- Email: [email protected]
Virgibacillus senegalensis sp. nov., a new moderately halophilic bacterium isolated from human gut
Accepted Manuscript Virgibacillus senegalensis sp. nov. A new moderately halophilic bacterium isolated from the human gut El hadji Seck, Jaishriram Rathored, Saber Khelaifia, Olivier Croce, Catherine Robert, Carine Couderc, Fabrizio Di Pinto, Cheikh Sokhna, Didier Raoult, Dr Jean-Christophe Lagier PII:
S2052-2975(15)00079-7
DOI:
10.1016/j.nmni.2015.09.014
Reference:
NMNI 78
To appear in:
New Microbes and New Infections
Received Date: 12 August 2015 Revised Date:
23 September 2015
Accepted Date: 24 September 2015
Please cite this article as: Seck Eh, Rathored J, Khelaifia S, Croce O, Robert C, Couderc C, Di Pinto F, Sokhna C, Raoult D, Lagier J-C, Virgibacillus senegalensis sp. nov. A new moderately halophilic bacterium isolated from the human gut, New Microbes and New Infections (2015), doi: 10.1016/ j.nmni.2015.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Virgibacillus senegalensis sp. nov. a new moderately halophilic bacterium isolated from the human gut El hadji Seck1, Jaishriram Rathored1, Saber Khelaifia1, Olivier Croce1, Catherine Robert1,
Lagier1* 1
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Carine Couderc1, Fabrizio Di Pinto1, Cheikh Sokhna2, Didier Raoult1,3 and Jean-Christophe
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS
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7278, IRD 198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, Aix-Marseille Université
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes IRD 198, CNRS
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7278, Aix-Marseille Université, Marseille, France; Campus Commun UCAD-IRD of Hann, Dakar, Senegal. 3
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz
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University, Jeddah, Saudi Arabia
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*Corresponding author: Dr Jean-Christophe LAGIER
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URMITE, UMR CNRS 7278, L’Institut de Recherche pour le Développement 198, INSERM U1095, Faculté de Médecine, Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Tel: 00 33 4 91 32 49 50, Fax: 00 33 4 91 38 77 72. E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract
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Virgibacillus senegalensis SK-1T (CSUR P1101 = DSM 28585), is the type strain of V.
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senegalensis sp. nov. It is an aerobic, Gram positive, moderately halophilic, motile bipolar
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flagellum, isolated from a healthy Senegalese male. Here, we describe the genomic and
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phenotypic characteristics of this isolate. The 3,755,098 bp long genome (1 chromosome, no
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plasmid) exhibits a G+C content of 42.9 % and contains 3,738 protein-coding and 95 RNA
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genes.
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Keywords: Keywords: Virgibacillus senegalensis; genome; moderately halophilic bacteria; human gut; culturomics; taxonogenomics.
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Abbreviations
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CSUR: Collection de Souches de l’Unité des Rickettsies
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DSM: Deutsche Sammlung von Mikroorganismen
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MALDI-TOF MS: Matrix-assisted laser-desorption/ionization time-of-flight mass
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spectrometry
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TE buffer: Tris-EDTA buffer
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SDS: sodium dodecyl sulfate
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URMITE: Unité des Maladies Infectieuses et Tropicales Emergentes
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Introduction The concept of "microbial culturomics" is based on the variation of physicochemical
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parameters of the culture conditions, so as to express the maximum of microbial diversity. It
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is based on rapid methods for identification as MALDI-TOF and 16SrRNA amplification and
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sequencing for unidentified colonies. This concept enriches considerably the gut microbiota
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repertoire including new species not previously isolated from humans [1, 2].
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This isolation is part of the "culturomics study", using high salt containing culture conditions
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to grow halophilic bacteria from human stool [1].
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The typical parameters used to define bacterial species comprise 16S rRNA
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sequencing and phylogeny, G + C content genomic diversity and DNA hybridization (DDH).
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However, some limitations have been noted [3-6]. Using the availability of data in genomics
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through the development of new tools for sequencing DNA, we introduced a new taxonomic
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method for the description of new bacterial species. This concept that we named
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taxonogenomics includes their genomic features [7] and proteomic information obtained by
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MALDI-TOF-MS analysis [8-17].
The genus Virgibacillus was first proposed by Heyndrickx in 1998 with the transfer of
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Bacillus pantothenticus to Virgibacillus pantothenticus. [18]. To date, there are more than 25
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recognized species [19]. These bacteria are positive, Gram variable rods which are ellipsoidal
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to oval endospores and contained the DNA G + C content ranging from 36 to 43%. [20].
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These species were isolated from sediments of a salt lake [20-23], fermented seafood in the
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traditional salt [24], a permafrost core collected from the Canadian High Arctic [25], a Navy
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solar salt marsh [26, 27], soil [28], seawater [29], field soil, a dairy product [30], residual
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wash water produced during processing wastewater, Spanish style green table olives [31],
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saline sample of mud, salt-crust [32], and Thaï fermented fish [33]. Here, we present a brief
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classification and a set of features for strain SK-1T (CSUR P1101 = DSM 28585), with the
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description of the complete genome sequence and annotation. We named Virgibacillus
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senegalensis this new isolate.
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Materials and methods
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Sample and culture condition The stool sample was collected from a healthy male Senegalese volunteer patient
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living in N’diop (a rural village in the Guinean-Sudanian zone in Senegal). After the patient
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gave signed informed consent, the sample was collected in a sterile pot and transported in our
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laboratory. The study and the assent procedure were approved by the National Ethics
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Committee of Senegal and by the Ethics Committees of the Institut Fédératif de Recherche
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48, Faculty of Medicine, Marseille, France, (agreement number 09-022). Salt concentration of
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the stool specimen was determined by a digital refractometer (Fisher scientific, Illkirch,
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France) and the pH using a pH-meter (Cyberscan510PH, Eutech instruments, Singapore)
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Strain SK-1T was isolated in February 2014 by aerobic culture on a home-made
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culture medium consisting of a Columbia agar culture medium (Sigma-Aldrich, Saint-Quentin
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Fallavier, France) modified by adding (per liter): MgCl2 6H2O, 5 g; MgSO4 7H2O, 5 g; KCl,
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2 g; CaCl2 2H2O, 1g; NaBr, 0,5 g; NaHCO3, 0,5 g, glucose, 2 g and 100g/L of NaCl. The pH
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was adjusted to 7.5 with 10M NaOH before autoclaving.
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MALDI-TOF identification
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An isolated colony was deposited in duplicate on a MALDI-TOF target to be
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analyzed. A matrix of 1,5µL (Saturated solution of α-cyano-4-hydroxycinnamic acid diluted
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in 500µl acetonitrile, 250µl of acid Tri-Fluoro-Acetic to 10%, and 250µl of HPLC water) was
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used on each spot. This solution enables ionization and desorption of the homogeneous
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biological sample with which it crystallizes. The analysis was performed by a Microflex
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(Bruker Daltonik) and protein spectra were compared with those of the hospital database. A
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score was assigned indicating the reliability of the identification of the bacteria: above 1.9 is
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considered a proper identification. Conversely if the bacterium is not referenced in the
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database, sequencing the 16S rRNA is used in order to achieve a correct identification [34].
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Identification by sequencing of 16S rRNA
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Colonies non identified by the MALDI-TOF after three tests were suspended in 200µl of distilled water for DNA extraction by EZ1 DNA Tissue Kit (QIAGEN,Venlo, Pays-Bas).
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The amplification of the 16S rRNA was done by standard PCR in a thermocycler using the
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universal primer pair FD1 and rp2 according to the following amplification program:
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activation of the polymerase (95°C for 5 min) followed by 40 cycles (95°C 30sec, 52°C
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45sec, 72°C 2min) followed by 5 min at 72°C. The DNA amplified by this reaction was
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revealed by electrophoresis on 1.5% agarose gel. Once validated, the PCR product was
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purified and sequenced using the Big Dye Terminator Sequencing Kit using the following
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internal primers: 536F, 536R, 800F, 800R, 1050F, 1050R, as previously described [2].
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Phylogenetic analysis
Phylogenetic analysis based on 16S rRNA of our isolates was performed to identify its
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phylogenetic affiliations with other near isolates, including other members of the genus
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Virgibacillus. The MEGA 6 (Molecular Evolutionary Genetics Analysis) software allowed us
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to construct a phylogenetic tree. Sequence alignment of the different species was performed
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using CLUSTAL W and the evolutionary distance was calculated with the Kimura two-
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parameter model [35].
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Biochemistry, atmospheric and antimicrobial susceptibility tests
ACCEPTED MANUSCRIPT Biochemical tests were performed using the commercially available Api ZYM
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(BioMerieux, Marcy-l’Etoile, France), API 50CH (BioMerieux, Marcy-l’Etoile, France), 20
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NE (BioMerieux, Marcy-l’Etoile, France) strips. The incubation time was 48 hours for the last
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two and 4h for Api ZYM. Growth of the strain SK-1T was tested in aerobic atmosphere, in
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the presence of 5% CO2, and also in anaerobic and microaerophilic atmospheres, created
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using AnaeroGenTM (Atmosphere Generation Systems, Dardily, France). Antibiotic
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susceptibility was determined by Mueller-Hinton agar in a Petri dish (BioMerieux, Marcy-
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l’Etoile, France). The following antibiotics were tested: doxycycline, rifampicin, vancomycin,
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nitrofurantoin, amoxicillin, erythromycin, ampicillin, ceftriaxone, ciprofloxacin, gentamicin,
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penicillin, trimethoprim / sulfamethoxazole imipenem, and metronidazole.
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Genome sequencing information
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Genomic DNA preparation
We cultured our strain in the home-made culture. After 48h, bacteria grown on four
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Petri dishes were resuspended in sterile water and centrifuged at 4oC at 2,000 xg for 20 min.
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Cell pellets were resuspended in 1 mL Tris/EDTA/NaCl [10mM Tris/HCl (pH7.0), 10 mM
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EDTA (pH8.0), and 300 mM [NaCl] and re-centrifuged under the same conditions. The
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pellets were then re-suspended in 200µL TE buffer and Proteinase K and kept overnight at
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37°C for cell lysis. DNA was purified with phenol/ chloroform/ isoamylalcohol (25:24:1),
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followed by an overnight precipitation with ethanol at -20°C. The DNA was resuspended in
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205µL TE buffer. DNA concentration was 155 ng/ µl as measured by by Qubit fluorometer,
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using the high sensitivity kit (Life technologies, Carlsbad, CA, USA).
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Genome sequencing and assembly
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Genomic DNA of Virgibacillus senegalensis was sequenced on the MiSeq Technology (Illumina Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in
ACCEPTED MANUSCRIPT order to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit
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(Illumina). gDNA was quantified by a Qubit assay with the high sensitivity kit (Life
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technologies, Carlsbad, CA, USA) to 155 ng/µl .The mate pair library was prepared with 1µg
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of genomic DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was
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simultaneously fragmented and tagged with a mate pair junction adapter. The pattern of the
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fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc,
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Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in size from
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1kb up to 11kb with an optimal size at 4.008 kb. No size selection was performed and 388.3
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ng of tagmented fragments were circularized. The circularized DNA was mechanically
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sheared to small fragments with an optimal at 634 bp on the Covaris device S2 in microtubes
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(Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity
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Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA) and the final
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concentration library was measured at 35.59 nmol/l. The libraries were normalized at 2nM
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and pooled. After a denaturation step and dilution at 15 pM, the pool of libraries was loaded
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onto the reagent cartridge and then onto the instrument along with the flow cell. Automated
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cluster generation and sequencing run were performed in a single 39-hours run in a 2x251-
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bp.Total information of 10.6 Gb was obtained from a 1326 K/mm2 cluster density with a
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cluster passing quality control filters of 99.1 % (24,492,260 clusters). Within this run, the
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index representation for Virgibacillus senegalensis was determined to be 7.06 %. The
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1,481,197 paired reads were filtered according to the read qualities. These reads were
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trimmed, and then assembled using the CLC genomicsWB4 software.
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Genome annotation and comparison
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Open Reading Frames (ORFs) were predicted using Prodigal [36] with default parameters but
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the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted
ACCEPTED MANUSCRIPT bacterial protein sequences were searched against the GenBank database [37] and the Clusters
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of Orthologous Groups (COG) databases using BLASTP. The tRNAScanSE tool [38] was
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used to find tRNA genes, whereas ribosomal RNAs were found using RNAmmer [39] and
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BLASTn against the GenBank database. Lipoprotein signal peptides and the number of
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transmembrane helices were predicted using SignalP [40] and TMHMM [41] respectively.
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ORFans were identified if their BLASTP E-value was lower than 1e-03 for alignment length
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greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used
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an E-value of 1e-05. Such parameter thresholds have already been used in previous works to
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define ORFans. Artemis [42] was used for data management and DNA Plotter [43] for
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visualization of genomic features. Mauve alignment tool (version 2.3.1) was used for multiple
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genomic sequence alignment [44]. To estimate the mean level of nucleotide sequence
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similarity at the genome level, we used the MAGI homemade software to calculate the
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average genomic identity of gene sequences (AGIOS) among compared genomes [45].
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Briefly, this software combines the Proteinortho software [45] for detecting orthologous
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proteins in pairwise genomic comparisons, then retrieves the corresponding genes and
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determines the mean percentage of nucleotide sequence identity among orthologous ORFs
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using the Needleman-Wunsch global alignment algorithm. Genomes from the genus
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Virgibacillus and closely related genera were used for the calculation of AGIOS values. Here,
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we compared the genome sequences of Virgibacillus senegalensis strain SK-1T (GenBank
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accession number PRJEB1962) with those of Virgibacillus kekensis strain YIM kkny9
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(NR_042744.1), Virgibacillus albus strain YIM 93624 (NR_109613.1), Aquibacillus
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salifodinae WSY08-1 (AB859945.1), Virgibacillus halodenitrificans DSM 10037
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(AY543169), Thalassobacillus devorans MSP14 ( JX518269.1), Halobacillus dabanensis
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HD 02 (HG931924.2), Halobacillus kuroshimensis DSM 18393 (AB195680.1),
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Thalassobacillus devorans strain XJSL7-8 (GQ903447.1), Bacillaceae bacterium EFN-4
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ACCEPTED MANUSCRIPT (EU817569.1), Virgibacillus marismortui strain M3-23 (GQ282501.1), Halobacillus salinus
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strain GSP59 ( AY505517.1), Virgibacillus alimentarius J18 (GU202420), Pseudomonas
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aeruginosa PAO1 (NR_074828.1) and Virgibacillus massiliensis (CCDP010000001).
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Results
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Phenotypic description
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Strain SK-1T was isolated in February 2014 (Table1) by aerobic culture on a homemade culture medium at 37° after 48h. No significant MALDI-TOF result for the strain SK-
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1T against our MALDI-TOF database was obtained, suggesting that our isolate was not a
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previously known species. We added the specter of SK-1T strain to our database (Figure 1).
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The gel view allows to observe the spectral differences with other members of Virgibacillus
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genus (Figure 2). PCR-based identification of the 16S rRNA of our newly isolate (GenBank
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accession number LK021111) yielded 96.3 % 16S rRNA sequence similarity with the
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reference Virgibacillus kekensis (GenBank accession number NR042744) the
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phylogenetically closest validated Virgibacillus species (Figure 3).
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After growth for 24 hour on our home-made culture medium at 37°C, the surface colonies
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were circular, grayish, shiny and smooth with a diameter of 1-2 mm in diameter V.
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senegalensis is Gram positive (figure 4).
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Growth is observed at temperatures ranging from 25 to 40°C, with an optimum at 37°C. The growth required a salinity ranging from 5 to 200 g/L of NaCl (optimum at 75 g/L).
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The optimum pH for growth was 7.5 (range between pH 5 to 9). Growth of the strain SK-1T
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was tested in an aerobic atmosphere, in the presence of 5% CO2, and also in anaerobic and
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microaerophilic atmospheres created using AnaeroGenTM (Atmosphere Generation Systems,
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Dardily, France), respectively. The strain was strictly aerobic and also grew in the presence of
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5% CO2 but did not grow in an anaerobic atmosphere. The size and ultrastructure of cells
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were determined by negative staining transmission electron microscopy 2 - 6 µm in length
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ACCEPTED MANUSCRIPT and 0.5 µm in diameter (Figure 5). Using the commercially available Api ZYM, Api 20NE
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(BioMerieux, Marcy-l’Etoile, France) to characterize the biochemical V. senegaiensis strain
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SK-1T , positive reactions were observed for urease, βglucosidase, protease, β-galactosidase,
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arginine hydrolase All other tested reactions were negative, notably nitrate reduction alkaline
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phosphatase, N-acetyl-β-glucosaminidase . The stain was also catalase and oxidase negative.
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Substrate oxidation and assimilation were examined with an API 50CH strip (BioMerieux,
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Marcy-l’Etoile, France) at 37°C. Negative reactions were obtained for D-mannose, D-lactose,
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L-arabinose, D-galactose, D-ribose, D-sucrose, D-fructose, D-glucose, D-mannitol, and D-
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maltose. Phenotypic characteristics were compared to those of the most closely related
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species (Table 2).
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Finally, antimicrobial susceptibility testing demonstrate that the strain SK-1T was susceptible to doxycycline, rifampicin, vancomycin, nitrofurantoin, amoxicillin,
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erythromycin, ampicillin, ceftriaxone, ciprofloxacin, gentamicin, penicillin,
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trimethoprim/sulfamethoxazole and imipenem, but resistant to metronidazole.
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Genome sequencing information
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Genome properties
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The draft genome of V. senegalensis consists of nine scaffolds with 59 contigs.
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The genome is 3,755,098 bp-long with a 42.9% G+C content (Table 3, Figure 6). Of the 3,833
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predicted genes, 3,738 (96.46%) were protein- coding genes, and 95 (2.44%) were RNAs (14
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genes are 5S rRNA, five genes are 16S rRNA, seven genes are 23S rRNA, 69 genes are tRNA
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genes). A total of 2,773 genes (62.82%) were assigned a putative function, 2,427 (65.43%)
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genes were assigned to COGs and 155 (4.04%) genes contain peptide signals whereas 980
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(25.56%) genes had transmembrane helices. A total of 245 genes were identified as ORFans
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(6.39%). The remaining genes were annotated as hypothetical proteins. The properties and
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statistics of the genome are summarized in Tables 3 and 4. The distribution of genes into
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COGs functional categories is presented in Table 5.
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Genome comparaison The draft genome of V. senegalensis SK-1T is smaller than those of Halobacillus
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kuroshimensis DSM 18393, Virgibacillus halodenitrificans DSM10037, Thalassobacillus
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devorans XJSL7-8, Thalassobacillus devorans XJSL7-8, Halobacillus dabanensis HD 02
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and Pseudomonas aeruginosa PAO1 (3.85, 3.92, 3.94, 4.1 and 6.26 Mb respectively) but
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larger than that of Virgibacillus alimentarius J18 (3.05 Mb). The G+C content of V.
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senegalensis SK-1T is smaller than those of Halobacillus kuroshimensis DSM 18393 and
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Pseudomonas aeruginosa PAO1 (47.0 and 66.60% respectively) and larger than those of
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Virgibacillus alimentarius J18, Virgibacillus halodenitrificans DSM10037, Halobacillus
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dabanensis HD 02 (37.1, 37.4, 41.5 % respectively) but equal to halassobacillus devorans
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XJSL7-8 (42.9 %). Protein coding genes of V. senegalensis SK-1T is smaller than those of
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Virgibacillus halodenitrificans DSM10037, Thalassobacillus devorans XJSL7-8,
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Halobacillus kuroshimensis DSM 18393, Halobacillus dabanensis HD 02 and Pseudomonas
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aeruginosa PAO1 (3,748, 3,752, 3,832, 3,835, 5,572 Mb respectively) but larger than those of
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Virgibacillus alimentarius J18 (2,889 Mb). Total gene content of V. senegalensis SK-1T
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(3883) is smaller than those of Halobacillus kuroshimensis DSM 18393, Halobacillus
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dabanensis HD 02 and Pseudomonas aeruginosa PAO1 (3,915, 4,011, and 5,697
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respectively) but larger than those of Thalassobacillus devorans XJSL7-8, Virgibacillus
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halodenitrificans DSM10037, Virgibacillus alimentarius J18 (3,840, 3,822 and 3,022
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respectively).
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Among species with standing in nomenclature, AGIOS values ranged from 66.41 between V. senegalensis SK-1T and Halobacillus kuroshimensis DSM 18393 to 73.39%
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evaluate the genomic similarity among studied strains, in addition to AGIOS [7], which was
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designed to be independent from DDH, we determined dDDH that exhibits a high correlation
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with DDH [46-47]. dDDH ranged from 18.4 to 27.2% between the different species tested
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(Table 6, Figure 7).
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Conclusion
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Based on phenotypic, genomic and phylogenetic analyzes, we formally propose the creation of Virgibacillus senegalensis sp. nov, represented here by the SK-1T strain. The
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strain was isolated from a stool sample of a Senegalese healthy individual.
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Taxonomic and nomenclatural proposals
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Description of Virgibacillus senegalensis sp. nov.
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Virgibacillus senegalensis (se.ne.ga. len′sis. L. masc. adj. senegalensis of Senegalia, the Roman name of Senegal, where the type strain was isolated). Growth occurred between 15
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to 45°C on a home-made culture medium herein described with optimal growth observed at
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37°C in aerobic atmosphere. Strain SK-1T required a salinity ranging from 5 to 200 g/L of
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NaCl (optimum at 100 g/L). The optimum pH for growth was 7.5 (range between pH 5 to 9).
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The strain SK-1T was strictly aerobic and also grew in the presence of 5% CO2 but no growth
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was observed under anaerobic and microaerophilic conditions. The colonies of the strain SK-
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1T were circular, grayish, shiny and smooth with a diameter of 2-6 mm. Cells stained Gram-
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positive. They were motile by polar flagella, spore forming (2 to 6 µm in length and 0.5 µm
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in diameter) and generally occurred individually or in pairs. Strain SK-1T is catalase and
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oxidase negative. Using API 50 CH and API20 NE (BioMerieux, Marcy-l’Etoile, France),
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strain SK-1T was positive for reduction of nitrates but negative for phosphatase alkaline
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activity, β-galactosidase, α N-acetyl-β-glucosaminidase, urease. Strain SK-1T was negative
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ACCEPTED MANUSCRIPT for ribose, L-arabinose and D-lactose assimilation and positive for D-glucose, D-fructose, D-
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mannose, D-mannitol, D-maltose and D-sucrose. The strain SK-1T was susceptible to
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doxycycline, rifampicin, vancomycin, nitrofurantoin, amoxicillin, erythromycin, ampicillin,
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ceftriaxone, ciprofloxacin, gentamicin, penicillin, trimethoprim/sulfamethoxazole and
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imipenem, but resistant to metronidazole.
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The G+C% content of the genome is 42.9%. The 16S rRNA and genome sequences are
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deposited in GenBank under accession numbers LK021111. The habitat of the microorganism
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is the human digestive tract. The type strain SK-1T (CSUR P1101, DSM28585) was isolated
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from a stool specimen of a Senegalese male individual.
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Authors’ contributions
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ES performed the phenotypic characterization of the bacterium and drafted the manuscript. JR
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and OC performed the genomic analyses. SK participated in the study coordination and
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helped to draft the manuscript. CR performed the genomic analyses and drafted the
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manuscript. CC performed the MALDI-TOF identification and figures. FDP performed the
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electronic microscopy visualization of the bacterium. CS participated in the study design and
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collected the sample. DR and JCL conceived the study and helped to draft the manuscript. All
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authors read and approved the final manuscript.
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Acknowledgements
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The authors thank the Xegen Company (www.xegen.fr) for automating the genomic
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annotation process. This study was funded by the Mediterranée-Infection Foundation.
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Figure legends.
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Figure 1. Reference mass spectrum from Virgibacillus senegalensis sp. nov. SK-1T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.
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Figure 2. Gel view comparing Virgibacillus senegalensis sp. nov. SK-1Tto the members of the family Virgibacillus and Oceanobacillus. The gel view displays the raw spectra of all 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 by a Gray scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. Displayed species are indicated on the left.
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Figure 3. Phylogenetic tree highlighting the position of Virgibacillus senegalensis strain SK1T (CSUR P1101 = DSM28585) relative to other type strains of Virgibacillus albus strain YIM 93624 (NR_109613.1) , Virgibacillus kekensis strain YIM kkny16 (NR_042744.1), Virgibacillus alimentarius strain J18 (NR_108710.1), Virgibacillus marismortui strain M3-23 (GQ282501.1), Virgibacillus necropolis strain LMG 19488 (NR_025472.1), Virgibacillus carmonensis strain LMG 20964 (NR_025481.1), Virgibacillus subterraneus strain H57B72 (FJ746573.1), Virgibacillus zhanjiangensis strain JSM 079157 (FJ425904.1), Virgibacillus litoralis strain JSM 089168 (FJ425909.1), Virgibacillus dokdonensis strain DSW-10 (NR_043206.1), Virgibacillus siamensis strain MS3-4 (AB365482.1), Virgibacillus salarius strain SA-Vb1 (NR_041270.1), Virgibacillus halophilus strain 5B73C (NR_041358.1), Virgibacillus natechei strain FarD (NR_132721.1), Virgibacillus chiguensis strain NTU-101 (NR_044086.1), Virgibacillus dokdonensis strain DSW-10 (NR_043206.1), Virgibacillus campisalis strain IDS-20 (GU586225.1), Virgibacillus pantothenticus strain NBRC 102447 (AB681789.1), Virgibacillus halodenitrificans strain NBRC 102361 (AB681753.1), Virgibacillus byunsanensis strain ISL-24 (FJ357159.1), Virgibacillus massiliensis strain Vm5 (HG931931.1) and Paenibacillus polymyxa strain KCTC3717 (AY359637.1). GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method within the MEGA software. Paenibacillus polymyxa was used as out group. The scale bar represents 0.005% nucleotide sequence divergence Figure 4. Gram staining of Virgibacillus senegalensis sp. nov. SK-1T
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Figure 5. Transmission electron microscopy of Virgibacillus senegalensis sp. nov. SK-1T. Cells are observed on a Tecnai G20 transmission electron microscope operated at 200 keV. The scale bar represents 500 nm.
Figure 6: Graphical circular map of the Virgibacillus senegalensis sp. nov. SK-1T. chromosome. From the outside in, the outer two circles show open reading frames oriented in the forward (colored by COG categories) and reverse (colored by COG categories) directions, respectively. The third circle marks the tRNA genes (green). The fourth circle shows the G+C% content plot. The inner-most circle shows GC skew, purple indicating negative values whereas olive for positive values
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Figure 7. Distribution of functional classes of predicted genes according to the clusters of orthologous groups of proteins
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Table 1. Classification of Virgibacillus senegalensis strain SK-1 Property Term Current classification Domain: Bacteria
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Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Bacillaceae Genus: Virgibacillus Species: Virgibacillus senegalensis Type strain: SK-1 Positive Rod shaped Motile by polar flagellum Endospore-forming Mesophile 37°C pH 5 to 9 7.5 0.5 to 10% 7.5% Aerobic
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Gram stain Cell shape Motility Sporulation Temperature range Optimum temperature pH Optimum pH Salinity Optimum salinity Oxygen requirement
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Table 2: Differential characteristics. V. V. V. senegalensis massiliensis olivae
V. V. V. V. V. V. V. V. salarius marismortuis sediminis xinjiangensis kekensis halodenitrificans proomii dokdonensis
Cell diameter (µm)
0.6-0.9
0.5-0.8
0.4-0.6 0.6-0.9
0.4-0.7
1.4~2.4
0.3–0.5 0.6-0.8
0.5-0.7
Oxygen requirement
Aerobic
Aerobic
Aerobic Aerobic Aerobic
Aerobic
Aerobic
Aerobic Aerobic
Aerobic Aerobic
Gram stain
+
+
+
+
+
+
+
+
+
+
+
Salt requirement
+
+
+
+
-
+
-
-
+
na
+
Motility
+
+
+
+
+
+
+
+
+
+
+
Endospore formation +
+
+
+
+
+
+
+
+
+
+
Indole
-
-
-
-
-
-
-
-
-
-
-
Alkaline phosphatase -
-
na
na
na
-
na
-
na
na
-
Catalase
-
+
+
Oxidase
-
+
+
Nitrate reductase
-
+
+
Urease
+
-
na
β-galactosidase
+
-
na
N-acetyl-glucosamine -
-
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+
+
+
+
+
+
+
+
+
+
+
-
+
+
na
+
-
+
+
+
+
+
+
-
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Production of
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Properties
-
na
-
-
-
+
na
-
-
-
-
-
-
+
+
-
na
+
-
na
-
na
+
-
na
-
-
-
-
-
-
-
-
+
L-Arabinose
-
-
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Ribose
-
-
na
na
na
+
-
-
na
+
+
D-mannose
-
+
-
+
+
-
-
+
+
+
+
Acid from
ACCEPTED MANUSCRIPT
Properties
V. V. V. senegalensis massiliensis olivae
V. V. V. V. V. V. V. V. salarius marismortuis sediminis xinjiangensis kekensis halodenitrificans proomii dokdonensis
D-mannitol
-
+
na
-
-
-
D-sucrose
-
+
-
-
-
-
D-glucose
-
+
-
+
+
+
D-fructose
-
+
+
+
+
D-maltose
-
+
-
+
-
D-lactose
-
-
-
-
-
343
+
-
+
-
-
+
+
+
+
+
+
+
+
-
+
+
+
+
-
+
+
+
-
-
-
-
+
+
+
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+
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Waste Salt lake washwater
w
+
Mural paintings
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Human gut
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Human gut
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Habitat
-
Salt lake
Salt lake
Salt lake
Solar saltern
Soil
Soil
ACCEPTED MANUSCRIPT Table 3. Nucleotide content and gene count levels of the genome Value
% of totala
Size (bp)
3,755,098
100
G+C content (%)
1,610,937
42.9
Coding region (bp)
3,129,675
83.34
Total genes
3,883
100
RNA genes
95
2.44
Protein-coding genes
3,738
98.46
Genes with function prediction
2,773
Genes assigned to COGs
2,421
Genes with peptide signals Genes with transmembrane helices CRISPRs repeats
a
SC 65.43
4.04
980
25.56
02
0.05
2,011
52.46
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The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome
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345 346
62.82
155
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Genes with Pfam Domains
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ACCEPTED MANUSCRIPT Table 4. Number of genes associated with the 25 general COG functional categories Value
% valuea
Description
J
172
4.60
Translation
A
0
0
RNA processing and modification
K
262
7.01
Transcription
L
196
5.24
Replication, recombination and repair
B
1
0.03
Chromatin structure and dynamics
D
30
0.80
Cell cycle control, mitosis and meiosis
Y
0
0
Nuclear structure
V
49
1.31
Defense mechanisms
T
153
4.09
Signal transduction mechanisms
M
156
4.17
Cell wall/membrane biogenesis
N
64
1.71
Cell motility
Z
0
0
Cytoskeleton
W
0
0
Extracellular structures
U
51
1.36
Intracellular trafficking and secretion
O
92
2.46
Posttranslational modification, protein turnover, chaperones
C
137
3.67
G
275
7.36
E
305
8.16
F
82
H
93
I
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Amino acid transport and metabolism
2.19
Nucleotide transport and metabolism
2.49
Coenzyme transport and metabolism Lipid transport and metabolism
202
5.40
Inorganic ion transport and metabolism
74
1.98
Secondary metabolites biosynthesis, transport and catabolism
448
11.99
General function prediction only
S
287
7.68
Function unknown
_
352
9.06
Not in COGs
R
349
Carbohydrate transport and metabolism
2.73
Q
a
Energy production and conversion
102
P
348
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Code
AC C
347
The total is based on the total number of protein coding genes in the annotated genome
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Halobacillus dabanensis
Halobacillus kuroshimensis
3,378
1,791
1,786
Halobacillus dabanensis
66.82
4,063
2,218
Halobacillus kuroshimensis
66.41
73.39
3,926
Thalassobacillus devorans
67.76
69.22
68.46
Virgibacillus alimentarius
67.16
66.25
Virgibacillus halodenitrificans
67.19
66.41
Pseudomonas aeruginosa
53.94
52.73
66.21
65.24
Virgibacillus halodenitrificans
Pseudomonas aeruginosa
Virgibacillus massiliensis
1,446
1,741
601
588
2,122
1,581
1,866
697
642
2,119
1,554
1,861
695
630
3880
1,596
1,923
687
618
64.45
66.55
3,119
1,663
607
604
64.71
66.67
71.39
3,876
661
676
55.86
53.96
49.86
50.02
5,681
244
63.48
65.57
69.67
70.87
50.45
1,768
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Virgibacillus massiliensis
1,776
Virgibacillus alimentarius
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Virgibacillus senegalensis
Thalassobacillus devorans
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Virgibacillus senegalensis
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Table 5. The numbers of orthologous proteins shared between genomes (upper right). Average percentage similarity of nucleotides corresponding to orthologous proteins shared between genomes (lower left) and numbers of proteins per genome (bold)
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350 351
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Halobacillus kuroshimensis Thalassobacillus devorans Virgibacillus alimentarius Virgibacillus halodenitrificans Pseudomonas aeruginosa Virgibacillus massiliensis
100% ± 00
26.7% ±2.54 26.3% ±2.55
100% ± 00
Virgibacillus alimentarius
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24.3% ± 2.55
Thalassobacillus devorans
Virgibacillus halodenitrificans
Pseudomonas aeruginosa
Virgibacillus massiliensis
22.8% ±2.54
26.4% ±2.54
21.8% ±2.56
20.8% ±2.53
22.8% ±2.53
24.4% ±2.56
27.7% ±2.55
27% ±2.55
21.2% ±2.57
20.0% ±2.53
24.6% ±2.55
27.2% ±2.55
24.7% ±2.57
21.8% ±2.54
19.5% ±2.53
100% ± 00
22.1% ±2.55
21.3% ±2.57
18.4% ±2.56
20.1% ±2.53
100% ± 00
23% ±2.56
21.1% ±2.53
20.0% ±2.54
100% ± 00
22.5% ±2.54
21.3% ±2.55
100% ± 00
20.3% ±2.52
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Halobacillus dabanensis
100% ± 00
Halobacillus kuroshimensis
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Virgibacillus senegalensis
Halobacillus dabanensis
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Virgibacillus senegalensis
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Table 6. Pairwise comparison of Virgibacillus senegalensis with eight other species using GGDC, formula 2 (DDH estimates based on identities / HSP length) *The confidence intervals indicate the inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size). These results are in accordance with the 16S rRNA (Figure 1) and phlogenomic analyses as well as the GGDC results.
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352 353 354 355
100% ± 00
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Figure 1
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In te n s . [a .u .]
x104
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1.5
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1.0
0.0 4000
6000
8000
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2000
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359 360
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0.5
10000
12000
14000
16000
18000
m/z
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363
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365
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Figure 5
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Figure 6
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ACCEPTED MANUSCRIPT Reference
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S2052-2975(15)00079-7
DOI:
10.1016/j.nmni.2015.09.014
Reference:
NMNI 78
To appear in:
New Microbes and New Infections
Received Date: 12 August 2015 Revised Date:
23 September 2015
Accepted Date: 24 September 2015
Please cite this article as: Seck Eh, Rathored J, Khelaifia S, Croce O, Robert C, Couderc C, Di Pinto F, Sokhna C, Raoult D, Lagier J-C, Virgibacillus senegalensis sp. nov. A new moderately halophilic bacterium isolated from the human gut, New Microbes and New Infections (2015), doi: 10.1016/ j.nmni.2015.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Virgibacillus senegalensis sp. nov. a new moderately halophilic bacterium isolated from the human gut El hadji Seck1, Jaishriram Rathored1, Saber Khelaifia1, Olivier Croce1, Catherine Robert1,
Lagier1* 1
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Carine Couderc1, Fabrizio Di Pinto1, Cheikh Sokhna2, Didier Raoult1,3 and Jean-Christophe
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS
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7278, IRD 198, Inserm 1095, Institut Hospitalo-Universitaire Méditerranée-Infection, Faculté de médecine, Aix-Marseille Université
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes IRD 198, CNRS
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7278, Aix-Marseille Université, Marseille, France; Campus Commun UCAD-IRD of Hann, Dakar, Senegal. 3
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz
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University, Jeddah, Saudi Arabia
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*Corresponding author: Dr Jean-Christophe LAGIER
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URMITE, UMR CNRS 7278, L’Institut de Recherche pour le Développement 198, INSERM U1095, Faculté de Médecine, Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Tel: 00 33 4 91 32 49 50, Fax: 00 33 4 91 38 77 72. E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract
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Virgibacillus senegalensis SK-1T (CSUR P1101 = DSM 28585), is the type strain of V.
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senegalensis sp. nov. It is an aerobic, Gram positive, moderately halophilic, motile bipolar
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flagellum, isolated from a healthy Senegalese male. Here, we describe the genomic and
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phenotypic characteristics of this isolate. The 3,755,098 bp long genome (1 chromosome, no
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plasmid) exhibits a G+C content of 42.9 % and contains 3,738 protein-coding and 95 RNA
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genes.
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Keywords: Keywords: Virgibacillus senegalensis; genome; moderately halophilic bacteria; human gut; culturomics; taxonogenomics.
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Abbreviations
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CSUR: Collection de Souches de l’Unité des Rickettsies
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DSM: Deutsche Sammlung von Mikroorganismen
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MALDI-TOF MS: Matrix-assisted laser-desorption/ionization time-of-flight mass
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spectrometry
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TE buffer: Tris-EDTA buffer
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SDS: sodium dodecyl sulfate
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URMITE: Unité des Maladies Infectieuses et Tropicales Emergentes
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Introduction The concept of "microbial culturomics" is based on the variation of physicochemical
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parameters of the culture conditions, so as to express the maximum of microbial diversity. It
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is based on rapid methods for identification as MALDI-TOF and 16SrRNA amplification and
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sequencing for unidentified colonies. This concept enriches considerably the gut microbiota
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repertoire including new species not previously isolated from humans [1, 2].
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This isolation is part of the "culturomics study", using high salt containing culture conditions
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to grow halophilic bacteria from human stool [1].
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The typical parameters used to define bacterial species comprise 16S rRNA
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sequencing and phylogeny, G + C content genomic diversity and DNA hybridization (DDH).
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However, some limitations have been noted [3-6]. Using the availability of data in genomics
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through the development of new tools for sequencing DNA, we introduced a new taxonomic
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method for the description of new bacterial species. This concept that we named
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taxonogenomics includes their genomic features [7] and proteomic information obtained by
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MALDI-TOF-MS analysis [8-17].
The genus Virgibacillus was first proposed by Heyndrickx in 1998 with the transfer of
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Bacillus pantothenticus to Virgibacillus pantothenticus. [18]. To date, there are more than 25
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recognized species [19]. These bacteria are positive, Gram variable rods which are ellipsoidal
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to oval endospores and contained the DNA G + C content ranging from 36 to 43%. [20].
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These species were isolated from sediments of a salt lake [20-23], fermented seafood in the
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traditional salt [24], a permafrost core collected from the Canadian High Arctic [25], a Navy
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solar salt marsh [26, 27], soil [28], seawater [29], field soil, a dairy product [30], residual
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wash water produced during processing wastewater, Spanish style green table olives [31],
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saline sample of mud, salt-crust [32], and Thaï fermented fish [33]. Here, we present a brief
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classification and a set of features for strain SK-1T (CSUR P1101 = DSM 28585), with the
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description of the complete genome sequence and annotation. We named Virgibacillus
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senegalensis this new isolate.
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Materials and methods
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Sample and culture condition The stool sample was collected from a healthy male Senegalese volunteer patient
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living in N’diop (a rural village in the Guinean-Sudanian zone in Senegal). After the patient
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gave signed informed consent, the sample was collected in a sterile pot and transported in our
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laboratory. The study and the assent procedure were approved by the National Ethics
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Committee of Senegal and by the Ethics Committees of the Institut Fédératif de Recherche
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48, Faculty of Medicine, Marseille, France, (agreement number 09-022). Salt concentration of
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the stool specimen was determined by a digital refractometer (Fisher scientific, Illkirch,
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France) and the pH using a pH-meter (Cyberscan510PH, Eutech instruments, Singapore)
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Strain SK-1T was isolated in February 2014 by aerobic culture on a home-made
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culture medium consisting of a Columbia agar culture medium (Sigma-Aldrich, Saint-Quentin
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Fallavier, France) modified by adding (per liter): MgCl2 6H2O, 5 g; MgSO4 7H2O, 5 g; KCl,
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2 g; CaCl2 2H2O, 1g; NaBr, 0,5 g; NaHCO3, 0,5 g, glucose, 2 g and 100g/L of NaCl. The pH
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was adjusted to 7.5 with 10M NaOH before autoclaving.
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MALDI-TOF identification
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An isolated colony was deposited in duplicate on a MALDI-TOF target to be
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analyzed. A matrix of 1,5µL (Saturated solution of α-cyano-4-hydroxycinnamic acid diluted
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in 500µl acetonitrile, 250µl of acid Tri-Fluoro-Acetic to 10%, and 250µl of HPLC water) was
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used on each spot. This solution enables ionization and desorption of the homogeneous
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biological sample with which it crystallizes. The analysis was performed by a Microflex
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(Bruker Daltonik) and protein spectra were compared with those of the hospital database. A
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score was assigned indicating the reliability of the identification of the bacteria: above 1.9 is
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considered a proper identification. Conversely if the bacterium is not referenced in the
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database, sequencing the 16S rRNA is used in order to achieve a correct identification [34].
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Identification by sequencing of 16S rRNA
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Colonies non identified by the MALDI-TOF after three tests were suspended in 200µl of distilled water for DNA extraction by EZ1 DNA Tissue Kit (QIAGEN,Venlo, Pays-Bas).
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The amplification of the 16S rRNA was done by standard PCR in a thermocycler using the
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universal primer pair FD1 and rp2 according to the following amplification program:
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activation of the polymerase (95°C for 5 min) followed by 40 cycles (95°C 30sec, 52°C
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45sec, 72°C 2min) followed by 5 min at 72°C. The DNA amplified by this reaction was
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revealed by electrophoresis on 1.5% agarose gel. Once validated, the PCR product was
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purified and sequenced using the Big Dye Terminator Sequencing Kit using the following
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internal primers: 536F, 536R, 800F, 800R, 1050F, 1050R, as previously described [2].
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Phylogenetic analysis
Phylogenetic analysis based on 16S rRNA of our isolates was performed to identify its
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phylogenetic affiliations with other near isolates, including other members of the genus
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Virgibacillus. The MEGA 6 (Molecular Evolutionary Genetics Analysis) software allowed us
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to construct a phylogenetic tree. Sequence alignment of the different species was performed
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using CLUSTAL W and the evolutionary distance was calculated with the Kimura two-
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parameter model [35].
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Biochemistry, atmospheric and antimicrobial susceptibility tests
ACCEPTED MANUSCRIPT Biochemical tests were performed using the commercially available Api ZYM
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(BioMerieux, Marcy-l’Etoile, France), API 50CH (BioMerieux, Marcy-l’Etoile, France), 20
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NE (BioMerieux, Marcy-l’Etoile, France) strips. The incubation time was 48 hours for the last
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two and 4h for Api ZYM. Growth of the strain SK-1T was tested in aerobic atmosphere, in
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the presence of 5% CO2, and also in anaerobic and microaerophilic atmospheres, created
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using AnaeroGenTM (Atmosphere Generation Systems, Dardily, France). Antibiotic
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susceptibility was determined by Mueller-Hinton agar in a Petri dish (BioMerieux, Marcy-
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l’Etoile, France). The following antibiotics were tested: doxycycline, rifampicin, vancomycin,
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nitrofurantoin, amoxicillin, erythromycin, ampicillin, ceftriaxone, ciprofloxacin, gentamicin,
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penicillin, trimethoprim / sulfamethoxazole imipenem, and metronidazole.
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Genome sequencing information
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Genomic DNA preparation
We cultured our strain in the home-made culture. After 48h, bacteria grown on four
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Petri dishes were resuspended in sterile water and centrifuged at 4oC at 2,000 xg for 20 min.
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Cell pellets were resuspended in 1 mL Tris/EDTA/NaCl [10mM Tris/HCl (pH7.0), 10 mM
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EDTA (pH8.0), and 300 mM [NaCl] and re-centrifuged under the same conditions. The
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pellets were then re-suspended in 200µL TE buffer and Proteinase K and kept overnight at
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37°C for cell lysis. DNA was purified with phenol/ chloroform/ isoamylalcohol (25:24:1),
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followed by an overnight precipitation with ethanol at -20°C. The DNA was resuspended in
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205µL TE buffer. DNA concentration was 155 ng/ µl as measured by by Qubit fluorometer,
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using the high sensitivity kit (Life technologies, Carlsbad, CA, USA).
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Genome sequencing and assembly
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Genomic DNA of Virgibacillus senegalensis was sequenced on the MiSeq Technology (Illumina Inc, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in
ACCEPTED MANUSCRIPT order to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit
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(Illumina). gDNA was quantified by a Qubit assay with the high sensitivity kit (Life
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technologies, Carlsbad, CA, USA) to 155 ng/µl .The mate pair library was prepared with 1µg
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of genomic DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was
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simultaneously fragmented and tagged with a mate pair junction adapter. The pattern of the
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fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc,
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Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in size from
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1kb up to 11kb with an optimal size at 4.008 kb. No size selection was performed and 388.3
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ng of tagmented fragments were circularized. The circularized DNA was mechanically
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sheared to small fragments with an optimal at 634 bp on the Covaris device S2 in microtubes
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(Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity
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Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA) and the final
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concentration library was measured at 35.59 nmol/l. The libraries were normalized at 2nM
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and pooled. After a denaturation step and dilution at 15 pM, the pool of libraries was loaded
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onto the reagent cartridge and then onto the instrument along with the flow cell. Automated
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cluster generation and sequencing run were performed in a single 39-hours run in a 2x251-
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bp.Total information of 10.6 Gb was obtained from a 1326 K/mm2 cluster density with a
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cluster passing quality control filters of 99.1 % (24,492,260 clusters). Within this run, the
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index representation for Virgibacillus senegalensis was determined to be 7.06 %. The
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1,481,197 paired reads were filtered according to the read qualities. These reads were
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trimmed, and then assembled using the CLC genomicsWB4 software.
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Genome annotation and comparison
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Open Reading Frames (ORFs) were predicted using Prodigal [36] with default parameters but
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the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted
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of Orthologous Groups (COG) databases using BLASTP. The tRNAScanSE tool [38] was
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used to find tRNA genes, whereas ribosomal RNAs were found using RNAmmer [39] and
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BLASTn against the GenBank database. Lipoprotein signal peptides and the number of
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transmembrane helices were predicted using SignalP [40] and TMHMM [41] respectively.
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ORFans were identified if their BLASTP E-value was lower than 1e-03 for alignment length
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greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used
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an E-value of 1e-05. Such parameter thresholds have already been used in previous works to
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define ORFans. Artemis [42] was used for data management and DNA Plotter [43] for
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visualization of genomic features. Mauve alignment tool (version 2.3.1) was used for multiple
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genomic sequence alignment [44]. To estimate the mean level of nucleotide sequence
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similarity at the genome level, we used the MAGI homemade software to calculate the
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average genomic identity of gene sequences (AGIOS) among compared genomes [45].
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Briefly, this software combines the Proteinortho software [45] for detecting orthologous
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proteins in pairwise genomic comparisons, then retrieves the corresponding genes and
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determines the mean percentage of nucleotide sequence identity among orthologous ORFs
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using the Needleman-Wunsch global alignment algorithm. Genomes from the genus
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Virgibacillus and closely related genera were used for the calculation of AGIOS values. Here,
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we compared the genome sequences of Virgibacillus senegalensis strain SK-1T (GenBank
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accession number PRJEB1962) with those of Virgibacillus kekensis strain YIM kkny9
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(NR_042744.1), Virgibacillus albus strain YIM 93624 (NR_109613.1), Aquibacillus
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salifodinae WSY08-1 (AB859945.1), Virgibacillus halodenitrificans DSM 10037
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(AY543169), Thalassobacillus devorans MSP14 ( JX518269.1), Halobacillus dabanensis
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HD 02 (HG931924.2), Halobacillus kuroshimensis DSM 18393 (AB195680.1),
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Thalassobacillus devorans strain XJSL7-8 (GQ903447.1), Bacillaceae bacterium EFN-4
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ACCEPTED MANUSCRIPT (EU817569.1), Virgibacillus marismortui strain M3-23 (GQ282501.1), Halobacillus salinus
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strain GSP59 ( AY505517.1), Virgibacillus alimentarius J18 (GU202420), Pseudomonas
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aeruginosa PAO1 (NR_074828.1) and Virgibacillus massiliensis (CCDP010000001).
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Results
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Phenotypic description
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Strain SK-1T was isolated in February 2014 (Table1) by aerobic culture on a homemade culture medium at 37° after 48h. No significant MALDI-TOF result for the strain SK-
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1T against our MALDI-TOF database was obtained, suggesting that our isolate was not a
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previously known species. We added the specter of SK-1T strain to our database (Figure 1).
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The gel view allows to observe the spectral differences with other members of Virgibacillus
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genus (Figure 2). PCR-based identification of the 16S rRNA of our newly isolate (GenBank
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accession number LK021111) yielded 96.3 % 16S rRNA sequence similarity with the
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reference Virgibacillus kekensis (GenBank accession number NR042744) the
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phylogenetically closest validated Virgibacillus species (Figure 3).
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After growth for 24 hour on our home-made culture medium at 37°C, the surface colonies
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were circular, grayish, shiny and smooth with a diameter of 1-2 mm in diameter V.
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senegalensis is Gram positive (figure 4).
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Growth is observed at temperatures ranging from 25 to 40°C, with an optimum at 37°C. The growth required a salinity ranging from 5 to 200 g/L of NaCl (optimum at 75 g/L).
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The optimum pH for growth was 7.5 (range between pH 5 to 9). Growth of the strain SK-1T
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was tested in an aerobic atmosphere, in the presence of 5% CO2, and also in anaerobic and
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microaerophilic atmospheres created using AnaeroGenTM (Atmosphere Generation Systems,
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Dardily, France), respectively. The strain was strictly aerobic and also grew in the presence of
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5% CO2 but did not grow in an anaerobic atmosphere. The size and ultrastructure of cells
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were determined by negative staining transmission electron microscopy 2 - 6 µm in length
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ACCEPTED MANUSCRIPT and 0.5 µm in diameter (Figure 5). Using the commercially available Api ZYM, Api 20NE
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(BioMerieux, Marcy-l’Etoile, France) to characterize the biochemical V. senegaiensis strain
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SK-1T , positive reactions were observed for urease, βglucosidase, protease, β-galactosidase,
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arginine hydrolase All other tested reactions were negative, notably nitrate reduction alkaline
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phosphatase, N-acetyl-β-glucosaminidase . The stain was also catalase and oxidase negative.
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Substrate oxidation and assimilation were examined with an API 50CH strip (BioMerieux,
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Marcy-l’Etoile, France) at 37°C. Negative reactions were obtained for D-mannose, D-lactose,
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L-arabinose, D-galactose, D-ribose, D-sucrose, D-fructose, D-glucose, D-mannitol, and D-
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maltose. Phenotypic characteristics were compared to those of the most closely related
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species (Table 2).
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Finally, antimicrobial susceptibility testing demonstrate that the strain SK-1T was susceptible to doxycycline, rifampicin, vancomycin, nitrofurantoin, amoxicillin,
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erythromycin, ampicillin, ceftriaxone, ciprofloxacin, gentamicin, penicillin,
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trimethoprim/sulfamethoxazole and imipenem, but resistant to metronidazole.
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Genome sequencing information
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Genome properties
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The draft genome of V. senegalensis consists of nine scaffolds with 59 contigs.
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The genome is 3,755,098 bp-long with a 42.9% G+C content (Table 3, Figure 6). Of the 3,833
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predicted genes, 3,738 (96.46%) were protein- coding genes, and 95 (2.44%) were RNAs (14
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genes are 5S rRNA, five genes are 16S rRNA, seven genes are 23S rRNA, 69 genes are tRNA
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genes). A total of 2,773 genes (62.82%) were assigned a putative function, 2,427 (65.43%)
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genes were assigned to COGs and 155 (4.04%) genes contain peptide signals whereas 980
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(25.56%) genes had transmembrane helices. A total of 245 genes were identified as ORFans
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(6.39%). The remaining genes were annotated as hypothetical proteins. The properties and
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statistics of the genome are summarized in Tables 3 and 4. The distribution of genes into
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COGs functional categories is presented in Table 5.
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Genome comparaison The draft genome of V. senegalensis SK-1T is smaller than those of Halobacillus
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kuroshimensis DSM 18393, Virgibacillus halodenitrificans DSM10037, Thalassobacillus
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devorans XJSL7-8, Thalassobacillus devorans XJSL7-8, Halobacillus dabanensis HD 02
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and Pseudomonas aeruginosa PAO1 (3.85, 3.92, 3.94, 4.1 and 6.26 Mb respectively) but
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larger than that of Virgibacillus alimentarius J18 (3.05 Mb). The G+C content of V.
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senegalensis SK-1T is smaller than those of Halobacillus kuroshimensis DSM 18393 and
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Pseudomonas aeruginosa PAO1 (47.0 and 66.60% respectively) and larger than those of
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Virgibacillus alimentarius J18, Virgibacillus halodenitrificans DSM10037, Halobacillus
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dabanensis HD 02 (37.1, 37.4, 41.5 % respectively) but equal to halassobacillus devorans
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XJSL7-8 (42.9 %). Protein coding genes of V. senegalensis SK-1T is smaller than those of
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Virgibacillus halodenitrificans DSM10037, Thalassobacillus devorans XJSL7-8,
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Halobacillus kuroshimensis DSM 18393, Halobacillus dabanensis HD 02 and Pseudomonas
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aeruginosa PAO1 (3,748, 3,752, 3,832, 3,835, 5,572 Mb respectively) but larger than those of
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Virgibacillus alimentarius J18 (2,889 Mb). Total gene content of V. senegalensis SK-1T
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(3883) is smaller than those of Halobacillus kuroshimensis DSM 18393, Halobacillus
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dabanensis HD 02 and Pseudomonas aeruginosa PAO1 (3,915, 4,011, and 5,697
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respectively) but larger than those of Thalassobacillus devorans XJSL7-8, Virgibacillus
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halodenitrificans DSM10037, Virgibacillus alimentarius J18 (3,840, 3,822 and 3,022
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respectively).
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Among species with standing in nomenclature, AGIOS values ranged from 66.41 between V. senegalensis SK-1T and Halobacillus kuroshimensis DSM 18393 to 73.39%
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evaluate the genomic similarity among studied strains, in addition to AGIOS [7], which was
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designed to be independent from DDH, we determined dDDH that exhibits a high correlation
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with DDH [46-47]. dDDH ranged from 18.4 to 27.2% between the different species tested
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(Table 6, Figure 7).
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Conclusion
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Based on phenotypic, genomic and phylogenetic analyzes, we formally propose the creation of Virgibacillus senegalensis sp. nov, represented here by the SK-1T strain. The
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strain was isolated from a stool sample of a Senegalese healthy individual.
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Taxonomic and nomenclatural proposals
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Description of Virgibacillus senegalensis sp. nov.
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Virgibacillus senegalensis (se.ne.ga. len′sis. L. masc. adj. senegalensis of Senegalia, the Roman name of Senegal, where the type strain was isolated). Growth occurred between 15
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to 45°C on a home-made culture medium herein described with optimal growth observed at
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37°C in aerobic atmosphere. Strain SK-1T required a salinity ranging from 5 to 200 g/L of
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NaCl (optimum at 100 g/L). The optimum pH for growth was 7.5 (range between pH 5 to 9).
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The strain SK-1T was strictly aerobic and also grew in the presence of 5% CO2 but no growth
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was observed under anaerobic and microaerophilic conditions. The colonies of the strain SK-
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1T were circular, grayish, shiny and smooth with a diameter of 2-6 mm. Cells stained Gram-
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positive. They were motile by polar flagella, spore forming (2 to 6 µm in length and 0.5 µm
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in diameter) and generally occurred individually or in pairs. Strain SK-1T is catalase and
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oxidase negative. Using API 50 CH and API20 NE (BioMerieux, Marcy-l’Etoile, France),
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strain SK-1T was positive for reduction of nitrates but negative for phosphatase alkaline
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activity, β-galactosidase, α N-acetyl-β-glucosaminidase, urease. Strain SK-1T was negative
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mannose, D-mannitol, D-maltose and D-sucrose. The strain SK-1T was susceptible to
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doxycycline, rifampicin, vancomycin, nitrofurantoin, amoxicillin, erythromycin, ampicillin,
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ceftriaxone, ciprofloxacin, gentamicin, penicillin, trimethoprim/sulfamethoxazole and
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imipenem, but resistant to metronidazole.
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The G+C% content of the genome is 42.9%. The 16S rRNA and genome sequences are
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deposited in GenBank under accession numbers LK021111. The habitat of the microorganism
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is the human digestive tract. The type strain SK-1T (CSUR P1101, DSM28585) was isolated
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from a stool specimen of a Senegalese male individual.
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Authors’ contributions
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ES performed the phenotypic characterization of the bacterium and drafted the manuscript. JR
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and OC performed the genomic analyses. SK participated in the study coordination and
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helped to draft the manuscript. CR performed the genomic analyses and drafted the
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manuscript. CC performed the MALDI-TOF identification and figures. FDP performed the
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electronic microscopy visualization of the bacterium. CS participated in the study design and
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collected the sample. DR and JCL conceived the study and helped to draft the manuscript. All
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authors read and approved the final manuscript.
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Acknowledgements
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The authors thank the Xegen Company (www.xegen.fr) for automating the genomic
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annotation process. This study was funded by the Mediterranée-Infection Foundation.
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Figure legends.
290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
Figure 1. Reference mass spectrum from Virgibacillus senegalensis sp. nov. SK-1T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.
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Figure 2. Gel view comparing Virgibacillus senegalensis sp. nov. SK-1Tto the members of the family Virgibacillus and Oceanobacillus. The gel view displays the raw spectra of all 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 by a Gray scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. Displayed species are indicated on the left.
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Figure 3. Phylogenetic tree highlighting the position of Virgibacillus senegalensis strain SK1T (CSUR P1101 = DSM28585) relative to other type strains of Virgibacillus albus strain YIM 93624 (NR_109613.1) , Virgibacillus kekensis strain YIM kkny16 (NR_042744.1), Virgibacillus alimentarius strain J18 (NR_108710.1), Virgibacillus marismortui strain M3-23 (GQ282501.1), Virgibacillus necropolis strain LMG 19488 (NR_025472.1), Virgibacillus carmonensis strain LMG 20964 (NR_025481.1), Virgibacillus subterraneus strain H57B72 (FJ746573.1), Virgibacillus zhanjiangensis strain JSM 079157 (FJ425904.1), Virgibacillus litoralis strain JSM 089168 (FJ425909.1), Virgibacillus dokdonensis strain DSW-10 (NR_043206.1), Virgibacillus siamensis strain MS3-4 (AB365482.1), Virgibacillus salarius strain SA-Vb1 (NR_041270.1), Virgibacillus halophilus strain 5B73C (NR_041358.1), Virgibacillus natechei strain FarD (NR_132721.1), Virgibacillus chiguensis strain NTU-101 (NR_044086.1), Virgibacillus dokdonensis strain DSW-10 (NR_043206.1), Virgibacillus campisalis strain IDS-20 (GU586225.1), Virgibacillus pantothenticus strain NBRC 102447 (AB681789.1), Virgibacillus halodenitrificans strain NBRC 102361 (AB681753.1), Virgibacillus byunsanensis strain ISL-24 (FJ357159.1), Virgibacillus massiliensis strain Vm5 (HG931931.1) and Paenibacillus polymyxa strain KCTC3717 (AY359637.1). GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method within the MEGA software. Paenibacillus polymyxa was used as out group. The scale bar represents 0.005% nucleotide sequence divergence Figure 4. Gram staining of Virgibacillus senegalensis sp. nov. SK-1T
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Figure 5. Transmission electron microscopy of Virgibacillus senegalensis sp. nov. SK-1T. Cells are observed on a Tecnai G20 transmission electron microscope operated at 200 keV. The scale bar represents 500 nm.
Figure 6: Graphical circular map of the Virgibacillus senegalensis sp. nov. SK-1T. chromosome. From the outside in, the outer two circles show open reading frames oriented in the forward (colored by COG categories) and reverse (colored by COG categories) directions, respectively. The third circle marks the tRNA genes (green). The fourth circle shows the G+C% content plot. The inner-most circle shows GC skew, purple indicating negative values whereas olive for positive values
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Figure 7. Distribution of functional classes of predicted genes according to the clusters of orthologous groups of proteins
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Table 1. Classification of Virgibacillus senegalensis strain SK-1 Property Term Current classification Domain: Bacteria
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Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Bacillaceae Genus: Virgibacillus Species: Virgibacillus senegalensis Type strain: SK-1 Positive Rod shaped Motile by polar flagellum Endospore-forming Mesophile 37°C pH 5 to 9 7.5 0.5 to 10% 7.5% Aerobic
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Gram stain Cell shape Motility Sporulation Temperature range Optimum temperature pH Optimum pH Salinity Optimum salinity Oxygen requirement
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Table 2: Differential characteristics. V. V. V. senegalensis massiliensis olivae
V. V. V. V. V. V. V. V. salarius marismortuis sediminis xinjiangensis kekensis halodenitrificans proomii dokdonensis
Cell diameter (µm)
0.6-0.9
0.5-0.8
0.4-0.6 0.6-0.9
0.4-0.7
1.4~2.4
0.3–0.5 0.6-0.8
0.5-0.7
Oxygen requirement
Aerobic
Aerobic
Aerobic Aerobic Aerobic
Aerobic
Aerobic
Aerobic Aerobic
Aerobic Aerobic
Gram stain
+
+
+
+
+
+
+
+
+
+
+
Salt requirement
+
+
+
+
-
+
-
-
+
na
+
Motility
+
+
+
+
+
+
+
+
+
+
+
Endospore formation +
+
+
+
+
+
+
+
+
+
+
Indole
-
-
-
-
-
-
-
-
-
-
-
Alkaline phosphatase -
-
na
na
na
-
na
-
na
na
-
Catalase
-
+
+
Oxidase
-
+
+
Nitrate reductase
-
+
+
Urease
+
-
na
β-galactosidase
+
-
na
N-acetyl-glucosamine -
-
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+
+
+
+
+
+
+
+
+
+
+
-
+
+
na
+
-
+
+
+
+
+
+
-
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Production of
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Properties
-
na
-
-
-
+
na
-
-
-
-
-
-
+
+
-
na
+
-
na
-
na
+
-
na
-
-
-
-
-
-
-
-
+
L-Arabinose
-
-
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Ribose
-
-
na
na
na
+
-
-
na
+
+
D-mannose
-
+
-
+
+
-
-
+
+
+
+
Acid from
ACCEPTED MANUSCRIPT
Properties
V. V. V. senegalensis massiliensis olivae
V. V. V. V. V. V. V. V. salarius marismortuis sediminis xinjiangensis kekensis halodenitrificans proomii dokdonensis
D-mannitol
-
+
na
-
-
-
D-sucrose
-
+
-
-
-
-
D-glucose
-
+
-
+
+
+
D-fructose
-
+
+
+
+
D-maltose
-
+
-
+
-
D-lactose
-
-
-
-
-
343
+
-
+
-
-
+
+
+
+
+
+
+
+
-
+
+
+
+
-
+
+
+
-
-
-
-
+
+
+
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+
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Waste Salt lake washwater
w
+
Mural paintings
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Human gut
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341
Human gut
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Habitat
-
Salt lake
Salt lake
Salt lake
Solar saltern
Soil
Soil
ACCEPTED MANUSCRIPT Table 3. Nucleotide content and gene count levels of the genome Value
% of totala
Size (bp)
3,755,098
100
G+C content (%)
1,610,937
42.9
Coding region (bp)
3,129,675
83.34
Total genes
3,883
100
RNA genes
95
2.44
Protein-coding genes
3,738
98.46
Genes with function prediction
2,773
Genes assigned to COGs
2,421
Genes with peptide signals Genes with transmembrane helices CRISPRs repeats
a
SC 65.43
4.04
980
25.56
02
0.05
2,011
52.46
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The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome
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345 346
62.82
155
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Genes with Pfam Domains
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Attribute
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ACCEPTED MANUSCRIPT Table 4. Number of genes associated with the 25 general COG functional categories Value
% valuea
Description
J
172
4.60
Translation
A
0
0
RNA processing and modification
K
262
7.01
Transcription
L
196
5.24
Replication, recombination and repair
B
1
0.03
Chromatin structure and dynamics
D
30
0.80
Cell cycle control, mitosis and meiosis
Y
0
0
Nuclear structure
V
49
1.31
Defense mechanisms
T
153
4.09
Signal transduction mechanisms
M
156
4.17
Cell wall/membrane biogenesis
N
64
1.71
Cell motility
Z
0
0
Cytoskeleton
W
0
0
Extracellular structures
U
51
1.36
Intracellular trafficking and secretion
O
92
2.46
Posttranslational modification, protein turnover, chaperones
C
137
3.67
G
275
7.36
E
305
8.16
F
82
H
93
I
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Amino acid transport and metabolism
2.19
Nucleotide transport and metabolism
2.49
Coenzyme transport and metabolism Lipid transport and metabolism
202
5.40
Inorganic ion transport and metabolism
74
1.98
Secondary metabolites biosynthesis, transport and catabolism
448
11.99
General function prediction only
S
287
7.68
Function unknown
_
352
9.06
Not in COGs
R
349
Carbohydrate transport and metabolism
2.73
Q
a
Energy production and conversion
102
P
348
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Code
AC C
347
The total is based on the total number of protein coding genes in the annotated genome
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Halobacillus dabanensis
Halobacillus kuroshimensis
3,378
1,791
1,786
Halobacillus dabanensis
66.82
4,063
2,218
Halobacillus kuroshimensis
66.41
73.39
3,926
Thalassobacillus devorans
67.76
69.22
68.46
Virgibacillus alimentarius
67.16
66.25
Virgibacillus halodenitrificans
67.19
66.41
Pseudomonas aeruginosa
53.94
52.73
66.21
65.24
Virgibacillus halodenitrificans
Pseudomonas aeruginosa
Virgibacillus massiliensis
1,446
1,741
601
588
2,122
1,581
1,866
697
642
2,119
1,554
1,861
695
630
3880
1,596
1,923
687
618
64.45
66.55
3,119
1,663
607
604
64.71
66.67
71.39
3,876
661
676
55.86
53.96
49.86
50.02
5,681
244
63.48
65.57
69.67
70.87
50.45
1,768
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Virgibacillus massiliensis
1,776
Virgibacillus alimentarius
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Virgibacillus senegalensis
Thalassobacillus devorans
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Virgibacillus senegalensis
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Table 5. The numbers of orthologous proteins shared between genomes (upper right). Average percentage similarity of nucleotides corresponding to orthologous proteins shared between genomes (lower left) and numbers of proteins per genome (bold)
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350 351
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Halobacillus kuroshimensis Thalassobacillus devorans Virgibacillus alimentarius Virgibacillus halodenitrificans Pseudomonas aeruginosa Virgibacillus massiliensis
100% ± 00
26.7% ±2.54 26.3% ±2.55
100% ± 00
Virgibacillus alimentarius
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24.3% ± 2.55
Thalassobacillus devorans
Virgibacillus halodenitrificans
Pseudomonas aeruginosa
Virgibacillus massiliensis
22.8% ±2.54
26.4% ±2.54
21.8% ±2.56
20.8% ±2.53
22.8% ±2.53
24.4% ±2.56
27.7% ±2.55
27% ±2.55
21.2% ±2.57
20.0% ±2.53
24.6% ±2.55
27.2% ±2.55
24.7% ±2.57
21.8% ±2.54
19.5% ±2.53
100% ± 00
22.1% ±2.55
21.3% ±2.57
18.4% ±2.56
20.1% ±2.53
100% ± 00
23% ±2.56
21.1% ±2.53
20.0% ±2.54
100% ± 00
22.5% ±2.54
21.3% ±2.55
100% ± 00
20.3% ±2.52
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Halobacillus dabanensis
100% ± 00
Halobacillus kuroshimensis
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Virgibacillus senegalensis
Halobacillus dabanensis
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Virgibacillus senegalensis
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Table 6. Pairwise comparison of Virgibacillus senegalensis with eight other species using GGDC, formula 2 (DDH estimates based on identities / HSP length) *The confidence intervals indicate the inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size). These results are in accordance with the 16S rRNA (Figure 1) and phlogenomic analyses as well as the GGDC results.
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352 353 354 355
100% ± 00
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Figure 1
358
In te n s . [a .u .]
x104
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1.5
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1.0
0.0 4000
6000
8000
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2000
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359 360
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0.5
10000
12000
14000
16000
18000
m/z
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365
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Figure 6
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ACCEPTED MANUSCRIPT Reference
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initiation
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