Markowitz4,
  • Philip Hugenholtz1,
  • Nikos C. Kyrpides1,
  • Hans-Peter Klenk2 and
  • Jonathan A. Eisen1, 8
Standards in Genomic Sciences20091:1030254

DOI: 10.4056/sigs.40645

Published: 31 December 2009

Abstract

Thermanaerovibrio acidaminovorans (Guangsheng et al. 1997) Baena et al. 1999 is the type species of the genus Thermanaerovibrio and is of phylogenetic interest because of the very isolated location of the novel phylum Synergistetes. T. acidaminovorans Su883T is a Gram-negative, motile, non-spore-forming bacterium isolated from an anaerobic reactor of a sugar refinery in The Netherlands. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from a member of the phylum Synergistetes. The 1,848,474 bp long single replicon genome with its 1765 protein-coding and 60 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords

strictly anaerobic amino acid fermentation thermophile oxidative decarboxylation lithotrophic co-culture with Methanobacterium thermoautotrophicum Synergistales Synergistetes

Introduction

Strain Su883T (= DSM 6589 = ATCC 49978) is the type strain of the species Thermanaerovibrio acidaminovorans, which represents the type species of the two species containing genus Thermanaerovibrio [1]. Strain SU883T is of particular interest because it is able to ferment quite a number of amino acids [2,3], and because its metabolism is greatly enhanced in the presence of the hydrogen scavenger Methanobacterium thermoautotrophicum, from which several single substrates solely hydrogen is formed as reduced fermentation product [3]. The physiological properties of the organism have been studied in detail [2,3].

Here we present a summary classification and a set of features for T. acidaminovorans strain SU883T, together with the description of the complete genome sequencing and annotation.

Classification and features

Until now, strain SU883T was the only strain known from this species. Uncultured clones with a rather high degree of 16S rRNA similarity to the sequence of strain SU883T (AF071414) have been obtained from mesophilic and thermophilic bioreactors treating pharmaceutical wastewater [4] (AF280844, 97.5%; AF280820, 97.7%). The sequence similarities to environmental metagenomic libraries [5,6] were below 81%, indicating a rather poor representation of closely related strains in the analyses habitats (status July 2009).

Figure 1 shows the phylogenetic neighborhood of T. acidaminovorans strain Su883T in a 16S rRNA based tree. The three 16S rRNA gene sequences in the genome of strain Su883T differed from each other by up to three nucleotides, and by up to 29 nucleotides (2%) from the previously published 16S rRNA sequence, generated from DSM 6589 (AF071414). The significant difference between the genome data and the reported 16S rRNA gene sequence, which contains ten ambiguous base calls, is most likely due to sequencing errors in the previously reported sequence data.
Figure 1.

Phylogenetic tree highlighting the position of T. acidaminovorans strain Su883T relative to the other type strains within the phylum Synergistetes. The tree was inferred from 1,333 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum likelihood criterion [9], and was rooted with the type strains of the genera within the phylum ‘Thermotogae’. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Strains with a genome sequencing project registered in GOLD [10] are printed in blue; published genomes in bold.

T. acidaminovorans cells are curved rods of 0.5–0.6 × 2.5–3.0 µm in size (Table 1 and Figure 2), with round ends, occur singly, in pairs, or in long chains when grown in a complex medium [3]. The organism is Gram-negative, non-spore-forming, moderately thermophilic, motile by means of a tuft of lateral flagella at the concave side, and strictly anaerobic for growth [1]. Interestingly, it tolerates flushing with air for at least one hour, and it produces catalase [3]. While being exposed to air, strain Su883T loses its motility [3]. Strain Su883T is able to grow by oxidative decarboxylation of succinate to propionate. A mechanism for reductive propionate formation could be excluded [3]. Glutamate, α-ketoglutarate, histidine, arginine, ornithine, lysine, and threonine are fermented to acetate and propionate. Serine, pyruvate, alanine, glucose, fructose, xylose, glycerol and citrate are fermented to acetate. Branched-chain amino acids are converted to branched-chain fatty acids. Hydrogen is the only reduced end product [3]. The growth and the substrate conversion are strongly enhanced by co-cultivation with methanogens, e.g., M. thermoautotrophicum [3]. Strain Su883T contains b-type cytochromes [3]. Originally, it was reported that in strain Su883T thiosulfate, nitrite, sulfur and fumarate are not reduced [3]. However, a more recent study shows that, although elemental sulfur (1%) inhibits the growth of strain Su883T on glucose, strain Su883T could grow lithoheterotrophically with H2 as electron donor, S0 as electron acceptor, and yeast extract as carbon source [16]. The catablolism of arginine has been studied in detail. Apparently, degradation of arginine occurs by the arginine deiminase (ADI) pathway [2]. No activity of arginase, a key enzyme of the arginase pathway, could be detected [2]. No growth was observed on glycine, aspartate, gelatin, xylose, ribose, galactose, lactose, sucrose, mannose, lactate, ethanol, methanol, acetoin, betaine, malonate, and oxalate [3]. With either succinate, α-ketoglutarate or glutamate, the following enzyme activities were measured in cell free extracts: propionyl CoA:succinate IISCoA transferase, propionate kinase, acetate kinase, glutamate dehydrogenase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, citrate lyase and hydrogenase [3]. The following enzymes were not detected: succinate thiokinase, fumarate reductase, succinate dehydrogenase, β-methylaspartase, hydroxyglutarate dehydrogenase, isocitrate dehydrogenase and formate dehydrogenase [3]. Unfortunately, no chemotaxonomic data are currently available for T. acidaminovorans strain Su883T.
Figure 2.

Scanning electron micrograph of T. acidaminovorans strain Su883T

Table 1.

Classification and general features of T. acidaminovorans strain Su883T according to the MIGS recommendations [11]

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [12]

 

Phylum Synergistetes

TAS [13]

 

Class Synergistia

TAS [13]

 

Order Synergistales

TAS [13]

 

Family Synergistaceae

TAS [13]

 

Genus Thermanaerovibrio

TAS [1]

 

Species Thermanaerovibrio acidamonovorans

TAS [1]

 

Type strain Su883

TAS [1]

 

Gram stain

negative

TAS [3]

 

Cell shape

curved rods, 0.5–0.6 × 2.5–3.0 µm

TAS [3]

 

Motility

motile, lateral flagella

TAS [3]

 

Sporulation

non-sporulating

TAS [3]

 

Temperature range

40–58°C

TAS [3]

 

Optimum temperature

55°C

TAS [3]

 

Salinity

no NaCl required for growth, upper tolerance border unknown

TAS [1]

MIGS-22

Oxygen requirement

strictly anaerobic

TAS [3]

 

Carbon source

succinate, glucose, fructose, amongst others (see text)

TAS [3]

 

Energy source

carbohydrates, amino acids

TAS [3]

MIGS-6

Habitat

granular methanogenic sludge

TAS [3]

MIGS-15

Biotic relationship

free living

NAS

MIGS-14

Pathogenicity

unknown

 
 

Biosafety level

1

TAS [14]

 

Isolation

sludge sample taken from an upflow anaerobic sludge bed (UASB) reactor of a sugar refinery

TAS [3]

MIGS-4

Geographic location

Breda, The Netherlands

TAS [3]

MIGS-5

Sample collection time

1992 or before

TAS [3]

MIGS-4.1

Latitude, Longitude

51.589, 4.774

NAS

MIGS-4.2

MIGS-4.3

Depth

not reported

 

MIGS-4.4

Altitude

not reported

 

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 [15]. If the evidence code is IDA, then the property should have been directly observed for a living isolate by one of the authors, or an expert mentioned in the acknowledgements.

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genomes OnLine Database [10] and the complete genome sequence in GenBank NOT YET. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Table 2.

Genome sequencing project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

Finished

MIGS-28

Libraries used

Three genomic libraries: two Sanger libraries (8 kb pMCL200 and fosmid pcc1Fos) and one 454 pyrosequence standard library

MIGS-29

Sequencing platforms

ABI3730, 454 GS FLX

MIGS-31.2

Sequencing coverage

9.7× Sanger; 9.9× pyrosequence

MIGS-30

Assemblers

Newbler version 1.1.02.15, phrap

MIGS-32

Gene calling method

Prodigal, GenePRIMP

 

INSDC ID

CP001818

 

Genbank Date of Release

November 19, 2009

 

GOLD ID

Gc01091

 

INSDC project ID

29531

 

Database: IMG-GEBA

2501651200

MIGS-13

Source material identifier

DSM 6589

 

Project relevance

Tree of Life, GEBA

Growth conditions and DNA isolation

T. acidaminovorans strain Su883T, DSM 6589, was grown anaerobically in DSMZ medium 104 (modified PYG medium) [17] at 55°C. DNA was isolated from 1–1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol without modification according to Wu et al. [18].

Genome sequencing and assembly

The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website (http://www.jgi.doe.gov/). 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 2,046 overlapping fragments of 1,000 bp and 1,838 of them entered into the final assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [19]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 401 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 19.6 ×coverage of the genome. The final assembly contains 19,461 Sanger and 358,573 pyrosequencing reads.

Genome annotation

Genes were identified using Prodigal [20] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline (http://geneprimp.jgi-psf.org/) [21]. 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. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (http://img.jgi.doe.gov/er) platform [22].

Genome properties

The genome is 1,848,474 bp long and comprises one main circular chromosome with a 63.8% GC content. (Table 3, Figure 3). Of the 1,825 genes predicted, 1,765 were protein coding genes, and 60 RNAs. In addition, 27 pseudogenes were identified. The majority of genes (79.3%) 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 4.
Figure 3.

Graphical circular map of the genome. 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, other RNAs black), GC content, GC skew.

Table 3.

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

1,848,474

100.00%

DNA Coding region (bp)

1,745,505

94.43%

DNA G+C content (bp)

1,179,189

63.79%

Number of replicons

1

 

Extrachromosomal elements

0

 

Total genes

1,825

100.00%

RNA genes

60

3.29%

rRNA operons

3

 

Protein-coding genes

1,765

96.71%

Pseudo genes

27

1.48%

Genes with function prediction

1,447

79.29%

Genes in paralog clusters

142

7.78%

Genes assigned to COGs

1,483

81.26%

Genes assigned Pfam domains

1,484

81.32%

Genes with signal peptides

275

15.07%

Genes with transmembrane helices

404

22.14%

CRISPR repeats

0

 
Table 4.

Number of genes associated with the general COG functional categories

Code

Value

%age

Description

J

150

8.5

Translation, ribosomal structure and biogenesis

A

0

0.0

RNA processing and modification

K

84

4.8

Transcription

L

71

4.0

Replication, recombination and repair

B

0

0.0

Chromatin structure and dynamics

D

26

1.5

Cell cycle control, mitosis and meiosis

Y

0

0.0

Nuclear structure

V

11

0.6

Defense mechanisms

T

101

5.7

Signal transduction mechanisms

M

97

5.5

Cell wall/membrane biogenesis

N

71

4.0

Cell motility

Z

0

0.0

Cytoskeleton

W

0

0.0

Extracellular structures

U

38

2.2

Intracellular trafficking and secretion

O

53

3.0

Posttranslational modification, protein turnover, chaperones

C

126

7.1

Energy production and conversion

G

86

4.9

Carbohydrate transport and metabolism

E

185

10.5

Amino acid transport and metabolism

F

66

3.7

Nucleotide transport and metabolism

H

97

5.5

Coenzyme transport and metabolism

I

32

1.8

Lipid transport and metabolism

P

63

3.6

Inorganic ion transport and metabolism

Q

18

1.0

Secondary metabolites biosynthesis, transport and catabolism

R

152

8.6

General function prediction only

S

104

5.9

Function unknown

-

282

16.0

Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Susanne Schneider (DSMZ) for DNA extraction and quality analysis. This work was performed under the auspices of the US Department of Energy 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, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, as well as German Research Foundation (DFG) INST 599/1-1.

Authors’ Affiliations

(1)
DOE Joint Genome Institute
(2)
DSMZ - German Collection of Microorganisms and Cell Cultures GmbH
(3)
Bioscience Division, Los Alamos National Laboratory
(4)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory
(5)
Oak Ridge National Laboratory
(6)
Lawrence Livermore National Laboratory
(7)
HZI - Helmholtz Centre for Infection Research
(8)
University of California Davis Genome Center

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Copyright

© The Author(s) 2009