Complete genome sequence of Thermanaerovibrio acidaminovorans type strain (Su883T)

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.


Introduction
Strain Su883 T (= 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 SU883 T 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 SU883 T , together with the description of the complete genome sequencing and annotation.

Classification and features
Until now, strain SU883 T was the only strain known from this species. Uncultured clones with a rather high degree of 16S rRNA similarity to the sequence of strain SU883 T (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 Su883 T in a 16S rRNA based tree. The three 16S rRNA gene sequences in the genome of strain Su883 T 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 Su883 T 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 Su883 T loses its motility [3]. Strain Su883 T 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. Hy-drogen 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 Su883 T contains b-type cytochromes [3]. Originally, it was reported that in strain Su883 T 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 Su883 T on glucose, strain Su883 T could grow lithoheterotrophically with H2 as electron donor, S 0 as electron acceptor, and yeast extract as carbon source [16]. The catabolism 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, αketoglutarate 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, βmethylaspartase, hydroxyglutarate dehydrogenase, isocitrate dehydrogenase and formate dehydrogenase [3]. Unfortunately, no chemotaxonomic data are currently available for T. acidaminovorans strain Su883 T .  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.  [18].

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All gen-eral 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 misassemblies 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.