bacillus subtilis was the first type species described in the genus bacillus ( cohn 1872 ) .
generally members of this genus are environmental bacteria present in soil , food , and fresh and sea water . in humans ,
some strains can be pathogenic , such as bacillus cereus ( associated mainly with food poisoning ) and bacillus anthracis ( the causative agent of anthrax ) , , . other strains are saprophytes .
recently high - throughput genome sequencing and mass spectrometry analyses of bacteria have given unprecedented access to an abundance of genetic and proteomic information , , .
currently a polyphasic approach is performed to describe new bacterial taxa , including their genome sequence , matrix - assisted laser - desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) spectrum , and major phenotypic characteristics such as gram staining , culture , metabolic characteristics , habitat and ( if applicable ) pathogenicity , .
bacillus ndiopicus strain ff3 (= csur p3025  =  dsm 27837 ) is designated as the type strain of bacillus ndiopicus .
this bacterium was isolated from the skin of a healthy senegalese man as part of a culturomics   study aiming at cultivating bacterial species from skin flora . here
in december 2012 , a skin specimen was sampled with a swab from a healthy senegalese volunteer living in ndiop , a rural village in the guinean  sudanian area in senegal ( table  1 ) .
this 39-year - old man was included in a research project approved by the national ethic committee for health research ( cners ) in senegal and the ethics committee of the institut fdratif de recherche ifr48 , faculty of medicine , marseille , france ( agreements 09 - 022 and 11 - 017 ) .
strain ff3 ( table  1 ) was isolated by cultivation on 5% blood 's sheep enriched columbia agar ( biomrieux , marcy letoile , france ) , under aerobic conditions , in december 2012 .
b.  ndiopicus strain ff3 exhibited a 96.8% nucleotide sequence similarity with bacillus massiliensis ( glazunova et  al . , 2006 ) , the phylogenetically closest bacillus species ( fig .  1 ) .
these values were lower than the 98.7% 16s rrna gene sequence threshold recommended by meier - kolthoff et  al . 
  to delineate a new species within the phylum firmicutes without carrying out dna - dna hybridization .
different growth temperatures ( 25 , 30 , 37 , 45 and 56c ) were tested .
optimal growth was observed at 37 and 45c after 24 hours of incubation ; weak growth was noticed at 30c .
colonies were 1  mm in diameter and transparent on 5% blood - enriched columbia agar .
growth of the strain was tested under anaerobic and microaerophilic conditions using the genbag anaer and genbag microaer systems , respectively ( biomrieux ) , and under aerobic conditions , with or without 5% co2 .
optimal growth was obtained under aerobic condition with 5% co2 and under microaerophilic condition at 37 and 45c .
cells grown on agar have a mean diameter of 1.2  m ( ranging from 0.8 to 1.6  m ) and a mean length of 2.5  m ( ranging from 1.8 to 3.2  m ) ( fig .  3 ) .
using the api zym strip ( biomrieux ) , positive reactions were observed with alkaline phosphatase , esterase , -chymotrypsin and lipase .
negative reactions were observed for leucine arylamidase , valine arylamidase , cystine arylamidase , phosphatase acid , trypsin , naphthol - as - bi - phosphohydrolase , -glucuronidase , -glucosidase , -glucosidase , n - acetyl--glucosaminidase , -mannosidase and -fucosidase . using the api 20e strip ( biomrieux ) , only the citrate test was positive ; all others tests were negative , including indole , -galactosidase , urease , ornithine decarboxylase , mannitol , sorbitol and rhamnose fermentation . using the api 50ch strip ( biomrieux ) , no positive reaction was observed , including for glycerol , d - arabinose , d - xylose , l - rhamnose , amygdalin , d - cellobiose , d - fucose , potassium 5-ketogluconate , l - arabitol , starch , d - maltose and d - mannose .
b.  ndiopicus was susceptible in  vitro to penicillin , amoxicillin , amoxicillin  clavulanic acid , ceftriaxone , imipenem , gentamicin , ciprofloxacin , erythromycin , doxycycline , rifampicin and vancomycin , but resistant to nitrofurantoin and metronidazole . when compared with representative species from the genus bacillus , b. ndiopicus strain ff3 exhibited several phenotypic differences , which are summarized in table  2 .
maldi - tof protein analysis was performed using a microflex lt ( bruker daltonics , leipzig , germany ) , as previously reported .
the scores previously established by bruker allowing validating ( or not ) the identification of species compared to the database of the instrument were applied .
briefly , a score of 2.000 with a species with a validly published name provided allows the identification at the species level ; a score of 1.700 and  < 2.000 allows the identification at the genus level ; and a score of < 1.700 does not allow any identification .
two microliters of matrix solution ( saturated solution of alpha - cyano-4-hydroxycinnamic acid ) in 50% acetonitrile and 2.5% trifluoroacetic acid were distributed on each smear and submitted at air drying for 5 minutes .
then the spectra from the 12 different colonies were imported into maldi biotyper 2.0 software ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 6252 bacterial spectra including 199 spectra from 104 bacillus species .
scores ranging from 1.2 to 1.4 were obtained for strain ff3 , suggesting that this isolate was not a member of any known species .
the reference mass spectrum from strain ff3 was incremented in our database ( fig .  4 ) .
the organism was selected for sequencing on the basis of its 16s rrna similarity , phylogenetic position and phenotypic differences with other members of the genus bacillus , which support that bacillus ndiopicus strain ff3 likely represents a new bacterial species .
this strain is part of a study aiming to characterize the skin flora of healthy senegalese people .
table  3 shows the project information and its association with minimum information about a genome sequence ( migs ) 2.0 compliance ; associated migs records are summarized .
bacillus ndiopicus strain ff3 (= csur p3025  =  dsm 27837 ) was grown aerobically on 5% sheep 's blood 
then we suspended all bacterial colonies in 500  l of tris - edta ( te ) buffer 10. we remove 100  l of this solution .
this volume is completed by 400  l te buffer 10 , 25  l proteinase k and 50  l sodium dodecyl sulfate and then incubated overnight at 56c for complete cells lysis . the next day
it is precipitated in absolute ethanol and incubated at  20c for at least 2 hours .
after a first centrifugation at 4c for 30 minutes at 8000  rpm , the pellet is taken up in 70% ethanol kept at  20c . a second centrifugation in the same conditions for 20 minutes is performed . after drying the tube in an oven at 37c for 5 minutes
the genomic dna concentration was measured at 47.7  ng/l by the qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) .
genomic dna of bacillus ndiopicus was sequenced on the miseq technology ( illumina , san diego , ca , usa ) with two applications , paired end and mate pair .
the paired - end and the mate - pair strategies were barcoded in order to be mixed with 11 other genomic projects prepared with the nextera xt dna sample prep kit ( illumina ) and 11 other projects with the nextera mate - pair sample prep kit ( illumina ) .
the genomic dna was diluted to 1  ng/l to prepare the paired - end library . the tagmentation step fragmented and tagged the dna with an optimal size distribution at 0.95  kb
. then limited - cycle pcr amplification ( 12 cycles ) completed the tag adapters and introduced dual - index barcodes .
after purification on ampure xp beads ( beckman coulter , fullerton , ca , usa ) , the libraries were then normalized on specific beads according to the nextera xt protocol ( illumina ) .
the pooled single strand library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell .
automated cluster generation and paired - end sequencing with dual index reads were performed in a single 39-hour run in 2    250  bp .
total information of 6.8  gb was obtained from a 807k / mm cluster density , with a cluster passing quality control filters of 90.88% ( 14  553  000 clusters ) . within this run ,
the index representation for bacillus ndiopicus was determined to 17.96% and present 2  375  297 reads filtered according to the read qualities .
the mate - pair library was prepared with 1  g of genomic dna using the nextera mate - pair illumina guide .
the genomic dna sample was simultaneously fragmented and tagged with a mate - pair junction adapter .
the profile of the fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 labchip .
the dna fragments were ranged in size from 1.5 to 13  kb , with an optimal size at 8  kb .
no size selection was performed , and 600  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments on a covaris device s2 in microtubes ( covaris , woburn , ma , usa ) .
after a denaturation step and dilution at 10  pm , the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell .
automated cluster generation and sequencing run were performed in a single 42-hour run in 2    250  bp .
open reading frames ( orfs ) prediction was carried out using prodigal   with default parameters .
functional assessment of protein sequences was performed by comparing them with sequences in the genbank   and clusters of orthologous groups ( cogs ) databases using blastp .
trnas , rrnas , signal peptides and transmembrane helices were identified using trnascan - se 1.21 , rnammer , signalp   and tmhmm , respectively .
artemis   was used for data management , and dna plotter   was used for visualization of genomic features . in - house
orfans were sequences which have no homology in a given database  that is , nonredundant ( nr ) or identified if their blastp e value was lower than 1e-03 for alignment lengths greater than 80 aa .
phast was used to identify , annotate and graphically display prophage sequences within bacterial genomes or plasmids . to estimate the nucleotide sequence similarity at the genome level between b.  ndiopicus and other members of bacillaceae family , orthologous proteins were detected by proteinortho software   ( with the following parameters : e value 1e-5 , 30% percentage of identity , 50% coverage and algebraic connectivity of 50% ) and genomes compared two by two . after fetching the corresponding nucleotide sequences of orthologous proteins for each pair of genomes
, we determined the mean percentage of nucleotide sequence identity using the needleman - wunsch global alignment algorithm .
the script created to calculate agios ( average genomic identity of orthologous gene sequences ) values was named magi ( marseille average genomic identity ) and is written in perl and bioperl modules .
the genome of b.  ndiopicus strain ff3 is 4  068  720  bp long ( one chromosome , no plasmid ) with a 37.03% g+c content ( fig . 
, we acknowledge the fact that because the genome of bacillus ndiopicus is a draft sequence , its exact size might be slightly different from that of our sequence , but given the fold coverage ( 52 ) , we are confident that the missing fragments are probably small and do not significantly influence the genome size . of the 3982 predicted genes , 3915 were protein - coding genes and 67 were rnas .
using phast software , three prophage regions were identified , including one complete and two incomplete prophages ( table  5 ) .
. today there are more than 277 sequenced genomes of bacillus species ( finished and draft ) available in genomes online database . here
we compared b.  ndiopicus genome sequence against other members of genus bacillus , including bacillus coagulans strain 2 - 6 , b.  coagulans strain 36d1 , lysinibacillus sphaericus strain c3 - 41 , bacillus bataviensis stain lmg 21833 , and bacillus isronensis strain b3w22 .
table  7 shows a comparison of genome size , g+c% content , and number of proteins for each genome selected for taxonogenomic study .
indeed , bacillus ndiopicus has a genome size of 4.06 mb higher than those of b.  coagulans 26 ( 3.07  mb ) , b.  coagulans
36d1 ( 3.55  mb ) and b.  isronensis b3w22 ( 4.02  mb ) but lower than those of b.  bataviensis lmg 21833 ( 5.37  mb ) and lysinibacillus sphaericus c3 - 41 ( 4.82  mb ) .
bacillus ndiopicus strain ff3 has a g+c content ( 37.03% ) lower than those of all the compared species such as b.  coagulans strain 2 - 6 ( 47.3% ) , b.  coagulans strain 36d1 ( 46.5% ) , b.  bataviensis strain lmg 21833 ( 39.6% ) , b.  isronensis strain b3w22 ( 38.8% ) and l.  sphaericus strain c3 - 41 ( 37.1% ) .
as it has been suggested in the literature that the g+c content deviation is at most 1% within species , these data are an additional argument for the creation of a new taxon .
the number of orthologous genes shared between b.  ndiopicus and other bacillus species as well as the average percentage nucleotide identity calculated using the magi method is tabulated in table  8 . on the basis of the analysis of magi , the agios ranged from 61.79 to 95.94% among the studied members .
the range of agios calculated using magi varies from 61.79 to 70.95% between b.  ndiopicus and other compared bacillus species .
antibiotic resistance genes were detected within the genome using the ardb website ( table  9 ) .
on the basis of phenotypic , phylogenetic and genomic analyses ( taxonogenomics ) , we formally propose the creation of bacillus ndiopicus sp . nov .
the strain was isolated from the skin of a 39-year - old healthy senegalese man living in ndiop , senegal .
n. ndiopicus , of ndiop , the name of the senegalese village where the man from whom strain ff3 was cultivated lives ) .
cells stain gram positive , are rod shaped and endospore forming , motile and have a mean diameter of 1.2  m and a mean length of 2.5  m .
colonies are 1  mm in diameter and transparent on 5% sheep 's blood . optimal growth is achieved at 37c in an aerobic atmosphere supplemented with 5% co2 .
positive reactions were obtained with citrate , alkaline phosphatase , esterase , lipase and -chymotrypsin .
negative reactions were observed for leucine arylamidase , valine arylamidase , cystine arylamidase , phosphatase acid , trypsin , naphthol - as - bi - phosphohydrolase , -glucuronidase , -glucosidase , -glucosidase , n - acetyl--glucosaminidase , -mannosidase and -fucosidase .
clavulanic acid , ceftriaxone , imipenem , gentamicin , ciprofloxacin , erythromycin , doxycycline , rifampicin and vancomycin , but resistant to nitrofurantoin and metronidazole .
the 16s rrna and genome sequences are deposited in genbank under accession numbers hg315675 and ccap000000000 , respectively .
the type strain ff3 (= csur p3025  =  dsm 27837 ) was isolated from the skin of a healthy 39-year - old senegalese man living in ndiop , senegal .
 strain ff3 t was isolated from the skin - flora of a 39-year - old healthy senegalese man . 
 matrix - assisted laser desorption / ionization time - of - flight mass spectrometry did not allow any identification . 
 this strain exhibited a 16s rrna sequence similarity of 96.8% with bacillus massiliensis , the phylogenetically closest species with standing nomenclature . using a polyphasic study made of phenotypic and genomic analyses , strain ff3 t was gram - positive , aeroanaerobic and rod shaped and exhibited a genome of 4  068  720  bp with a g+c content of 37.03% that coded 3982 protein - coding and 67 rna genes ( including four rrna operons ) . 
 on the basis of these data , we propose the creation of bacillus ndiopicus sp . nov . 
Introduction
Organism information
Genome sequencing information
Conclusion
Description of 