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, referring 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.

The 16S rRNA gene sequence of strain TGB-C1^T revealed an only distant relationship with the other representatives of the family Syntrophomonadaceae [1] (Figure1), 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.

Figure 1 Phylogenetic tree highlighting the position of S. lipocalidus TGB-C1T relative to the type strains within the family Syntrophomonadaceae. The trees were inferred from 1,434 aligned characters [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].

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 pH_25°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].

Figure 2 Scanning electron micrograph of S. lipocalidus TGB-C1T

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 protein-coding 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.

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.