Non-contiguous finished genome sequence and description of Anaerococcus vaginalis
© The Author(s) 2012
Published: 30 July 2012
We report the properties of a draft genome sequence of the bacterium Anaerococcus vaginalis strain PH9, a species within the Anaerococcus genus. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. A. vaginalis is an obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,048,125-bp long (one chromosome but no plasmid) and contains 2,095 protein-coding and 38 RNA genes, including three rRNA genes.
Anaerococcus vaginalis strain PH9 (= CSUR P188= DSM25446) was isolated from the stool of a 26-year-old woman suffering from morbid obesity as part of a study aiming at cultivating all species within human feces. It is a Gram-positive, anaerobic, indole-negative coccus.
The genus Anaerococcus (Ezaki et al. 2001) was created in 2001  and to date, this genus consist of saccharolytic, butyrate-producing anaerobic and non-motile gram-positive cocci. Seven species are validated, including A. hydrogenalis, A. lactolyticus, A. murdochii, A. octavius, A. prevotii, A. tetradius and A. vaginalis [2,3]. Members of the genus Anaerococcus have mostly been isolated from the human vagina, but have also occasionally been identified in the nasal cavity, on the skin, and in various infectious processes including ovarian, peritoneal, sacral, digital and cervical abscesses, vaginoses, bacteremias, foot ulcers, a sternal wound, and a knee arthritis [1–5]. 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 .
A. vaginalis (Li et al. 1992) was first isolated from vaginal discharges and ovarian abscesses . Initially, it was classified in the genus Peptostreptococcus but later reclassified within the genus Anaerococcus .
To the best of our knowledge, we first report the isolation of Anaerococcus sp. from the fecal flora of a patient suffering from morbid obesity. Herein, we present a set of features for of A. vaginalis strain PH9 together with the description of the complete genomic sequence and annotation.
Classification and features
Classification and general features of Anaerococcus vaginalis strain PH9
Family Clostridiales family XI Incertae sedis
Species Anaerococcus vaginalis
Type strain PH9
growth in BHI medium + 5% NaCl
Sample collection time
0 m above sea level
Strain PH9 exhibited catalase activity but no oxidase activity. Using API Rapid ID 32A, a positive reaction was observed for arginine dihydrolase, histidine arylamidase, leucine arylamidase and mannose fermentation. A weak activity was observed for glycine arylamidase. A. vaginalis is susceptible to penicillin G, imipeneme, amoxicillin + clavulanic acid, vancomycin, clindamycin and metronidazole.
Genome sequencing and annotation
Genome project history
One 454 paired end 3-kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
NCBI project ID
Genbank Date of Release
Study of the human gut microbiome
Growth conditions and DNA isolation
A. vaginalis strain PH9 (DSM25446, CSUR P188) was grown anaerobically on 5% sheep blood-enriched Columbia agar at 37°C. Six petri dishes were spread and resuspended in 6x100µl 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) for 40 seconds. DNA was then incubated for a lysozyme treatment (30 minutes at 37°C) and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified using the Qiamp kit (Qiagen). The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 115.2ng/µl.
Genome sequencing and assembly
Five µg of DNA were mechanically fragmented on the Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3–4kb. The DNA fragmentation was visualized through the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 2.92 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 415 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 1,440 pg/µL. The library concentration equivalence was calculated as 6.36E+09 molecules/µL. The library was stored at −20°C until further use.
The library was clonally amplified with 0.25cpb and 1cpb respectively in 2x8 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were quite high at 17.78% but in the range of 5 to 20% from the Roche procedure.
Approximately 790,000 beads were loaded on the GS Titanium PicoTiterPlate PTP Kit 70x75 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 191,750 passed filter wells were obtained and generated 59.42 Mb with an average length of 309 bp. The passed filter sequences were assembled Using Newbler with 90% identity and 40bp as overlap. The final assembly identified 93 large contigs (>1500bp).
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) databases using BLASTP. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer  and BLASTn against the NR database. ORFans were identified if their BLASTP E-value were 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 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
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 Anaerococcus species
To date, two genomes from Anaerococcus species have been published. Here, we compared the genome sequence of A. vaginalis strain PH9 with those of A. prevotii strain PC1T  and A. senegalensis strain JC48T .
The draft genome sequence of A. vaginalis has a similar size to that of A. prevotii (2.04 vs 1.99 Mb, respectively), but a slightly larger than A. senegalensis (1.79Mb). The G+C content of A. vaginalis is comparable to A. senegalensis (29.60 vs 28.56%, respectively) and smaller than that of A. prevotii (35.64%). The gene content of A. vaginalis is larger than those of A. prevotii and A. senegalensis (2,133, 1,916 and 1,774, respectively). The ratio of genes per Mb of A. vaginalis is larger to those of A. senegalensis and A. prevotii (1,045, 991 and 962, respectively). Moreover, the distribution of genes into COG categories (Table 4) was highly similar in the three genomes.
A. vaginalis shared a mean 84.8% (range 71.10-100%) and 88.38% (range 70.3-100%) sequence similarity with A. prevotii and A. senegalensis respectively at the genome level.
We describe the phenotypic, phylogenetic and genomic characteristics of Anaerococcus vaginalis strain PH9. This bacterial strain has been found in Marseille, France.
Nucleotide sequence accession numbers
The A. vaginalis strain PH9 whole-genome shotgun (WGS) project and 16SrRNA gene sequence have been deposited in GenBank under accession numbers CAGU00000000 and JN837489, respectively.
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