Non-contiguous finished genome sequence and description of Holdemania massiliensis sp. nov.

Holdemania massiliensis strain AP2T sp. nov. is the type strain of H. massiliensis sp. nov., a new species within the genus Holdemania. This strain, whose genome is described here, was isolated from the fecal flora of a 21-year-old French Caucasian female suffering from severe restrictive anorexia nervosa. H. massiliensis is a Gram-positive, anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,795,625 bp-long genome (one chromosome but no plasmid) contains 3,461 protein-coding and 49 RNA genes, including 3 rRNA genes.


Introduction
Holdemania massiliensis strain AP2 T (= CSUR P195 = DSM 26143) is the type strain of H. massiliensis sp. nov. This bacterium is a Gram-positive, nonspore-forming, indole negative, anaerobic and non-motile bacillus that was isolated from the stool of a 21-year-old woman suffering from anorexia as part of a "culturomics" study aiming to individually cultivate all species within human feces [1][2][3]. The current prokaryotic species classification, known as polyphasic taxonomy, is based on a combination of genomic and phenotypic properties [4]. The number of sequenced genomes is increasing exponentially and in parallel with the decreasing cost of sequencing. To date, more than 6,000 bacterial genomes have been published and approximately 25,000 genomes project are anticipated to be completed in a near future [5]. We recently proposed to integrate genomic information in the taxonomic framework and description of new bacterial species .
Here we present a summary classification and a set of features for H. massiliensis sp. nov. strain AP2 T (= CSUR P195 = DSM 26143), together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species H. massiliensis.
The genus Holdemania (Willems et al. 1997) was created in 1997 on the basis of 16S rDNA gene sequence, biochemical tests, fatty acid and cell wall murein analysis [28]. To date, this genus includes a single species, H. filiformis, which was isolated from feces of healthy humans [29].

Classification and features
A stool sample was collected from a 21-year-old French Caucasian female suffering from severe restrictive anorexia nervosa, who had been hospitalized for recurrent weight loss and aggravation of her general state. She had an eight year history of mental anorexia. The patient gave an informed and signed consent. Both this study and the assent procedure were approved by the Ethics Committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France and the agreement of the ethics committee of the IFR48 (Marseille, France) was obtained under reference 09-022. Several other new bacterial species were isolated from diverse stool samples using microbial culturomics . The fecal specimen from the patient was preserved at -80°C immediately after collection. Strain AP2 T (Table 1) was isolated in November 2011 after preincubation in an anaerobic blood culture bottle with the addition of 5ml of thioglycolate, and inoculation in Columbia agar (BioMerieux, Marcy l'Etoile, France). This strain exhibited a 97% nucleotide sequence similarity with H. filiformis [28], and a range of 90-91% nucleotide sequence similarity to most closely related members of the genus Erysipelothrix [29] (Figure 1). This value was lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridization [40]. , 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 Ontolog y project [39]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the a uthors or an expert mentioned in the acknowledg ements. Different growth temperatures (25, 30, 37, 45°C) were tested. Growth occurred at 25 and 30°C after 72 hours of inoculation and the optimal growth was observed at 37°C after 24 hours of inoculation. Colonies were 0.2 mm in diameter, light grey, with α-haemolysis on blood-enriched Columbia agar. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMérieux), and under aerobic conditions with or without 5% CO2. The strain growth was obtained only in anaerobic condition. The motility test was negative. Cells grown on agar are Gram-positive rods ( Figure 2). The mean dimensions by electron microscopy were 0.57 µm in width and 1.75 µm in length ( Figure 3).  Strain AP2 T exhibited a positive oxidase but no catalase activity. Using an API rapid 32A strip (Biomerieux), positive reactions were obtained for β-galactosidase, α-glucosidase, α-fucosidase and pyroglutamic acid arylamidase. Substrate oxidation and assimilation was examined using an API 50CH strip (Biomerieux) at 37°C. Positive reactions were observed for glycerol, D-ribose, Dgalactose, D-glucose, D-fructose, D-mannose, inositol, D-mannitol, D-sorbitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose, D-melezitose, gentiobiose, D-tagatose and postassium gluconate. H. massiliensis is susceptible to amoxicillin, metronidazole, vancomycin, clindamycin and imipenem. The phenotypic characteristics that differentiate H. massiliensis from other species are summarized in Table 2. Matrix-assisted laser-desorption/ionization timeof-flight (MALDI-TOF) MS protein analysis was carried out as previously described [41] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve individual colonies were deposited on a MTP 384 MALDI-TOF target plate (Bruker). The twelve AP2 T spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 4,706 bacteria. A score enabled the presumptive identification and discrimination of the tested species from those in a database: a score > 2 with a validated species enabled the identification at the species level; and a score < 1.7 did not enable any identification. For strain AP2 T , no significant score was obtained, suggesting that our isolate was not a member of any known species (Figures 4 and 5). We added the spectrum from strain AP2 T to our database ( Figure 4). Oxygen requirement anaerobic anaerobic anaerobic facultative anaerobes Pigment production + ---   The gel view displays the raw spectra of loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed as a g ray scale. The color bar and the right y-axis indicate the relation between the color with which a peak is displayed and the peak intensity in arbitrary units. Displayed species are indicated on the left.

Genome sequencing information 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 Holdemania genus, and is part of a "culturomics" study of the human digestive flora aiming at isolating all bacterial species within human feces. It was the second draft genome of a Holdemania species and the first genome of Holdemania massiliensis sp. nov. A summary of the project information is shown in Table 3. The Genbank accession number is CALK00000000 and consists of 66 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance [30].

Growth conditions and DNA isolation
H. massiliensis sp. nov. strain AP2 T , (= CSUR P195= DSM 26143), was grown anaerobically on Columbia agar medium at 37°C. Five Petri dishes were spread and resuspended in 3x100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed using glass powder in the Fastprep-24 device (Sample Preparation system, MP Biomedicals, USA) for 2×20 seconds. DNA was treated with 2.5 µg/µL of lysozyme (30 minutes at 37°C) and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration of DNA was 69.3 ng/µl as measured by using Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer.
Genome sequencing and assembly DNA (5 µg) was mechanically fragmented for the paired-end sequencing, using a Covaris device (Covaris Inc., Woburn, MA,USA) with an enrichment size of 3-4 kb. The DNA fragmentation was visualized through an Agilent 2100 BioAnalyzer on a DNA Labchip 7500 which yielded an optimal size of 3.4 kb. The library was constructed using the 454 GS FLX Titanium paired-end rapid library protocol (Roche, Meylan, France). Circularization and nebulization were performed which generated a pattern of optimal size of 589 bp. PCR amplification was performed for 17 cycles followed by double size selection. The single-stranded pairedend library was quantified using a Quant-it Ribogreen Kit (Invitrogen) using the Genios Tecan fluorometer. The library concentration equivalence was calculated as 1.42× 10 10 molecules/µL. The library was stored at -20°C until further use. For the shotgun sequencing, DNA (500 ng) was mechanically fragmented using a Covaris device (Covaris Inc.) as described by the manufacturer. The DNA fragmentation was visualized using an Agilent 2100 BioAnalyzer on a DNA Labchip 7500 which yielded an optimal size of 1.7 kb. The library was constructed using the GS Rapid library Prep kit (Roche) and quantified using a TBS 380 mini fluorometer (Turner Biosystems, Sunnyvale, CA, USA). The library concentration equivalence was calculated as 2.8× 10 9 molecules/µL. The library was stored at -20°C until further use.
The shotgun library was clonally amplified with 1 and 2 cpb in two emPCR reactions each, and the paired-end library was amplified with 0.5 cpb in three emPCR reactions using the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 6.8 and 9.8%, respectively, for the shotgun library, and 11.29% for the paired-end library. These yields fall into the expected 5 to 20% range according to Roche protocol.
For each library, approximately 790,000 beads for a quarter region were loaded on the GS Titanium PicoTiterPlate PTP kit and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and analyzed on a cluster using the gsRunBrowser and Newbler assembler (Roche

Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [42] 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 [43] and the Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAScanSE tool [44] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [45] and BLASTn against the GenBank database. Lipoprotein signal peptides and numbers of transmembrane helices were predicted using SignalP [46] and TMHMM [47] respectively. 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 Evalue of 1e -05 . Such parameter thresholds have already been used in previous works to define ORFans. Ortholog sets composed of one gene from each of the four genomes H. massiliensis strain AP2 T , Holdemania filiformis strain ATCC 51649 (GenBank accession number ACCF00000000), Solobacterium moorei strain F0204 (AECQ00000000), and Erysipelothrix rhusiopathiae strain Fujisawa (AP012027) were identified using Proteinortho software (version 1.4) [48] by using cut-off values of 30% protein identity and an E-value of 1e -05 . The average percentages of nucleotide sequence identity between corresponding orthologous sets were determined using the Needleman-Wunsch algorithm global alignment technique. Artemis [49] was used for data management and DNA Plotter [50] was used for visualization of genomic features. The Mauve alignment tool was used for multiple genomic sequence alignment and visualization [51].

Genome properties
The genome of H. massiliensis strain AP2 T is 3,795,625 bp long (1 chromosome, no plasmids) with a 47.1% G + C content ( Figure 6 and Table  4). Of the 3,510 predicted genes, 3,461 were protein-coding genes, and 49 were RNAs. Three rRNA genes (one 16S rRNA, one 23S rRNA and one 5S rRNA) and 46 predicted tRNA genes were identified in the genome. A total of 2,581 genes (74.57%) were assigned a putative function. Two hundred thirteen genes were identified as ORFans (6.06%). The remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 4 and Table 5. The distribution of genes into COGs functional categories is presented in Table 5.  The total is based on either the size of the g enome in base pairs or the total number of protein coding genes in the annotated g enome  1,124, 1,035 and 994, respectively). However, the distribution of genes into COG categories was almost similar in all compared genomes (Figure 7). The nucleotide sequence identity ranged from 62.02 to 84.32% among compared genomes. Table  6 summarizes the numbers of orthologous genes and the average percentage of nucleotide sequence identity between the different genomes studied.

Conclusion
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Holdemania massiliensis sp. nov. that contains the strain AP2 T . This bacterial strain has been found in Marseille, France.