Open Access

Complete genome sequence of Spirochaeta smaragdinae type strain (SEBR 4228T)

  • Konstantinos Mavromatis
  • , Montri Yasawong
  • , Olga Chertkov,
  • , Alla Lapidus
  • , Susan Lucas
  • , Matt Nolan
  • , Tijana Glavina Del Rio
  • , Hope Tice
  • , Jan-Fang Cheng
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Natalia Ivanova
  • , Roxanne Tapia,
  • , Cliff Han,
  • , David Bruce,
  • , Lynne Goodwin,
  • , Amrita Pati
  • , Ami Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , John C. Detter,
  • , Manfred Rohde
  • , Evelyne Brambilla
  • , Stefan Spring
  • , Markus Göker
  • , Johannes Sikorski
  • , Tanja Woyke
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Hans-Peter Klenk
  • and Nikos C. Kyrpides
Corresponding author

DOI: 10.4056/sigs.1143106

Received: 18 October 2010

Published: 31 October 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.


spiral shapedcorkscrew-like motilitychemoorganotrophstrictly anaerobeobligately halophilerhodanese-like proteinSpirochaetaceaeGEBA


Strain SEBR 4228T (= DSM 11293 = JCM 15392) is the type strain of the species Spirochaeta smaragdinae. Currently, there are eighteen species [1] 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 [3] and described in 1997 by Magot et al. as ‘Spirochaeta smaragdinae’ [3]. 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 [4], with the type strain of S. bajacaliforniensis [5], 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 [3]. Several type strains from the genus Treponema show the highest degree of similarity for non-Spirochaeta strains (82.9-83.6%) [4]. A representative genomic 16S rRNA sequence of strain SEBR 4228T was compared using BLAST with the most recent release of the Greengenes database [6] 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.

Figure 1

Phylogenetic tree highlighting the position of S. smaragdinae SEBR 4228T relative to the type strains of the other species within the genus and of the other genera within the genus Spirochaeta. The tree was inferred from 1,385 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum likelihood criterion [9] and rooted in accordance with the current taxonomy [10]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 500 bootstrap replicates [11] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [12] are shown in blue, published genomes in bold.

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 [3]. 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 [3]. 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 [3]. 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 [3]. The periplasmic, non-extracellular location of the flagella make the Spirochaeta a valuable candidate for the study of flagella evolution [26]. The enlarged spherical bodies, which are typical for spirochetes, are also observed in strain SEBR 4228T [3]. The temperature range for growth is from 20°C to 40°C, with an optimum temperature at 37°C [3]. The pH range for growth is between 5.5 and 8.0, with an optimum pH of 7.0 [3]. Strain SEBR 4228T is obligately halophilic [3] and is able to grow on media that contains 1-10% of NaCl, with an optimum salinity at 5% NaCl [3]. Under optimum growth conditions, the doubling time is approximately 25 h in the presence of glucose and thiosulfate [3]. 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 [3]. Yeast extract is required for growth and cannot be replaced by a vitamin mixture [3]. Strain SEBR 4228T ferments fumarate to acetate and succinate [3]. The major end-product of glucose fermentation of strain SEBR 4228T is lactate with traces of H2 and ethanol [3]. S. smaragdinae is the only species of Spirochaeta known to reduce thiosulfate or elemental sulfur to sulfide [3]. Strain SEBR 4228T produces lactate, acetate, CO2 and H2S as the end-products of glucose oxidation when thiosulfate is present in the growth medium [3]. The strain contains a rhodanese-like protein which expresses rhodanese activity [27]. This enzyme is able to reduce thiosulfate to sulfide [28]. Rhodanese is also widely found in other members of the domain Bacteria [29-31].

Figure 2

Scanning electron micrograph of S. smaragdinae SEBR 4228T

Table 1

Classification and general features of S. smaragdinae SEBR 4228T according to the MIGS recommendations [13].




    Evidence code

   Current classification

   Domain Bacteria

    TAS [14]

   Phylum Spirochaetae

    TAS [15,16]

   Class Spirochaetes

    TAS [16]

   Order Spirochaetales

    TAS [17,18]

   Family Spirochaetaceae

    TAS [18,19]

   Genus Spirochaeta

    TAS [18,20-22]

   Species Spirochaeta smaragdinae

    TAS [3,23]

   Type strain SEBR 4228

    TAS [3]

   Gram stain


    TAS [3]

   Cell shape


    TAS [3]



    TAS [3]




   Temperature range

   between 20°C and over 40°C

    TAS [3]

   Optimum temperature


    TAS [3]


   1-10% NaCl (optimum 5%)

    TAS [3]


   Oxygen requirement

   obligately anaerobic

    TAS [3]

   Carbon source


    TAS [3]

   Energy source


    TAS [3]




    TAS [3]


   Biotic relationship


    TAS [3]





   Biosafety level


    TAS [24]


   oil-injection water sample in the   production system of an oil field

    TAS [3]


   Geographic location

   Emerald oil fields in Congo

    TAS [3]


   Sample collection time

   1997 or before

    TAS [3]



   not reported



   not reported



   not reported



   not reported

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


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 [33], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [34]. The genome project is deposited in the Genome OnLine Database [12] 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.

Table 2

Genome sequencing project information





   Finishing quality



   Libraries used

   Three genomic libraries:   454 pyrosequence standard and   PE (12 kb insert size) libraries and one   Illumina standard library


   Sequencing platforms

   454 GS FLX Titanium, Illumina GAii


   Sequencing coverage

   58.8 × pyrosequence, 6.9 × Illumina



   Newbler version 2.0.0-PostRelease-   11/04/2008, phrap,


   Gene calling method

   Prodigal 1.4, GenePRIMP



   Genbank Date of Release

   August 6, 2010



   NCBI project ID


   Database: IMG-GEBA



   Source material identifier

   DSM 11293

   Project relevance

   Tree of Life, GEBA

Growth conditions and DNA isolation

S. smaragdinae SEBR 4228T, DSM 11293, was grown anaerobically in medium 819 (Spirochaeta smaragdinae medium) [35] 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. [34].

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 (Web Site). 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 [36] 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 (Web Site) 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 (Web Site), Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [37]. 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 [38]. 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.

Genome annotation

Genes were identified using Prodigal [39] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [40]. 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 [41].

Genome properties

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.

Table 3

Genome Statistics



   % of Total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of replicons


Extrachromosomal elements


Total genes



RNA genes



rRNA operons


Protein-coding genes



Pseudo genes



Genes with function prediction



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


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.

Table 4

Number of genes associated with the general COG functional categories








    Translation, ribosomal structure and biogenesis




    RNA processing and modification








    Replication, recombination and repair




    Chromatin structure and dynamics




    Cell cycle control, cell division, chromosome partitioning




    Nuclear structure




    Defense mechanisms




    Signal transduction mechanisms




    Cell wall/membrane/envelope biogenesis




    Cell motility








    Extracellular structures




    Intracellular trafficking and secretion, and vesicular transport




    Posttranslational modification, protein turnover, chaperones




    Energy production and conversion




    Carbohydrate transport and metabolism




    Amino acid transport and metabolism




    Nucleotide transport and metabolism




    Coenzyme transport and metabolism




    Lipid transport and metabolism




    Inorganic ion transport and metabolism




    Secondary metabolites biosynthesis, transport and catabolism




    General function prediction only




    Function unknown




    Not in COGs



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.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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