Non-contiguous finished genome sequence and description of Anaerococcus vaginalis

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.


Introduction
Anaerococcus vaginalis strain PH9 (= CSUR P188= DSM25446) was isolated from the stool of a 26-yearold woman suffering from morbid obesity as part of a study aiming at cultivating all species within human feces. It is a Gram-positive, anaerobic, indolenegative coccus.
The genus Anaerococcus (Ezaki et al. 2001) was created in 2001 [1] 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][2][3][4][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 [6]. A. vaginalis (Li et al. 1992) was first isolated from vaginal discharges and ovarian abscesses [7]. Initially, it was classified in the genus Peptostreptococcus but later reclassified within the genus Anaerococcus [1]. 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
A stool sample was collected from a 26-year-old woman living in Marseille, France, who suffered from morbid obesity: BMI=48.2 (118.8 kg, 1.57 meter). At the time of stool sample collection, she was not a drug-user and was not on a diet. The patient gave an informed and signed consent, and the agreement of local ethics committee of the IFR48 (Marseille, France) were obtained under agreement 11-017. The fecal specimen was preserved at -80°C after collection. Strain PH9 (Table 1) was isolated in 2011 by anaerobic cultivation on 5% sheep bloodenriched Columbia agar (BioMerieux, Marcy l'Etoile, France) after 4 days of preincubation of the stool sample with addition of thioglycolate in a blood culture bottle. This strain exhibited a 98.8% 16S rRNA sequence similarity with A. vaginalis (Li et al. 1992), the phylogenetically closest validated Anaerococcus species ( Figure 1). This value was higher than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species [18]. As a consequence, strain PH9 belongs to A. vaginalis.
Growth at different temperatures (25, 30, 37, 45°C) was tested; no growth occurred at 25°C and 45°C, growth occurred between 30 and 37°C, and optimal growth was observed at 37°C. Colonies were 0.5 mm to 1 mm in diameter on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar.
Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMérieux), and in the presence of air, with or without 5% CO 2 and in aerobic conditions. Optimal growth was obtained anaerobically, with weak growth being observed in microaerophilic condition, and no growth occurring in aerobic conditions and with 5% CO 2 . Gram staining showed Gram positive cocci. A motility test was negative. Cells grown on agar are Gram-positive ( Figure 2) and have a mean diameter of 0.71 µm by electron microscopy and are mostly grouped in pairs, short chains or small clumps (Figure 3). , 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 the Gene Ontology project [17]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.   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.
Matrix-assisted laser-desorption/ionization timeof-flight (MALDI-TOF) MS protein analysis was carried out as previously described [19]. Briefly, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate, and to spread it as a thin film on a MTP 384 MALDI-TOF target plate (Bruker Daltonics, Germany). Twelve distinct deposits were done for strain PH9 from twelve isolated colonies. Each smear was overlaid with 2µL of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50% acetonitrile, 2.5% tri-fluoracetic acid, and allowed to dry for five minutes. Measurements were performed with a Microflex spectrometer (Bruker). Spectra were recorded in the positive linear mode for the mass range of 2,000 to 20,000 Da (parameter settings: ion source 1 (ISI), 20kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 675 shots at a variable laser power. The time of acquisition was between 30 seconds and 1 minute per spot. The twelve PH9 spectra were imported into the MALDI Bio Typer software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 2,843 bacteria, including spectra from seven validated Anaerococcus species used as reference data, in the Bio Typer database. The method of identification includes the m/z from 3,000 to 15,000 Da. For every spectrum, 100 peaks at most were taken into account and compared with the spectra in the database. A score enabled the presumptive identification and discrimination of the tested species from those in the database: a score ≥ 2 with a validated species enabled the identification at the species level; a score ≥ 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification. Spectra were compared with the Bruker database that contained spectra from the seven validated Anaerococcus species. The score and spectra obtained were similar to those of A. vaginalis, thus confirming that our isolate was a member of the A. vaginalis species. We incremented our database with the spectrum from strain PH9 (Figure 4). Standards in Genomic Sciences

Genome sequencing and annotation Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the Anaerococcus genus, and is part of a study of the human digestive flora aiming at isolating all bacterial species within human feces. It is the third published genome from an Anaerococcus species and the first genome from the A. vaginalis species. A summary of the project information is shown in Table 2. The Genbank accession number is CAGU00000000. The genome consists of 93 contigs. Table 2 shows the project information and its association with MIGS version 2.0 compliance [1].

Growth conditions and DNA isolation
A. vaginalis strain PH9 (DSM25446, CSUR P188) was grown anaerobically on 5% sheep bloodenriched 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).

Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [20] 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 [21] and the Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAScanSE tool [22] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [23] 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.

Genome properties
The genome of A. vaginalis strain PH9 is 2,048,125 bp long (1 chromosome, but no plasmid) with a 29.6% G + C content of ( Figure 5 and Table 3). Of the 2,133 predicted genes, 2,095 were protein-coding genes, and 38 were RNAs. Three rRNA genes (one 16S rRNA, one 23S rRNA and one 5S rRNA) and 35 predicted tRNA genes were identified in the genome.
A total of 1,546 genes (72.48%) were assigned a putative function. Eighty-one genes were identified as ORFans (3.8%). The remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3.
The distribution of genes into COGs functional categories is presented in Table 4.

Conclusion
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.