Non-contiguous finished genome sequence and description of Anaerococcus senegalensis sp. nov.
© The Author(s) 2012
Published: 19 March 2012
Anaerococcus senegalensis strain JC48T sp. nov. is the type strain of A. senegalensis sp. nov. a new species within the genus Anaerococcus. This strain whose genome is described here was isolated from the fecal flora of a healthy patient. A. senegalensis is an obligate anaerobic coccus. Here we describe the features of this organism together with the complete genome sequence and annotation. The 1,790,835 bp long genome (1 chromosome but no plasmid) contains 1,721 protein-coding and 53 RNA genes including 5 rRNA genes
KeywordsAnaerococcus senegalensis genome
Anaerococcus senegalensis strain JC48T (= CSUR P156 = DSM25366) is the type strain of A. senegalensis sp. nov. This bacterium is a Gram-positive, anaerobic, indole-negative coccus that was isolated from the stool of a healthy Senegalese patient as part of a “culturomics” study aimed at cultivating individually all species within human feces.
Defining bacterial species is a matter of debate. This is notably due to the elevated cost and poor reproducibility and inter-laboratory comparability of the “gold standard” DNA-DNA hybridization and G+C content determination . In contrast, the development of PCR and sequencing methods is now widely available and cost-effective, which profoundly changes the way Archaea, Bacteria and are classified. Using 16S rRNA sequences with internationally-validated cutoff values enabled the taxonomic classification or reclassification of hundreds of taxa . More recently, high throughput genome sequencing and mass spectrometric analyses of bacteria gave unprecedented access to a wealth of genetic and proteomic information . As a consequence, we propose to use a polyphasic approach  to describe new bacterial taxa that includes their genome sequence, MALDI-TOF spectrum and main phenotypic characteristics (habitat, Gram-stain reaction, culture and metabolic characteristics, and when applicable, pathogenicity).
The genus Anaerococcus (Ezaki et al. 2001) was created in 2001 . To date, this genus, comprised of saccharolytic and butyrate-producing anaerobic and non-motile Gram-positive cocci, contains seven species including A. hydrogenalis (Ezaki et al. 1990) Ezaki et al. 2001, A. lactolyticus (Li et al. 1992) Ezaki et al. 2001, A. murdochii (Song et al. 2010), A. octavius (Murdoch et al. 1997) Ezaki et al. 2001, A. prevotii (Foubert and Douglas 1948) Ezaki et al. 2001, A. tetradius (Ezaki et al. 1983) Ezaki et al. 2001, and A. vaginalis (Li et al. 1992) Ezaki et al. 2001. Members of the genus Anaerococcus have mainly been isolated from the human vagina, but have also occasionally been identified in the nasal cavity, on the skin, and various infectious processes including ovarian, peritoneal, sacral, digital and cervical abscesses, vaginoses, bacteremias, foot ulcers, a sternal wound, and a knee arthritis [5–9]. In addition, uncultured bacteria with 16S rRNA sequences highly similar to members of the Anaerococcus genus have been detected in metagenomes from the human skin flora . However, to the best of our knowledge, our report is the first to describe the isolation of a member of the genus Anaerococcus from the normal fecal flora.
Here we present a summary classification and a set of features for A. senegalensis sp. nov. strain JC48T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species A. senegalensis.
Classification and features
Classification and general features of Anaerococcus senegalensis strain JC48T
Family Clostridiales Incertae sedis XI
Species Anaerococcus senegalensis
Type strain JC48T
Growth in BHI medium + 5% NaCl
Sample collection time
51 m above sea level
Strain 48T exhibited catalase activity but not oxidase activity. Using API Rapid ID 32A, a positive reaction was obtained for urease, arginine dihydrolase, indole production, β glucuronidase, mannose fermentation, alkaline phosphatase, arginine arylamidase, leucyl glycine arylamidase, histidine arylamidase. A weak reaction was obtained for pyroglutamyl arylamidase. A. senegalensis is susceptible to penicillin G, imipeneme, amoxicillin + clavulanic acid, vancomycin, clindamycin and metronidazole. By comparison with A. vaginalis, strain 48T differed in urease and pyroglutamyl arylamidase production .
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
Study of the human gut microbiome
Growth conditions and DNA isolation
A. senegalensis sp. nov. strain JC48T, CSUR P156, was grown anaerobically on 5% sheep blood-enriched Columbia agar at 37°C. Four petri dishes were spread and resuspended in 3×100µl 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; MP Biomedicals, USA) using 2×20 seconds cycles. DNA was then treated with 2.5µg/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 the Genios_Tecan fluorometer at 50ng/µl.
Genome sequencing and assembly
DNA (5 µg) was mechanically fragmented on a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3–4 kb. The DNA fragmentation was visualized through the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 3.785 kb. The library was constructed according to the 454 GS FLX Titanium paired end protocol. Circularization and nebulization were performed and generated a pattern with an optimal at 614 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 96 pg/µL. The library concentration equivalence was calculated as 2,87E+08 molecules/µL. The library was stored at −20°C until further use.
The library was clonally amplified with 0.5 cpb and 1 cpb respectively in 2×8 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were lower than expected at 3.92%, compared to the range of 5 to 20% from the Roche procedure.
Approximately 790,000 beads were loaded on the GS Titanium PicoTiterPlate PTP Kit 70×75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 310,172 passed filter wells were obtained and generated 99 Mb with a length average of 320 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40bp as overlap. The final assembly identified 4 scaffolds and 39 contigs (>100bp).
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. 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 Anaerococcus species, we compared the ORFs only using BLASTN at a query coverage of ≥ 70% and a minimum nucleotide length of 100 bp.
Nucleotide content and gene count levels of the genome
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
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 Anaerococcus prevotii
To date, the genome from A. prevotii strain PC1T is the only genome from Anaerococcus species that has been sequenced . By comparison with A. prevotii, A. senegalensis exhibited a lower G + C content (35.64% vs 28.56%, respectively) and a smaller number of genes (1,913 vs 1,774) and genes with peptide signals (337 vs 142). In contrast, A. senegalensis had higher ratios of genes per Mb (957 vs 990) and genes assigned to COGs (74.28% vs 79. 26%). However, the distribution of genes into COG categories (Table 4) was highly similar in both genomes.
In addition, A. senegalensis shared mean nucleotide sequence similarities at the genome level of 76.6% (range 62.5-100%) and 75.4% (range 62.7-100%) with A. prevotii strains PC1T and ACS065-V-Col13 (GenBank accession number AEXM00000000), respectively.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Anaerococcus senegalensis sp. nov. that contains the strain JC48T. This bacterium has been found in Senegal.
Description of Anaerococcus senegalensis sp. nov.
Anaerococcus senegalensis (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 0.5 mm to 1 mm in diameter on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Cells are coccoid with a mean diameter of 0.87 µm, occurring mostly in pairs, short chains or small clumps. Optimal growth is achieved anaerobically. Weak growth is observed in microaerophilic conditions and with 5% CO2. No growth is observed in aerobic conditions. Growth occurs between 30–37°C, with optimal growth observed at 37°C, in BHI medium + 5% NaCl. Cells stain Gram-positive, are non-endospore-forming, and non-motile. Catalase, urease, arginine dihydrolase, β glucuronidase, alkaline phosphatase, arginine arylamidase, leucyl glycine arylamidase, and histidine arylamidase activity are present. Mannose fermentation and indole production are also present. A weak reaction is obtained for pyroglutamyl arylamidase. Oxidase activity is absent. Cells are susceptible to penicillin G, imipeneme, amoxicillin + clavulanic acid, vancomycin, clindamycin and metronidazole. The G + C content of the genome is 28.56%.
The type strain JC48T (= CSUR P156 = DSM 25366) was isolated from the fecal flora of a healthy patient in Senegal.
European Molecular Biology Laboratory
National Center for Biotechnology Information (Bethesda, MD, USA)
Ribosomal Database Project (East Lansing, MI, USA)
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