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Complete genome sequence of Spirochaeta smaragdinae type strain (SEBR 4228T)
Standards in Genomic Sciences volume 3, pages 136–144 (2010)
Spirochaeta smaragdinae Magot et al. 1998 belongs to the family Spirochaetaceae. The species is Gram-negative, motile, obligately halophilic and strictly anaerobic and is of interest because it is able to ferment numerous polysaccharides. S. smaragdinae is the only species of the family Spirochaetaceae known to reduce thiosulfate or element sulfur to sulfide. This is the first complete genome sequence in the family Spirochaetaceae. The 4,653,970 bp long genome with its 4,363 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain SEBR 4228T (= DSM 11293 = JCM 15392) is the type strain of the species Spirochaeta smaragdinae. Currently, there are eighteen species  and two subspecies in the genus Spirochaeta [1,2]. The generic name derives from the Greek word ‘speira’ meaning ‘a coil’ and the Greek word ‘chaitê’ meaning ‘hair’, referring to the spiral shape of bacterial cell. The species epithet is derived from the Latin word ‘smaragdinae’ meaning ‘from Emerald’, referring to the name Emerald of an oil field in Congo. Strain SEBR 4228T was isolated from an oil-injection production water sample of a Congo offshore oilfield  and described in 1997 by Magot et al. as ‘Spirochaeta smaragdinae’ . Here we present a summary classification and a set of features for S. smaragdinae SEBR 4228T, together with the description of the complete genomic sequencing and annotation.
Classification and features
Strain SEBR 4228T shares 82.2–99.0% 16S rRNA gene sequence identity with the type strains from the other members of genus Spirochaeta , with the type strain of S. bajacaliforniensis , isolated from a mud sample in Laguna Figueroa (Baja California, Mexico) showing the highest degree of sequence similarity (99%). Notwithstanding the high degree of 16S rRNA gene sequence identity, these two strains are characterized by low genomic similarity (38%) in DNA-DNA hybridization studies and differ by numerous differences in carbon source utilization . Several type strains from the genus Treponema show the highest degree of similarity for non-Spirochaeta strains (82.9-83.6%) . A representative genomic 16S rRNA sequence of strain SEBR 4228T was compared using BLAST with the most recent release of the Greengenes database  and the relative frequencies of taxa and keywords, weighted by BLAST scores, were determined. The three most frequent genera were Spirochaeta (76.4%), ‘Sphaerochaeta’ (15.8%) and Cytophaga (7.8%). Within the five most frequent keywords in the labels of environmental samples were ‘microbial’ (11.7%), ‘mat’ (10.5%), ‘hypersaline’ (7.7%), and ‘sediment’ (1.7%). The environmental samples database (env_nt) contains the marine metagenome genomic clone 1061006082084 (EK988302) that is 92% identical to the 16S rRNA gene sequence of SEBR 4228T. No phylotypes from genomic surveys could be linked to the species S. smaragdinae or even the genus Spirochaeta, indicating a rather rare occurrence of these in the habitats screened so far (as of August 2010).
Figure 1 shows the phylogenetic neighborhood of S. smaragdinae SEBR 4228T in a 16S rRNA based tree. The sequences of the two 16S rRNA gene copies differ from each other by up to one nucleotide, and differ by up to five nucleotides from the previously published 16S rRNA sequence generated from DSM 11293 (U80597), which contains two ambiguous base calls.
Strain SEBR 4228T is a Gram-negative, chemoorganotrophic and strictly anaerobic bacterium with spiral shaped, 0.3–0.5 × 5–30 µm long cells (Figure 2 and Table 1). It possesses a multilayer, crenulating, Gram-negative cell envelope, which consists of an outer membrane and an inner membrane adjoining the cytoplasmic membrane . Sillons, which are the contact point between the protoplasmic cylinder, the inner membrane and the outer membrane, are also observed from the cells of S. smaragdinae SEBR 4228T . Strain SEBR 4228T forms translucent colonies with regular edges (0.5 mm of diameter) after two weeks of incubation on SEM agar plates at 37°C . The strain is motile with a corkscrew-like motion, which is characteristic for the typical 1-2-1 periplasmic flagellar arrangement of the members of the genus Spirochaeta . The periplasmic, non-extracellular location of the flagella make the Spirochaeta a valuable candidate for the study of flagella evolution . The enlarged spherical bodies, which are typical for spirochetes, are also observed in strain SEBR 4228T . The temperature range for growth is from 20°C to 40°C, with an optimum temperature at 37°C . The pH range for growth is between 5.5 and 8.0, with an optimum pH of 7.0 . Strain SEBR 4228T is obligately halophilic  and is able to grow on media that contains 1–10% of NaCl, with an optimum salinity at 5% NaCl . Under optimum growth conditions, the doubling time is approximately 25 h in the presence of glucose and thiosulfate . Strain SEBR 4228T is able to utilize biotrypcase, fructose, fumarate, galactose, D-glucose, glycerol, mannitol, mannose, ribose, D-xylose and yeast extract, but not acetate, D-arabinose, butyrate, casamino acids, lactate, maltose, propionate, pyruvate, rhamnose, sorbose, sucrose and L-xylose . Yeast extract is required for growth and cannot be replaced by a vitamin mixture . Strain SEBR 4228T ferments fumarate to acetate and succinate . The major end-product of glucose fermentation of strain SEBR 4228T is lactate with traces of H2 and ethanol . S. smaragdinae is the only species of Spirochaeta known to reduce thiosulfate or elemental sulfur to sulfide . Strain SEBR 4228T produces lactate, acetate, CO2 and H2S as the end-products of glucose oxidation when thiosulfate is present in the growth medium . The strain contains a rhodanese-like protein which expresses rhodanese activity . This enzyme is able to reduce thiosulfate to sulfide . Rhodanese is also widely found in other members of the domain Bacteria [29–31].
No cellular fatty acids profiles are currently available for S. smaragdinae SEBR 4228T. However, C16:0 dimethyl acetate is the major cellular fatty acids of the type strains of the closely related S. dissipatitropha, S. asiatica and S. americana, and C16:0 fatty acid methyl ester is the major cellular fatty acids of S. africana [20,32].
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 Genome OnLine Database  and the complete 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.
Growth conditions and DNA isolation
S. smaragdinae SEBR 4228T, DSM 11293, was grown anaerobically in medium 819 (Spirochaeta smaragdinae medium)  at 35°C. DNA was isolated from 0.5–1 g of cell paste using MasterPure Gram Positive DNA Purification Kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer, with modification st/LALMice for cell lysis as described in Wu et al. .
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. Pyrosequencing reads were assembled using the Newbler assembler version 2.0.0-PostRelease-11/04/2008 (Roche). The initial Newbler assembly consisted of 51 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 was assembled with Velvet  and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 273 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 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, Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) . Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 147 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to improve the final consensus quality using an in-house developed tool - the Polisher . The error rate of the completed genome sequence is 0.2 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 65.7× coverage of the genome.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . 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 (IMG-ER) platform .
The genome consists of a 4,653,970 bp long chromosome with a 49.0% GC content (Table 3 and Figure 3). Of the 4,363 genes predicted, 4,306 were protein-coding genes, and 57 RNAs; eighty seven pseudogenes were also identified. The majority of the protein-coding genes (74.2%) 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.
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We would like to gratefully acknowledge the help of Maren Schröder (DSMZ) for growing cultures of S. smarasgdinae. This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-1 and Thailand Research Fund Royal Golden Jubilee Ph.D. Program No. PHD/0019/2548 for MY.
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Mavromatis, K., Yasawong, M., Chertkov, O. et al. Complete genome sequence of Spirochaeta smaragdinae type strain (SEBR 4228T). Stand in Genomic Sci 3, 136–144 (2010). https://doi.org/10.4056/sigs.1143106
- spiral shaped
- corkscrew-like motility
- strictly anaerobe
- obligately halophile
- rhodanese-like protein