planococcus massiliensis strain es2 (= csur p1103  =  dsm 28915 ) is the type strain of p.  massiliensis sp .
this isolation is part of a wider culturomics study , with an aim to maximize the culture conditions to explore the human microbiota in depth .
for this project , several hundred samples from healthy individuals , antibiotic - treated individuals , or people with , for example , obesity , anorexia nervosa , or malnutrition were analysed by culturomics in order to extend our knowledge of gut microbes . in this case
, we created media containing a high salt concentration in order to cultivate halophilic microorganisms .
furthermore , the availability of genomic data for many bacterial species   inspired us to propose a new concept for the description of new species of bacteria by adding proteomic information obtained by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof )   and genomic analyses .
this concept changes the current methods of defining a new bacterial species , which are based on the genetic , phenotypic and chemotaxonomic criteria that are not reproducible and can not be applied to all the bacterial genus , , . here
we present a summary classification and a set of features for the type strain planococcus massiliensis sp .
strain es2 , together with the description of the complete genomic sequence and its annotation .
planococcus massiliensis is the first representative member of the genus of planococcus isolated from a human subject . to date , 17 recognized species are representatives of planococcus ( p.  alkanoclasticus , p. antarcticus , p. citreus , p. columbae , p. donghaensis , p. halocryophilus , p. kocurii , p. maitriensis , p. maritimus , p. mcmeekini , p. okeanokoites , p. plakortidis , p. psychrophylus , p. rifietoensis , p. salinarum , p. stackebrandtii ) ( http://www.bacterio.net ) .
all these species are gram - positive , aerobic cocci that are able to grow at moderately low temperatures and high salt concentrations , and have been predominantly isolated from saline environments , .
the study and the assent procedure were approved by the national committee of senegal and the ethics committee of federative research institute 48 ( faculty of medicine , marseille , france ) under agreement 09 - 022 .
the sample was obtained from a native senegalese man living in ndiop , a rural village in the guinean  sudanese zone in senegal .
the specimen was collected in sterile plastic containers , formed into aliquots and stored at  80c until use . for each sample , ph and salinity were systematically determined with a ph meter ( thermofisher scientific , saint aubin , france ) and a digital refractometer ( thermofisher scientific ) before any analysis was performed
. then it was cultured in a liquid colombia broth culture medium ( sigma - aldrich , saint - quentin fallavier , france ) modified by adding ( per litre ) : 5  g mgcl2 6h2o ; 5  g mgso4 7h2o ; 2  g kcl ; 1  g cacl2 2h2o ; 0.5  g nabr ; 0.5  g nahco3 ; 2  g glucose ; and 100  g nacl .
each colony was deposited in duplicate on a maldi - tof msp96 target and covered with 1.5 l of a matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid in 50% acetonitrile , 2.5% trifluoroacetic acid ) to allow crystallisation of molecules .
maldi - tof was performed using the lt microflex spectrometer ( bruker daltonics , leipzig , germany ) .
all spectra were recorded in positive linear mode for the mass range from 2000 to 20  000 da ( parameters : ion source 1 ( isi ) , 20  kv ; is2 , 18.5  kv lens , 7  kv ) .
the generated spectra were then compared to the bruker database , which was supplemented with the new species found through the culturomics project .
the resulting score enabled the identification or not of tested species : a score of 2 with a validly published species enables identification at the species level , a score of 1.7 but < 2 enables identification at the genus level and a score of < 1.7 does not enable any identification .
isolated colonies were suspended in 200  l distilled water for dna extraction using an ez1 dna tissue kit ( qiagen , venlo , netherlands ) .
the amplification of the 16s rrna was performed by a standard  pcr using the universal primer pair fd1 5-agagtttgatcctggctcag-3 and rp2 5-acggctaccttgttacgactt-3 .
the pcr product was purified and sequenced using the big dye terminator sequencing kit v.1.1 ( perkin - elmer , courtaboeuf , france ) with the following internal primers : 536f , 536r , 800f , 800r , 1050f and 1050r ; 16s rrna amplification and sequencing were carried out as previously described by steven et  al . .
the 16s rrna nucleotide sequence was corrected using chromas pro 1.34 software ( technelysium , tewantin , australia ) , and the blastn searches were performed by national center for biotechnology information ( http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/blast.cgi ) .
mega6 ( molecular evolutionary genetics analysis ) software   allowed us to construct a phylogenetic tree .
sequences alignment of the different species was performed by clustalw , and calculation of the evolutionary distance was done with the kimura two - parameter model , .
growth of the strain es2 was tested under aerobic atmosphere , in the presence of 5% co2 , and also in anaerobic and microaerophilic atmospheres created using anaerogen and campygen respectively ( thermofisher scientific ) .
gram staining and motility were observed by the use of the dm1000 photonic microscope ( leica microsystems , nanterre , france ) .
cell morphology was examined with a tecnai g20 ( fei , limeil - brevannes , france ) transmission electron microscope .
biochemical tests were realized by using the commercially available api zym , api 50ch and api 20 ne strips ( biomrieux , marcy ltoile , france ) .
oxidase and catalase reactions were determined by using a bd bbl dryslide ( becton dickinson , franklin lakes , nj , usa ) according to the manufacturer 's instructions .
sensitivity to antibiotics was determined by using sircan discs ( i2a , montpellier , france ) on mueller - hinton agar in a petri dish ( biomrieux ) .
doxycycline , rifampicin , vancomycin , nitrofurantoin , amoxicillin , erythromycin , ampicillin , ceftriaxone , ciprofloxacin , gentamicin , penicillin , trimethoprim / sulfamethoxazole , imipenem and metronidazole activity were tested on our strain .
after 48 hours of culture , the bacteria were resuspended in sterile water and centrifuged at 4c at 2000 
cell pellets were resuspended in 1  ml tris / edta / nacl solution ( 10  mm tris / hcl ( ph7.0 ) , 10  mm edta ( ph8.0 ) and 300  mm nacl ) and recentrifuged under the same conditions .
the pellets were then resuspended in 200 l of tris - edta ( te ) buffer and proteinase k and kept overnight at 37c for cell lysis .
dna was purified with phenol / chloroform / isoamylalcohol ( 25:24:1 ) , followed by an overnight precipitation with ethanol at  20c .
it was then resuspended in 205 l te buffer and quantified ( 155  ng/l ) by a qubit fluorometer using the high - sensitivity kit ( thermofisher scientific ) .
genomic dna of planococcus massiliensis was sequenced on the miseq technology ( illumina , san diego , ca , usa ) with the mate pair strategy .
the gdna was barcoded in order to be mixed with 11 other projects with the nextera mate pair sample prep kit ( illumina ) .
the mate pair library was prepared with 1  g of gdna using the nextera mate pair illumina guide .
the pattern of fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 lab chip .
the dna fragments ranged in size from 1 to 11  kb , with an optimal size at 4.008  kb .
no size selection was performed , and 388.3  ng of tagmented fragments were circularized .
the circularized dna was mechanically sheared to small fragments , with an optimum of 634  bp , on the covaris device s2 in microtubes ( covaris , woburn , ma , usa ) .
the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent technologies ) , and the final concentration library was measured at 35.59 
after a denaturation step and dilution at 15  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 the sequencing run were performed in a single 39-hour run at a 2    251  bp read length .
total information of 10.6 gb was obtained from a 1326k / mm cluster density with a cluster passing quality control filters of 99.1% ( 24  492  260 clusters ) . within this run ,
the 1  481  197 paired reads were filtered according to the read qualities .
open reading frames ( orfs ) were predicted using prodigal   with default parameters , but the predicted orfs were excluded if they spanned a sequencing gap region .
the predicted bacterial protein sequences were searched against the genbank database   and the clusters of orthologous groups ( cogs ) database using blastp .
the trnascanse tool   was used to find trna genes , whereas ribosomal rnas were found using rnammer   and blastn against the genbank database .
lipoprotein signal peptides and the number of transmembrane helices were predicted using signalp   and tmhmm   respectively .
orfans were identified if their blastp e value was lower than 1e-03 for alignment length greater than 80 amino acids .
if alignment lengths were smaller than 80 amino acids , we used an e value of 1e-05 .
artemis   was used for data management and dna plotter   for visualization of genomic features .
the mauve 2.3.1 alignment tool was used for multiple genomic sequence alignment . to estimate the mean level of nucleotide sequence similarity at the genome level
, we used the magi homemade software to calculate the average genomic identity of gene sequences ( agios ) among compared genomes .
briefly , this software combines the proteinortho software   for detecting orthologous proteins in pairwise genomic comparisons , then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous orfs using the needleman - wunsch global alignment algorithm .
genomes from the genus planococcus and closely related genera were used for the calculation of agios values .
ccxs00000000 ) was compared to the one of planomicrobium glaciei strain chr43 ( nts ) ( genbank accession no .
aepb01000000 ) and bacillus subtilis subsp . spizizenii strain tu - b-10 ( genbank accession no .
cp002905 ) . to estimate the overall similarity between the genomes , the genome - to - genome distance calculator ( ggdc ) was used , .
the system calculates the distances by comparing the genomes to obtain high - scoring segment pairs ( hsp ) and interfering distances from a set of formulas : 1 , hsp length / total length ; 2 , identities / hsp length ; and 3 , identities / total length .
we did not obtain a significant maldi - tof score for strain es2 against the bruker database , suggesting that our isolate was not a known species .
the gel view highlighted the spectral differences with other members of the genus planococcus ( figure  2 ) .
pcr - based identification of the 16s rrna of our new isolate ( genbank accession no .
this indicated a 97.95% 16s rrna sequence similarity with planococcus halocryophilus ( genbank accession no .
the other closest species were p.  donghaensis ( 97.72% ) , planococcus glaciei ( 97.06% ) and b.  subtilis ( 91.92% ) .
the species p.  massiliensis , p. halocryophilus and p.  donghaensis shared a single cluster , whereas p.  glaciei was present in a distant clade in the phylogenetic tree ( figure  3 ) . this value of similarity remains lower than the 98.7% 16s rrna gene sequence threshold recommended by stackebrandt and ebers to delineate a new species without carrying out dna - dna hybridization .
thus , this bacterium was considered to be a new species called planococcus massiliensis strain es2 sp .
strain es2 is able to grow at temperatures between 25 and 40c ( optimum 37c ) and ph 69 ( optimum ph 7.08.0 ) , and it tolerates nacl concentrations between 5 to 200  g / l ( optimum 75 
we tested the planococcus massiliensis growth on 5% sheep 's blood  enriched columbia agar ( biomrieux , marcy ltoile , france , at 37c ) but we observed weak growth with colonies measuring about 0.3 to 0.6  mm after 48 hours of growth .
it is a motile , non - spore - forming and gram - positive bacterium ( figure  4 ) .
atmospheric testing demonstrated that planococcus massiliensis was strictly aerobic and grew in the presence of 5% co2 but did not grow in an anaerobic atmosphere .
colonies that grow on our homemade culture medium were orange , circular , entire , smooth and convex , and they had a diameter of 1.0 to 2.0  mm after 48 hours .
individual cells exhibited a diameter of 0.6 to 0.9  m and had a slightly curved form with a flagellum under electron microscopy ( figure  5 ) . using api galleries , we observed positive reactions for esterase , lipase , trypsin , naphthol - as - bi - phosphohydrolase , -glucosidase , d - glucose , d - fructose , d - mannose , d - ribose and d - arabinose .
negative reactions were observed for leucine arylamidase , valine arylamidase , -galactosidase , alkaline phosphatase , cystine arylamidase , -chymotrypsin , acid phosphatase , -galactosidase , -glucuronidase , -glucosidase , -mannosidase , -fucosidase , arginine dihydrolase , n - acetyl--glucosaminidase , nitrate , d - galactose , d - mannitol and urease .
phenotypic characteristics were compared to those of the most closely related species ( table  1 ) .
antimicrobial susceptibility testing demonstrate that strain es2 was susceptible to doxycycline , rifampicin , vancomycin , nitrofurantoin , amoxicillin , erythromycin , ampicillin , ceftriaxone , ciprofloxacin , gentamycin , penicillin , trimethoprim / sulfamethoxazole and imipenem , but it was resistant to metronidazole .
the draft genome of p.  massiliensis es2 consists of six scaffolds with 32 contigs and generated a genome size of 3  357  017  bp with a 46.0% g+c content ( table  2 , figure  6 ) .
of the 3405 predicted genes , 3357 are protein - coding genes and 48 are rnas ( eight genes are 5s rrna , two are 16s rrna , three are 23s rrna and 35 are trna ) . a total of 2601 genes ( 66.90% ) were assigned a putative function .
the draft genome of planococcus massiliensis strain es2 is smaller than those of planomicrobium glaciei , planococcus halocryophilus and bacillus subtilis subsp .
spizizenii ( 3.35 , 3.9 , 3.43 and 4.21 mb respectively ) but larger than those of planococcus donghaensis ( 3.30 mb ) .
the g+c content of planococcus massiliensis is smaller than those of p.  glaciei ( 46.0 and 47.0% respectively ) but larger than those of planococcus halocryophilus , p. donghaensis and bacillus subtilis ( 39.9 , 39.7 and 43.8% respectively ) .
the gene content of p.  massiliensis is smaller than those of p.  glaciei , p. halocryophilus and b.  subtilis ( 3405 , 3967 , 3429 and 4307 respectively ) but larger than that of p.  donghaensis ( 3251 ) . the number of rrna genes varied from four for p.  donghaensis , 13 for p.  massiliensis , 30 for b.  subtilis , 60 for p.  halocryophilus and 62 for p.  glaciei respectively
. a large number of genes assigned to cogs functional categories for amino acid transport and metabolism , transcription , carbohydrate transport and metabolism and translation were identified .
nevertheless , we observed a relative lower number of genes assigned for amino acid transport and metabolism in p.  massiliensis compared to other species ( figure  7 ) .
the genes for rna processing and modification , nuclear structure and extracellular structures were absent in all the genomes . finally , the genes coding for cogs category cytoskeleton were present only in p.  massiliensis and p.  glaciei ( figure  7 ) .
in addition , p.  massiliensis shared 3880 , 2775 , 3146 and 4099 orthologous genes with p.  glaciei , p. halocryophilus , p. donghaensis and b.  subtilis ( table  4 ) .
the average nucleotide sequence identity ranged from 85.84% between p.  donghaensis and p.  halocryophilus to 56.69% between p.  halocryophilus and b.  subtilis ( table  4 ) .
the genomic similarity level between strain es2 and closely related planomicrobium and planococcus species was also estimated using the ggdc ( table  5 ) .
this comparison of the genomes using ggdc revealed that p.  massiliensis shows a slightly higher dna - dna hybridization ( ddh ) estimate with p.  glaciei compared to those with p.  halocryophilus and b.  subtilis ( table  5 ) . for b.  subtilis ,
a higher ddh value was estimated with p.  halocryophilus but did not vary much to the other genomes .
however , given the confidence intervals ( table 5 ) , the ddh estimates do not show significant differences .
in the context of culturomics studies , several new bacterial species are isolated and then characterized .
it is in this context that we studied the phenotypic and phylogenetic characteristics and conducted genomic analyses on strain es2 .
massiliensis represents the eighth halophilic bacterium isolated from human stool . because the colon is not a high - salinity environment
, it would be interesting to examine the role of salt or salty products as a potential source of any unusual taxon , such as halophilic .
adj . , massiliensis for massilia , the old roman name for marseille , where the strain was isolated ) .
strain es2 grows at an optimum temperature of 37c , at ph 7.08.0 and at nacl concentration of 75 
cells are gram - positive , strictly aerobic , straight or curved rods ( 0.60.9  m ) , motile , and nonendospore forming .
colonies are orange , circular , entire , smooth and convex , 1.02.2  mm in diameter .
p.  massiliensis shows positive reactions for esterase , lipase , trypsin , naphthol - as - bi - phosphohydrolase , -glucosidase , d - glucose , d - fructose , d - mannose , d - ribose and d - arabinose . the strain is also oxidase positive but catalase negative .
strain es2 is susceptible to doxycycline , rifampicin , vancomycin , nitrofurantoin , amoxicillin , erythromycin , ampicillin , ceftriaxone , ciprofloxacin , gentamicin , penicillin , trimethoprim / sulfamethoxazole and imipenem .
massiliensis es2 (= csur p1103 ,  =  dsm 28915 ) was isolated from a stool sample of a healthy senegalese man .
it exhibited a genome size of 3  357  017  bp with a 46.0% g+c content .
the 16s rrna sequence was deposited in genbank under accession number lk021122 , and the whole genome shotgun sequence has been deposited in genbank under accession number ccxs00000000 .
 we propose the main phenotypic characteristics and the complete genome sequence and annotation of planococcus massiliensis strain es2 t (= csur p1103  =  dsm 28915 ) , the type strain of p.  massiliensis sp . 
 nov . 
 , isolated from a faeces sample collected from a healthy senegalese man . 
 it is an aerobic , gram - positive , moderately halophilic , motile and rod - shaped bacterium . 
 the 3  357  017  bp long genome exhibits a g+c content of 46.0% and contains 3357 protein - coding genes and 48 rna genes . 
Introduction
Material and Methods
Results
Conclusion
Taxonomic and nomenclatural proposals