Non-contiguous finished genome sequence and description of Clostridium senegalense sp. nov.
© The Author(s) 2012
Published: 30 July 2012
Clostridium senegalense strain JC122T, is the type strain of Clostridium senegalense sp. nov., a new species within the genus Clostridium. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. C. senegalense strain JC122T is an obligate anaerobic Gram-positive rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,893,008 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 26.8% and contains 3,704 protein-coding and 57 RNA genes, including 6 rRNA genes.
Clostridium senegalense strain JC122T (= CSUR P152 = DSM 25507), is the type strain of Clostridium senegalense sp. nov. This bacterium is a Gram-positive, anaerobic, spore-forming, indole negative rod-shaped bacterium that was isolated from the stool of a healthy Senegalese patient as part of a “culturomics” study aiming at cultivating individually all species within human feces.
Since 1995 and the sequencing of the first bacterial genome, that of Haemophilus influenzae, more than 2,000 bacterial genomes have been sequenced . This was permitted by technical improvements as well as increased interest in having access to the complete genetic information encoded by bacteria. In the same time, biological tools for the definition of new bacterial species have not evolved, DNA-DNA hybridization still being considered as the gold standard  despite its drawbacks and the taxonomic revolution that has resulted from the comparison of 16S rDNA sequences . In this manuscript, we propose to use genomic data, in addition to phenotypic information , to describe a new Clostridium species.
Here we present a summary classification and a set of features for C. senegalense sp. nov. strain JC122T (= CSUR P152= DSM 25507) together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species C. senegalense.
The genus Clostridium (Prazmowski, 1880) was created in 1880  and consists of obligate anaerobic rod-shaped bacilli capable of producing endospores . More than 180 species have been described to date. Members of the genus Clostridium are mostly environmental bacteria or associated with the commensal digestive flora of mammals. However, several are major human pathogens, including C. botulinum, C. difficile and C. tetani [6,7]. Few species, such as C. butyricum and C. pasteurianum, fix nitrogen and have gained importance in agricultural and industrial applications [8,9].
Classification and features
Classification and general features of Clostridium senegalense strain JC122T
Species Clostridium senegalense
Type strain JC122T
growth in BHI medium + 5% NaCl
Sample collection time
51 m above sea level
Strain JC122T exhibited neither catalase nor oxidase activities. Using API Rapid ID 32A, a positive reaction was observed for arginine dihydrolase, N-acetyl-β-glucosanimidase and pyroglutamic acid arylamidase. Negative reactions were observed for urease, indole and nitrate reduction. C. senegalense is susceptible to amoxicillin, imipenem, metronidazole, rifampicin and vancomycin but resistant to trimethoprim/sulfamethoxazole.
Genome sequencing information
Genome project history
One 454 paired end 3-kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
Genbank Date of Release
July 25, 2011
Study of the human gut microbiome
Growth conditions and DNA isolation
C. senegalense sp. nov. strain JC122T, CSUR P152 = DSM 25507, was grown on blood agar medium at 37°C. Five petri dishes were spread and resuspended in 5x100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (Sample Preparation system) from MP Biomedicals, USA) using 2x20 seconds cycles. DNA was then treated with 2.5 µg/µL lysozyme (30 minutes at 37°C) and extracted through the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on a Genios_Tecan fluorometer at 70.7 ng/µl.
Genome sequencing and assembly
This project was loaded twice on a 1/4 region for the paired end application and once on a 1/8 region for the shotgun on PTP Picotiterplates. The shotgun library was constructed with 500ng of DNA as described by the manufacturer Roche with the GS Rapid library Prep kit. For the paired-end sequencing, DNA (5µg) was mechanically fragmented on the Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size of 3–4kb. The DNA fragmentation was visualized using an Agilent 2100 BioAnalyzer on a DNA labchip 7500 to yield an optimal size of 3.6 kb. The library was constructed according to the 454_Titanium paired end protocol and manufacturer. Circularization and nebulization were performed and generated a pattern with an optimum at 561 bp. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired end library was then quantified on the Quant-it Ribogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 52pg/µL. The library concentration equivalence was calculated as 1.7E+08 molecules/µL. The library was held at −20°C until use.
The shotgun library was clonally amplified with 3cpb in 3 emPCR reactions and the paired end library was amplified with lower cpb (1cpb) in 4 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2. The yield of the emPCR was 5.37% for the shotgun and 19.27% for the paired end according to the quality expected by the range of 5 to 20% from the Roche procedure. A total of 340,000 beads for the 1/8 region for the shotgun and 790,000 beads on the 1/4 region for the paired end were loaded on the GS Titanium PicoTiterPlates (PTP Kit 70×75) and sequenced with the GS Titanium Sequencing Kit XLR70.
The runs were performed overnight and then analyzed on the cluster through the gsRunBrowser and gsAssembler_Roche. The global 383,079 passed filter sequences generated 96.50 Mb with a length average of 277bp. These sequences were assembled using the Newbler software from Roche with 90% identity and 40 bp as overlap. Fourteen scaffolds and 120 large contigs (>1500bp) were obtained, for a genome size of 3,893,008 bp.
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters but the predicted ORFs were excluded if they were spanning a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank database  and the Clusters of Orthologous Groups (COG) database using BLASTP. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer  and BLASTn against the GenBank database. ORFans were identified if their BLASTP E-value was lower than 1e-03 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e-05. Such parameter thresholds have already been used in previous works to define ORFans.
To estimate the mean level of nucleotide sequence similarity at the genome level between Clostridium species, we compared the ORFs only using BLASTN and the following parameters: a query coverage of ≥ 70% and a minimum nucleotide length of 100 bp.
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Genes with function prediction
Genes assigned to COGs
Genes with peptide signals
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
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
Not in COGs
Comparison with the genomes from other Clostridium species
Seventy-three genomes are currently available for Clostridium species. Here, we compared the genome sequence of C. senegalense strain JC122T with those of C. botulinum strain ATCC 19397 and C. cellulovorans strain, ATCC 35296.
The draft genome sequence of C. senegalense strain JC122T has a similar size to that of C. botulinum (3.89 and 3.94 Mb, respectively), but a smaller size than C. cellulovorans (5.2 Mb). The G+C content of C. senegalense was lower than C. botulinum and C. cellulovorans (26.8% vs 28.2 and 31.2%, respectively). The gene content of C. senegalense is comparable to that of C. botulinum (3,761 and 3,750, respectively) but is smaller to that of C. cellulovorans (4,500). The ratios of genes per Mb and numbers of genes assigned to COGs of C. senegalense and C. botulinum are similar (974 vs 946 and 2,560 vs 2,549, respectively), but larger than the ratio of genes per Mb (844) and smaller than the number of genes assigned to COGs of C. cellulovorans (2,927). However, the distribution of genes into COG categories (Table 4) was similar in all the three compared genomes.
In addition, C. senegalense shared a mean 84.9% (range 77.4-95%) and 82.79% (range 77.2-92.3%) sequence similarity with C. botulinum and C. cellulovorans respectively at the genome level.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Clostridium senegalense sp. nov. which contains the strain JC122T. This bacterium has been found in Senegal.
Description of Clostridium senegalense sp. nov.
Clostridium senegalense (se.ne.gal.e′n.sis. L. gen. masc. n. senegalensis, pertaining to, or originating from Senegal, the country from which the specimen was isolated).
Colonies are 2 mm in diameter on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Cells are rod-shaped with a mean diameter of 1.1 µm. Optimal growth is achieved anaerobically. No growth is observed in aerobic conditions. Growth occurs between 25–37°C, with optimal growth observed at 37°C, in BHI medium + 5% NaCl. Cells stain Gram-positive, are endospore-forming, and motile. Catalase, oxidase, urease, indole and nitrate reduction activity are absent. Arginine dihydrolase, N-acetyl-β-glucosanimidase and pyroglutamic acid arylamidase activity are present. Cells are susceptible to amoxicillin, imipenem, metronidazole, rifampicin and vancomycin but resistant to trimethoprim/sulfamethoxazole. The G+C content of the genome is 26.8%.
The type strain is JC122T (= CSUR P152 = DSM 25507) was isolated from the fecal flora of a healthy patient in Senegal.
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