Genome sequence of Oceanobacillus picturae strain S1, an halophilic bacterium first isolated in human gut

Oceanobacillus picturae is a strain of a moderately halophilic bacterium, first isolated from a mural painting. We demonstrate, for the first time, the culture of human Oceanobacillus picturae, strain S1T, whose genome is described here, from a stool sample collected from a 25-year-old Saoudian healthy individual. We used a slightly modified standard culture medium adding 100 g/L of NaCl. We provide a short description of this strain including its MALDI-TOF spectrum, the main identification tool currently used in clinical microbiology. The 3,675,175 bp long genome exhibits a G + C content of 39.15 % and contains 3666 protein-coding and 157 RNA genes. The draft genome sequence of Oceanobacillus picturae has a similar size to the Oceanobacillus kimchii (respectively 3.67 Mb versus 3.83 Mb). The G + C content was higher compared with Oceanobacillus kimchii (respectively 39.15 % and 35.2 %). Oceanobacillus picturae shared almost identical number of genes (3823 genes versus 3879 genes), with a similar ratio of genes per Mb (1041 genes/Mb versus 1012 genes/Mb). The genome sequencing of Oceanobacillus picturae strain S1 isolated for the first time in a human, will be added to the 778 genome projects from the gastrointestinal tract listed by the international consortium Human Microbiome Project.


Introduction
A pure culture remains essential in microbiology. Nevertheless, metagenomics studies replaced culture methods entirely with regards to the exploration of complex ecosystems. The Human Microbiome Project (HMP) is an initiative with the goal of identifying and characterizing the microorganisms which are found in association with both healthy and diseased humans. To date (25 March 2015), 778 genome projects from the gastrointestinal tract are listed by HMP [1]. Since 2012, we applied microbial culturomics (based on the multiplication of the culture condition with a rapid identification method by MALDI-TOF) in order to extend the human gut composition. Testing more than 500,000 colonies by MALDI-TOF, we isolated more than 700 different bacterial species including more than 90 new bacterial species and 180 previously known bacterial species but first isolated in humans [1]. Each new bacterial species was described by taxonogenomics, a polyphasic approach adding genome sequencing and MALDI-TOF comparison in addition to classic phenotypic characteristics [2]. In addition, in order to make the genome sequencing data available to the international scientific community, we propose the sequencing of the genomes of all bacterial species we isolated in humans, for which no genome sequencing was previously available [1]. This will facilitate the future analysis of metagenomics studies. These strains are available for the scientific community (Collection de Souches de l'Unité des Rickettsies = CSUR). Herein, we report the genome sequencing of Oceanobacillus picturae strain S1 isolated for the first time in humans.
In this study we isolated for the O. picturae from humans for the first time. Strain S1 was isolated from a stool sample of a 25 year-old obese Saudi individual (BMI = 51 kg/m 2 ) using a modified Columbia agar (Becton Dickinson, Pont de Claix, France) adding 100 g/L of NaCl. We described here the genome sequencing of this bacterium.

Classification and features
A stool specimen was collected from a 25-year-old Saudi obese patient. The patient gave informed and signed consent. The study and the assent procedure were approved by the Ethics Committees of the King Abdulaziz University, King Fahd medical Research Center, Saudi , 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 [27]. 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  [28] was used, wherein sequences were aligned using MUSCLE [29] alignment curation by Gblocks [30] and construction of phylogenic tree performed using PhyML [31]. Numbers at the nodes are bootstrap values obtained by repeating the analysis 100 times. The scale bar represents a 2 % nucleotides sequences divergence Fig. 2 Gram staining of Oceanobacillus picturae strain S1 T Fig. 3 Transmission electron microscopy of Oceanobacillus picturae strain S1 T , using a Morgani 268D (Philips) at an operating voltage of 60 kV. The scale bar represents 500 nm reference strain O. picturae strain LMG19492 T (Genbank accession number NR_028952) (Fig. 1). This strain was deposited in the CSUR (under number P887). Strain S1 colonies were observed on sheep blood agar (Biomérieux, Marcy l'Etoile, France) after 24 h of aerobic incubation at 37°C. The colonies were greyish, 3-4 mm in diameter. Gram staining revealed Gram-positive bacilli (Fig. 2) and electron microscopy performed using a Morgani 268D (Phillips) showed rods with a mean length of 1.5 μm and a mean width of 0.5 μm (Fig. 3). Fig. 4 Reference mass spectrum from Oceanobacillus picturae strain S1 T . Spectra from 10 individual colonies were compared and a reference spectrum was generated Optimal growth was observed at 37°C and strain S1 grew only under aerobe conditions.

Extended feature descriptions
We added in the description the MALDI-TOF spectra of this bacterium. Indeed, mass spectrometry has become the reference identification method in clinical microbiology [1]. Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out. 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 MALDI-TOF target plate (Bruker Daltonics, Germany). Twelve distinct deposits were done for the strain S1 from twelve isolated colonies. After air-drying, 2 μl matrix solution (saturated solution of α-cyanohydroxycinnaminic acid in 50 % aqueous acetonitrile containing 2.5 % trifluoroacetic acid) per spot was applied. MALDI-TOF MS was conducted using the Microflex LT spectrometer (Bruker Daltonics). All spectra were recorded in the positive linear mode for the mass range of 2000 to 20,000 Da (parameter settings: ion source 1 (ISI), 20 kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 675 shots with variable laser power. The time of acquisition was between 30 s and 1 min per spot. The twelve spectra of strain S1 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 over 4108 bacteria including the spectra from the most closely related species including Oceanobacillus oncorhynchi CIP108867T, Oceanobacillus profundus CIP 109535T, Oceanobacillus chironomi CIP 109536T, Oceanobacillus iheyensis CIP 107618T, and Oceanobacillus oncorhynchi subsp. incaldanensis CIP 109235T, and Oceanobacillus sojae. In addition to these validly published species, we compared the O. picturae spectrum with spectra of Oceanobacillus massiliensis strain N'Diop and 'Oceanobacillus manasiensis' (HG931336). The resulting score was > 2, matching with Oceanobacillus picturae CIP 108264 T. The identification method included the m/z from 3000 to 15,000 Da. For every spectrum, a maximum of 100 peaks were compared with spectra in the database. We added the spectrum from strain S1 T to our database (Fig. 4).

Genome project history
The O. picturae genome was sequenced as part of a culturomics study aiming to isolate all bacterial species colonizing the human gut [1] (Table 2). To the best of our knowledge, O. picturae represent the fifth genome sequenced into the Oceanobacillus genus and the first genome of O. picturae. The genome accession number is CCAX00000000 and consists of 5 contigs without gaps. Table 3 shows the project information and its association with MIGS version 2.0 compliance.

Growth conditions and genomic DNA preparation
O. picturae strain S1 T (CSUR P1091 = DSM 28586) was grown at 37°C in an aerobic atmosphere on ten Petri dishes. The bacteria were harvested and resuspended in 4 × 100 μL of TE buffer. Then, 200 μL of this suspension was diluted in 1 mL TE buffer for lysis treatment that included a 30-min incubation with 2.5 μg/μL lysozyme at 37°C, followed by an overnight incubation with 20 μg/μL proteinase-K at 37°C. Extracted DNA was then purified using 3 successive phenol-chloroform extractions and ethanol precipitation at −20°C overnight. After centrifugation, the DNA was resuspended in 160 μL TE buffer. The yield and concentration were measured by the Quant-it Picogreen kit (Invitrogen) on the Genios-Tecan fluorometer at 88.6 ng/μl.

Genome sequencing and assembly
Genomic DNA of Oceanobacillus picturae was sequenced using MiSeq Technology (Illumina Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit (Illumina). The gDNA was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 40.5 ng/μl. The mate pair library was prepared with 1 μg of genomic DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in size from 1 kb up to 10 kb. No size selection was performed and only 14 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with an optimum at 696 bp on the Covaris device S2 in microtubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA). The libraries were normalized at 2 nM and pooled. After a denaturation step and dilution at 10pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing runs were performed in a single 42-h run in a 2x251-bp. Total information of 4.7 Gb was obtained from a 488 K/mm2 cluster density with a cluster passing quality control filters of 97.2 % (9,590,000 clusters). Within this run, the index representation for O. picturae was determined to be 11.16 %. Illumina reads were trimmed using Trimmomatic [32], then assembled through Spades software [33,34]. Contigs obtained were combined together by SSpace [35] and Opera software v1.2 [36] helped by GapFiller V1.10 [37] to reduce the set. Some manual refinements using CLC Genomics v7 software

Genome properties
The genome of Oceanobacillus picturae contained 3,675,175 bp with a G + C content of 39.15 % (Fig. 5, Tables 3 and 4

Conclusion
Oceanobacillus picturae strain S1 was the first strain of this bacterial species isolated from the human gut. The G + C % content of the genome was 39.15 %. The 16S rRNA and genome sequences were deposited in EMBL/ EBI database under accession numbers HG931335 and CCAX00000000 respectively.

Additional files
Additional file 1: