Complete genome sequence of Syntrophothermus lipocalidus type strain (TGB-C1T)

Syntrophothermus lipocalidus Sekiguchi et al. 2000 is the type species of the genus Syntrophothermus. The species is of interest because of its strictly anaerobic lifestyle, its participation in the primary step of the degradation of organic maters, and for releasing products which serve as substrates for other microorganisms. It also contributes significantly to maintain a regular pH in its environment by removing the fatty acids through β-oxidation. The strain is able to metabolize isobutyrate and butyrate, which are the substrate and the product of degradation of the substrate, respectively. This is the first complete genome sequence of a member of the genus Syntrophothermus and the second in the family Syntrophomonadaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,405,559 bp long genome with its 2,385 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain TGB-C1 T (= DSM 12680) is the type strain of Syntrophothermus lipocalidus [1] which in turn is the type species of the genus Syntrophothermus [2]. Currently, this is the only species placed in the genus Syntrophothermus. The genus name derives from the Greek words "syn", together with, "trophos", one who feeds, and "thermus", hot, referring to a thermophilic bacterium growing in syntrophic association with hydrogenotrophic organisms at high temperature of around 55°C [1]. The species epithet derives from the Greek word "lipos", fat, and from the Latin adjective "calidus", expert, re-ferring to the organisms trait of specifically utilizing fatty acids [1]. Strain TGB-C1 T was isolated from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) [1]. No further cultivated strains belonging to the species S. lipocalidus have been described so far. Here we describe the features of this organism, together with the complete genome sequence and annotation.

Classification and features
The 16S rRNA gene sequence of strain TGB-C1 T revealed an only distant relationship with the other representatives of the family Syntrophomonadaceae [1] (Figure 1), with Thermosyntropha lipolytica [10] showing the highest degree of sequence similarity (88.1%). The sequence distances of strain TGB-C1 T to other members of this family were 13.6% with Syntrophomonas wolfei subsp. wolfei, 14.0% with S. bryantii, and 14.8% with S. sapovorans, respectively [1]. Further analysis showed 98% 16S rRNA gene sequence identity with an uncultured bacterium represented by clone AR80B63 (AB539943) from the high-temperature Yabase oil field in Japan. The sequence of the 16S rRNA gene of strain TGB-C1 T is identical with two unclassified sequences from an hydrothermal vent metagenome LCHCB.C3615 [11] and from human gut metagenome DNA (contig sequence: F2-Y_011332) [12] (status August 2010), indicating that members of the species, genus and even family are widely represented in the habitats screened so far.
A representative genomic 16S rRNA sequence of S. lipocalidus TGB-C1 T was also compared using BLAST with the most resent release of the Greengenes database [13] and the relative frequencies of taxa and keywords, weighted by BLAST scores, were determined. The five most frequent genera were Moorella (44.1%), Syntrophomonas (33.8%), Clostridium (6.0%), Syntrophothermus (5.6%) and Carboxydocella (3.5%). The species yielding the highest score was Moorella thermoautotrophica. The five most frequent keywords within the labels of environmental samples which yielded hits were 'microbial' (5.5%), 'anaerobic' (4.2%), 'rice' (2.9%), 'soil' (2.8%) and 'populations' (2.8%). The three most frequent keywords within the labels of environmental samples which yielded hits of a higher score than the highest scoring species were 'temperature'(8.2%), 'acetate, coupled, evidence, field, hydrogenotrophic, methanogenesis, oil, oxidation, petroleum, reservoir, syntrophic, yabase' (5.0%) and 'dependent, hot, muddy, reducing , sediment, southwestern, spring, succession, sulfate, taiwan' (3.2%). These keywords largely fit to what is known about the ecology and physiology of strain TGB-C1 T [1]. Figure 1 shows the phylogenetic neighborhood of S. lipocalidus TGB-C1 T , in a 16S rRNA based tree. The sequences of the two 16S rRNA gene copies in the genome differ from each other by up to two nucleotides, and differ by up to two nucleotides from the previously published 16S rRNA sequence (AB021305).
Cells of strain TGB-C1 T are Gram-negative, slightly curved rods with round ends and weakly motile with flagella, 2.4 -4.0 µm long and 0.4 -0.5 µm wide ( Figure 2 and Table 1) [1], occurring singly or in pairs. Roll-tube isolation revealed the presence of small white colonies, lens-shaped and 0.1 -0.2 mm in diameter [1]. The growth rate of the strain TGB-C1 T on 10 mM crotonate was 0.93 ± 0.01 d -1 . Strain TGB-C1 T is strictly anaerobic [1]. It grows on crotonate at temperatures between 45°C and 60°C, with the optimum at 55°C. The pH25°C range for growth is 5.8-7.5, with an optimum at 6.5-7.0 [1]. Strain TGB-C1 T metabolizes in two ways, in pure culture only in the presence of the unsaturated fatty acid crotonate and in co-culture with Methanobacterium thermoautotrophicum strain ΔH in the presence of saturated fatty acids [1]. In pure culture, the fermentation products are acetate and butyrate in equimolar amounts. In co-culture with M. thermoautotrophicum, the substrates used are butyrate, straight-chain fatty acids from C4 to C10 and isobutyrate [1]. By oxidizing fatty acids, S. lipocalidus produces acetate and hydrogen [1], the latter of which is then scavenged by the syntrophic methanogen M. thermoautotrophicum [1]. Syntrophic hydrogenotrophic interactions with bacteria from the genus Methanobacterium have been also observed in the genome sequenced bacterium Aminobacterium colombiense strain ALA-1 T from the phylum Synergistetes [26]. S. lipocalidus is the only species in the family Syntrophomonadaceae that is able to metabolize isobutyrate [2]. Neither yeast extract nor tryptone significantly stimulates growth [1]. In the presence of butyrate as electron donor, the following compounds do not serve as electron acceptors: sulfate, nitrate, sulfite, thiosulfate, fumarate, Fe(III)-nitrilotriacetate [1]. Cell growth is inhibited by ampicillin, chloramphenicol, kanamycin, neomycin, rifampin or vancomycin (each 50 µg ml -1 ) [1].

Chemotaxonomy
To date, no experimental reports have specified the lipid composition of the cell envelope of strain TGB-C1 T . Nevertheless, the cell envelope of the strain TGB-C1 T was Gram-negative stained, although electron micrographs and the 16S rRNA analysis showed that the strain was affiliated to the Gram-positive bacteria [1]. This feature was also observed for another member of the family Syntrophomonadaceae, S. bryantii [22,27]. The cell envelope is composed of the cytoplasmic membrane, an electrondense layer, which is most probably made of peptidoglycan, and an electron-dense outermost wall [1].  [3,4] of the 16S rRNA gene sequence under the maximum likelihood criterion [5] and rooted in accordance with the current taxonomy [6]. 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 [7] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [8] are shown in blue, published genomes in bold [9].  Altitude not reported NAS 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 of the Gene Ontology project [25]. If the evidence code is IDA, then the property was directly observed 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 [28], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [29]. The genome project is deposited in the Genome OnLine Database [8] and the com-plete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [31]. Pyrosequencing reads were assembled using the Newbler assembler version 2.1-PreRelease-4-28-2009-gcc-3.4.6-threads (Roche). The initial Newbler assembly consisting of 16 contigs in one scaffold was converted into a phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired end library. Illumina GAii sequencing data (704 Mb) was assembled with Velvet [32] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. 454 draft assembly was based on 248.9 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [33] was used for sequence assembly and quality assessment in the following finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [31], Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [34]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 37 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [35]. The error rate of the completed genome sequence is less than 1 in 100,000.

Genome annotation
Genes were identified using Prodigal [36] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI [37]. 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 (IMG-ER) platform [38].

Genome properties
The genome consists of a 2,405,559 bp long chromosome with a 51.0% GC content (Table 3 and Figure  3). Of the 2,440 genes predicted, 2,385 were proteincoding genes, and 55 RNAs; 72 pseudogenes were also identified. The majority of the protein-coding genes (70.7%) were assigned with 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.