Open Access

Complete genome sequence of Thermanaerovibrio acidaminovorans type strain (Su883T)

  • Mansi Chovatia
  • , Johannes Sikorski
  • , Maren Schröder
  • , Alla Lapidus
  • , Matt Nolan
  • , Hope Tice
  • , Tijana Glavina Del Rio
  • , Alex Copeland
  • , Jan-Fang Cheng
  • , Susan Lucas
  • , Feng Chen
  • , David Bruce,
  • , Lynne Goodwin,
  • , Sam Pitluck
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Galina Ovchinnikova
  • , Amrita Pati
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , Patrick Chain,
  • , Elizabeth Saunders
  • , John C. Detter,
  • , Thomas Brettin,
  • , Manfred Rohde
  • , Markus Göker
  • , Stefan Spring
  • , Jim Bristow
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Nikos C. Kyrpides
  • , Hans-Peter Klenk
  • and Jonathan A. Eisen,
Corresponding author

DOI: 10.4056/sigs.40645

Received: 22 November 2009

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 anaerobicamino acid fermentationthermophileoxidative decarboxylationlithotrophicco-culture with Methanobacterium thermoautotrophicumSynergistalesSynergistetes

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.

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 MIGS-4.2

Latitude, Longitude

  51.589, 4.774

NAS

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.

Figure 2

Scanning electron micrograph of T. acidaminovorans strain Su883T

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

  9.7x 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 (Web Site). 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 (Web Site) [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 (Web Site) 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.

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

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 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.


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References

  1. Baena S, Fardeau ML, Woo TH, Ollivier B, Labat M and Patel BK. Phylogenetic relationships of three amino-acid-utilizing anaerobes, Selenomonas acidaminovorans, 'Selenomonas acidaminophila' and Eubacterium acidaminophilum, as inferred from partial 16S rDNA nucleotide sequences and proposal of Thermanaerovibrio acidaminovorans gen. nov., comb. nov. and Anaeromusa acidaminophila gen. nov., comb. nov. Int J Syst Bacteriol. 1999; 49:969-974PubMed
  2. Plugge CM and Stams AJM. Arginine catabolism by Thermanaerovibrio acidaminovorans. FEMS Microbiol Lett. 2001; 195:259-262 View ArticlePubMed
  3. Guangsheng C, Plugge CM, Roelofsen W, Houwen FP and Stams AJM. Selenomonas acidaminovorans sp. nov., a versatile thermophilic proton-reducing anaerobe able to grow by decarboxylation of succinate to propionate. Arch Microbiol. 1992; 157:169-175
  4. LaPara TM, Nakatsu CH, Pantea L and Alleman JE. Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Appl Environ Microbiol. 2000; 66:3951-3959 View ArticlePubMed
  5. Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, Takami H, Morita H, Sharma VK and Srivastava TP. Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes. DNA Res. 2007; 14:169-181 View ArticlePubMed
  6. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen J, Nelson KE and Nelson W. Environmental genome shotgun sequencing of the Sargasso Sea. Science. 2004; 304:66-74 View ArticlePubMed
  7. Lee C, Grasso C and Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics. 2002; 18:452-464 View ArticlePubMed
  8. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000; 17:540-552PubMed
  9. Stamatakis A, Hoover P and Rougemont J. A Rapid Bootstrap Algorithm for the RAxML Web Servers. Syst Biol. 2008; 57:758-771 View ArticlePubMed
  10. Liolios K, Mavromatis K, Tavernarakis N and Kyrpides NC. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2008; 36:D475-D479 View ArticlePubMed
  11. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  12. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  13. Jumas-Bilak E, Roudiere L and Marchandin H. Description of 'Synergistetes' phyl. nov. and emended description of the phylum 'Deferribacteres' and of the family Syntrophomonadaceae, phylum 'Firmicutes'. Int J Syst Evol Microbiol. 2009; 59:1028-1035 View ArticlePubMed
  14. Anonymous. Biological Agents: Technical rules for biological agents TRBA 466.Web Site
  15. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  16. Zavarzina DG, Zhilina T, Tourova T, Kuznetsov B, Kostrikina N and Bonch-Osmolovskaya EA. Thermanaerovibrio velox sp. nov., a new anaerobic, thermophilic, organotrophic bacterium that reduces elemental sulfur, and emended description of the genus Thermanaerovibrio. Int J Syst Evol Microbiol. 2000; 50:1287-1295PubMed
  17. List of growth media used at DSMZ: Web Site
  18. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova N, Kunin V, Goodwin L, Wu M and Tindall BJ. A phylogeny-driven genomic encyclopedia of Bacteria and Archaea. Nature. 2009; 462:1056-1060 View ArticlePubMed
  19. Sims D, Brettin T, Detter JC, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F and Lucas S. Complete genome of Kytococcus sedentarius type strain (541T). Stand Genomic Sci. 2009; 1:12-20 View Article
  20. Anonymous. Prodigal Prokaryotic Dynamic Programming Genefinding Algorithm. Oak Ridge National Laboratory and University of Tennessee 2009 Web Site
  21. Pati A, Ivanova N, Mikhailova, N, Ovchinikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. (Submitted).
  22. Markowitz VM, Mavromatis K, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed