Strain S 6022^T (DSM 20745 = ATCC 49802 = NCIMB 13125) is the type strain of the species Sphaerobacter thermophilus, representing the type species of the genus Sphaerobacter. S. thermophilus was described by Demharter et al. in 1989 [1]. It is Gram-positive, non-motile and non-sporeforming. It was originally isolated from thermal treated municipal sewage sludge from München-Grosslappen, Germany [2]. Cells of S. thermophilus were also identified in three other municipal sludge stabilization plants spread across Germany (Isenbüttel, Nettetal, and Gemmingen) using an immunolabelling procedure. From the operating parameters of these plants a minimum temperature growth range of 40-65°C can be predicted [2]. Here we present a summary classification and a set of features for S. thermophilus strain S 6022^T, together with the description of the complete genomic sequencing and annotation.

The closest related cultivated organism with a 16S rRNA sequence recorded in Genbank is Thermomicrobium roseum (DSM 5159) [3,4], which shares a mere 87% sequence similarity with strain S 6022^T, indicating that S. thermophilus is phylogenetically one of the most isolated bacterial species. Only some uncultivated bacterial clones show a slightly closer relationship, e.g. clone Amb_16S_1237 (EF018775) isolated from Populus tremula (trembling aspen, 92%), EU035785 and EF643378 from soil in a radish-rich area in Jaunpur (India), clone AKYG1722 from farm soil adjacent to a silage storage bunker in Minnesota (89%), and AM935838 from a pilot-scale bioremediation process of hydrocarbon-contaminated soil in France (88%). None of the phylotypes sequenced during environmental screenings or genomic surveys surpassed 82% sequence similarity with strain S 6022^T, expressly underlining the phylogenetically isolated and rare occurrence of S. thermophilus (status May 2009).

Figure 1 shows the phylogenetic neighborhood of S. thermophilus strain S 6022^T in a 16S rRNA based tree. The sequence of the sole 16S rRNA gene in the genome of strain S 6022^T differs by six nucleotides (0.4%) from the previously published 16S rRNA sequence generated from DSM 20745 (AJ420142). The difference between the genome data and the previously reported 16S rRNA in GenBank gene sequence is most likely due to sequencing errors in the latter.

Figure 1 Phylogenetic tree of S. thermophilus strain S 6022T and all type strains of the phylum Chloroflexi, inferred from 1,304 aligned characters [5,6] of the 16S rRNA gene sequence under the maximum likelihood criterion [7]. The tree was rooted with the members of Anaerolineae and Caldilineae within the Chloroflexi. 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%. Lineages with type strain genome sequencing projects registered in GOLD [8] are shown in blue, published genomes in bold.

S. thermophilus S 6022^T cells are coccoid (Figure 2), but are also described as coccoid rods, 1-1.5 by 1.5-3 µm, in older cultures or in glucose-free medium irregular club- or dumb-bell shaped forms [1]. Branched cells are not observed. Colonies on Ottow Medium (DSMZ Medium No. 467) [9] are opaque, circular with entire margin and reach a diameter of 1-2 mm after 3 days of incubation at 60°C. The strain grows strictly aerobically with optimal growth at 55°C and pH 8.5 (Table 1). There is no acid production from glucose. Strain S 6022^T possesses catalase and oxidase and hydrolyzes starch but not gelatin, casein or cellulose [1]. Strain S 6022^T shares many features such as thermophilia, optimal pH for growth, and lack of motility with its closest relative, T. roseum (DSM 5159) [3,4]. The genome sequence as presented here might contribute to the solution of the question if S. thermophilus, like T. roseum encodes a complete flagellar system [4], although neither strain is motile. Interestingly, none of the other species in the Chloroflexi for which a genome sequence currently exists encode for any flagellar structural components [4].

Figure 2 Scanning electron micrograph of S. thermophilus S strain 6022T

The two replicons containing genome is 3,993,764 bp long with a 68.1% GC content (Table 3 and Figure 3). Of the 3,582 genes predicted, 3525 were protein coding genes, and 57 RNAs; 40 pseudogenes were also identified. The majority of the protein-coding genes (72.3%) were assigned a putative function while those remaining were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 4.

Figure 3 Graphical circular map of the genome. Chromosome (left), plasmid (right), drown not in scale. 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.