Complete genome sequence of Sphaerobacter thermophilus type strain (S 6022T)

Sphaerobacter thermophilus Demharter et al. 1989 is the sole and type species of the genus Sphaerobacter, which is the type genus of the family Sphaerobacteraceae, the order Sphaerobacterales and the subclass Sphaerobacteridae. Phylogenetically, it belongs to the genomically little studied class of the Thermomicrobia in the bacterial phylum Chloroflexi. Here, the genome of strain S 6022T is described which is an obligate aerobe that was originally isolated from an aerated laboratory-scale fermentor that was pulse fed with municipal sewage sludge. We describe the features of this organism, together with the complete genome and annotation. This is the first complete genome sequence of the thermomicrobial subclass Sphaerobacteridae, and the second sequence from the chloroflexal class Thermomicrobia. The 3,993,764 bp genome with its 3,525 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
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 nonsporeforming. 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.

Classification and features
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 sur-passed 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.  [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 s equencing 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].

Genome sequencing information 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 [8] and the complete genome sequence 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.

Growth conditions and DNA isolation
S. thermophilus S 6022 T , DSM 20745, was grown in DSMZ medium 467 [20] 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 instructions with modification st/FT for cell lysis according to Wu et al. [21]. Genome sequencing and assembly. The genome was sequenced using a combination of Sanger, 454 and Illumina sequencing platforms.

Genome sequencing and assembly
All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. 454 Pyrosequencing reads were assembled using the Newbler assembler ver-sion 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,435 overlapping fragments of 1,000 bp and entered into the 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 Arachne assembler. Possible mis-assemblies were corrected and gaps between contigs were closed by custom primer walks from sub-clones or PCR products. A total of 109 Sanger finishing reads were produced. Illumina reads were used to improve the final consensus quality using an inhouse developed tool (the Polisher -publication in preparation). The final assembly consists of 35,091 Sanger and 516,954 Roche/454 reads. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 35.9× coverage of the genome.

Genome annotation
Genes were identified using Prodigal [22] 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/) [23]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro data-bases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (http://img.jgi.doe.gov/er) platform [24]. Altitude 518 m 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 [18]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Genome properties
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.