Antonie van Leeuwenhoek DOI 10.1007/s10482-016-0677-6

ORIGINAL PAPER

Risungbinella massiliensis sp. nov., a new member of isolated from human gut

Gre´gory Dubourg . Jean-Christophe Lagier . Catherine Robert . Nicholas Armstrong . Carine Couderc . Pierre-Edouard Fournier . Didier Raoult

Received: 21 September 2015 / Accepted: 8 March 2016 Ó Springer International Publishing Switzerland 2016

Abstract A novel filamentous bacterium, desig- genome sequence and annotation. The G?C content of nated GD1T, was isolated from the gut microbiota of the genomic DNA was determined to be 40.1 mol %. a 38-year-old male who suffered from a Coxiella The major fatty acids of strain GD1T were identified as burnetii vascular for which he received multiple a iso-C15:0, iso-C17:0, anteiso-C15:0, iso-C14:0 and broad-spectrum antibiotic cocktail at the time of the C16:0. The 3,440,191 bp long genome contains 3540 stool collection. The strain was isolated as a part of protein-coding and 67 RNA genes, including three culturomics study by cultivation on 5 % sheep blood rRNA genes. Strain GD1T (= DSM 46691 = CSUR agar in aerobic condition at 28 °C, after 14 days of P1082) sp. nov. is here classified as the type strain of a incubation. Strain GD1T shows 16S rRNA gene new , Risungbinella massiliensis, within the sequence similarities of 98.01 % to the type strain of family Thermoactinomycetaceae. To date, strain Risungbinella pyongyangensis. We describe here the GD1T is the first member of the family Thermoacti- features of this bacterium, together with the complete nomycetaceae isolated from human gut and the fourth from a human specimen.

Electronic supplementary material The online version of Keywords Genome Á Culturomics Á Taxono- this article (doi:10.1007/s10482-016-0677-6) contains supple- genomics mentary material, which is available to authorized users.

G. Dubourg Á J.-C. Lagier Á C. Robert Á D. Raoult N. Armstrong Á P.-E. Fournier Á D. Raoult (&) Special Infectious Agents Unit, King Fahd Medical Unite´ de Recherche sur les Maladies Infectieuses et Research Center, King Abdul Aziz University, Jeddah, Tropicales Emergentes, UM 63, CNRS 7278, IRD 198, Saudi Arabia Inserm 1095, Institut Hospitalo-Universitaire Me´diterrane´e-Infection, Faculte´ de me´decine, Aix- Marseille Universite´, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France e-mail: [email protected]

G. Dubourg Á C. Couderc Á P.-E. Fournier Á D. Raoult Poˆle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fe´de´ration de Bacte´riologie–Hygie`ne– Virologie, University, Hospital Centre Timone, Institut Hospitalo-Universitaire (IHU) Me´diterrane´e Infection, Assistance Publique–Hoˆpitaux de Marseille, Marseille, France 123 Antonie van Leeuwenhoek

Introduction Risungbinella Strain GD1T (= DSM 46691 = CSUR P1082) sp. nov. is here classified as the type strain of a A novel Gram-positive, aerobic bacterium designated T new species, Risungbinella massiliensis sp. nov., strain GD1 (= DSM 46691 = CSUR P1082) was together with the description of the complete genomic isolated as a part of a culturomics study (Lagier et al. sequencing and annotation. 2012) from the gut microbiota of a 38-year-old male who suffered from a Coxiella burnetii vascular infection complicated by an esophageal fistula, for Methods which he received a multiple broad-spectrum antibi- otic cocktail at the time of the stool collection Growth conditions and phenotypic tests (Dubourg et al. 2013, 2014). Currently, the classification of prokaryotes is A stool sample was collected from a 38-year-old male mainly founded on a combination of phenotypic and who suffered from a C. burnetii vascular infection genotypic characteristics (Stackebrandt and Ebers complicated by an esophageal fistula, for which he 2006; Tindall et al. 2010) including 16S rRNA gene received a multiple broad-spectrum antibiotic cocktail phylogeny, DNA–DNA hybridization (DDH) and at the time of the stool collection (Dubourg et al. G?C content. However, these methods suffer several 2014). The study was approved by the Ethics Com- pitfalls, even if they remain considered as a ‘‘gold mittee of the Institut Fe´de´ratif de Recherche IFR48, standard’’ (Moore et al. 1987; Rossello´-Mora 2006). Faculty of Medicine, Marseille, France, under agree- Thus, we recently proposed to add genomic informa- ment number 09-002. The faecal specimen was tion to phenotypic criteria for the description of new preserved at -80 °C after collection. Strain GD1T bacterial species, considering the declining cost of was isolated in March 2012 by cultivation on 5 % sequencing and the rapid growth in the number of sheep blood agar in aerobic conditions at 28 °C, after bacterial genomes being sequenced (Lagier et al. 14 days of incubation. 2015). Growth at different temperatures (22, 30, 37 and To date, the family Thermoactinomycetaceae accom- 56 °C) was tested. Growth of the strain was tested in modates 18 genera containing 31 species (http://www. 5 % sheep blood-enriched Columbia agar (BioMer- bacterio.net/thermoactinomycetaceae.html). Microor- ieux) and Tryptic Soy agar (Becton–Dickinson) under ganisms that belong to Thermoactinomycetaceae are anaerobic and microaerophilic conditions using the aerobic, yield filamentous growth and are Gram-pos- GENbag anaer and GENbag microaer systems, itive. Most of these strains have been isolated from respectively (BioMerieux), and under aerobic condi- environmental sources such as marine sediments, tions, with or without 5 % CO2. Growth was tested for sugar cane, soil or mushroom compost (Addou et al. salt tolerance, with 0–5, 50 and 100 % (w/v) NaCl. 2012, 2013; Chen et al. 2012; Han et al. 2013; The pH range for growth was tested at pH 6, 7, and 8 Hatayama et al. 2005; Kim et al. 2015; Li et al. 2012, using Tryptic Soy agar. Phenotypic tests were per- 2013; Matsuo et al. 2006; Park et al. 2007; Tsubouchi formed using an API ZYM strip (BioMerieux), an API et al. 2013; Wu et al. 2015; Yang et al. 2013, 2015; 50 CH strip (BioMe´rieux), and API 20 NE (BioMe´r- Yao et al. 2014; Yoon et al. 2005; Yu et al. 2012; ieux). Tests for hydrolysis of gelatin, starch were Zarparvar et al. 2014; Zhang et al. 2013; Zhang et al. performed as described by Gonzalez et al. (1978). 2015; Zhang et al. 2007; Zhou et al. 2014). However, In vitro susceptibility to antibiotics was determined activia has been cultured from sputa from using the disk-diffusion method on Mueller–Hinton a patient for which tuberculosis was suspected (Yassin agar with 5 % blood. et al. 2009). In the same manner, Kroppenstedtia Electronic microscopy was performed with detec- eburnea or Hazenella coriaceae were isolated from tion Formvar coated grids which were deposited on a various clinical samples, most of which were blood 40 lL bacterial suspension drop and incubated at (Buss et al. 2013; von Jan et al. 2011). 37 °C for 30 min, followed by a 10 s incubation on Risungbinella pyongyangensis was recently iso- ammonium molybdate 1 %. Grids were then observed lated from an agricultural soil sample (Kim et al. 2015) using a Morgagni 268D transmission electron micro- and is to date the sole member of the genus scope (Philips) at an operating voltage of 60 kV.

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MALDI-TOF analysis phylogenetic inferences were obtained using the max- imum-likelihood method within the MEGA6 pro- Sample preparation gramme. Actinomyces polynesiensis strain MS2T (HF952919) was used as an outgroup. Two or three freshly grown colonies were transferred with a plastic loop into a polypropylene microtube Fatty acid methyl ester (FAME) analysis by GC/ 1.5 ml containing 300 lL of ultra-pure water; the MS microtubes were then centrifuged at 13,0009g for 2 min and the supernatant was discarded. Formic acid Approximately 40 mg of bacterial biomass was har- (70 %) and acetonitrile (ACN) were added in a 1:1 (v/ vested from a 48 h pure culture on Columbia agar with v) ratio to the bacterial pellet. The mixture was 5 % sheep blood (BioMe´rieux). Cellular fatty acid vortexed for 30 s and centrifuged for 2 min. An methyl esters were prepared as previously described aliquot of 1 lL of the supernatant was transferred to a (Sasser 2006). GC/MS analyses were carried out on a spot onto a 96- well stainless steel MALDI target Clarus 500 gas chromatograph equipped with a SQ8S plate. Applied cell supernatants were air-dried for MS detector (Perkin Elmer, Courtaboeuf, France). 2 10 min following deposition of 1 lL of the a-cyano-4- lL of duplicate FAME extracts was volatised at hydroxycinnamic (CHCA, Sigma-Aldrich, Sa˜o Paulo, 250 °C (split 20 mL/min) in a Focus liner with wool Brazil) matrix prepared in an organic solvent mixture and separated on an Elite-5MS column (30 m, to a final saturation concentration in a 50 % acetoni- 0.25 mm i.d., 0.25 mm film thickness) using a linear trile/2.5 % trifluoroacetic acid (TFA) solution that was temperature gradient (70–290 °Cat6°C/min) overlaid and allowed to dry. Each sample was spotted enabling the detection of C4 to C24 fatty acid methyl 16 times. Mass spectra acquisition and data analysis esters. Helium flowing at 1.2 mL/min was used as MALDI-TOF MS analysis of all strains was per- carrier gas. MS inlet line was set at 250 °C and the EI formed on a MicroFlex mass spectrometer (Bruker source at 200 °C. Full scan monitoring was performed Daltonics, Bremen, Germany). The spectra were from 45 to 500 m/z. All data was collected and recorded in the linear positive mode at a laser processed using Turbomass 6.1 (Perkin Elmer, frequency of 60 Hz within a mass range from m/z Courtaboeuf, France). Fatty acid methyl esters were 2000 to 20,000. identified by spectral database search using MS Search 2.0 operated with the Standard Reference Database 1A Phylogenetic analysis (NIST, Gaithersburg, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK). A 37 A phylogenetic tree was generated to highlight the component FAME mix (Supelco, Sigma-Aldrich, position of strain GD1T relative to the following type Saint-Quentin Fallavier, France) was used for reten- strains: Thermoactinomyces thalpophilus KCTC 9789 tion time correlations with estimated non polar (Genbank accession AF138738), sediminis retention indexes from the NIST database; FAME strain RHA1 (GenBank accession FJ422144), Laceyella identifications were confirmed using this index. sacchari (AJ251779), Thermoactinomyces putidus KCTC 3666 (AF138736), Lihuaxuella thermophila Genome sequencing and assembly YIM 77831T (Genbank accession JX045707), Ther- moactinomyces daqus strain H-18 (Genbank accession Genomic DNA of strain GD1T was sequenced on a KF590624), H. coriacea strain 23436 (Genbank acces- MiSeq Technology platform (Illumina Inc, San Diego, sion JQ798970), kribbensis strain A 9500T CA, USA) with the mate pair strategy (Supplementary (Genbank accession AB049939), R. pyongyangensis Tables 1, 2). The genomic DNA (gDNA) was bar- strain MC 210T (Genbank accession KJ729272), Bacil- coded in order to be mixed with 11 others projects with lus endophyticus strain 2DT(T) (Genbank accession the Nextera Mate Pair sample prep kit (Illumina). The AF295302), Bacillus megaterium strain IAM 13418 gDNA was quantified by a Qubit assay with the high (Genbank accession D16273) and Bacillus pumilus sensitivity kit (Life technologies, Carlsbad, CA, USA) strain ATCC 7061 (Genbank accession AY876289). to 42 ng/ll. The mate pair library was prepared with Sequences were aligned using CLUSTALW and 1 lg of genomic DNA using the Nextera mate pair 123 Antonie van Leeuwenhoek

Thermoactinomyces thalpophilus AF138738.1strain KCTC 9789 AF138738 Laceyella sediminis FJ422144.1strain RHA1 FJ422144 AJ251779AJ251779.1 Thermoactinomyces putidusstrain AF138736 KCTC 3666 AF138736 Lihuaxuella thermophila JX045707.1strain YIM 77831T JX045707 Thermoactinomyces daqusstrain KF590624.1 H-18 KF590624 Hazenella coriacea JQ798970|strain 23436 JQ798970 Shimazuella kribbensisstrain AB049939.1 A 9500T AB049939 Risungbinella massiliensis HF952921.1strain GD1T HF952921 Risungbinella pyongyangensis MC210Tstrain MC KJ729272.1 210T KJ729272 Bacillus endophyticus strainAF295302.1 2DT(T) AF295302 Bacillus megaterium D16273.1strain IAM 13418 D16273 Bacillus pumilus AY876289.1strain ATCC 7061 AY876289 Actinomyces polynesiensis HF952919.1strain MS2T HF952919

Fig. 1 Phylogenetic tree highlighting the position of Risung- majority consensus tree. Actinomyces polynesiensis strain MS2T binella massiliensis strain GD1T relative to other type strains. (HF952919) was used as an outgroup. The scale bar represents a Numbers at the nodes are percentages of bootstrap values 5 % nucleotide sequence divergence obtained by repeating 1000 times the analysis to generate a

Fig. 2 Transmission electron microscope analysis of R. massiliensis strain GD1T showing an isolated cell (3A) and branched cells (3B). The scale bar represents 200 nm

Illumina guide. The genomic DNA sample was 680 bp on the Covaris device S2 in microtubes simultaneously fragmented and tagged with a mate (Covaris, Woburn, MA, USA).The library profile pair junction adapter. The pattern of the fragmentation was visualised on a High Sensitivity Bioanalyzer was validated on an Agilent 2100 BioAnalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, (Agilent Technologies Inc, Santa Clara, CA, USA) USA).The libraries were normalised at 2 nM and with a DNA 7500 labchip. The DNA fragments were pooled. After a denaturation step and dilution at determined to range in size from 1 kb up to 11 kb, 10 pM, the pool of libraries was loaded onto the with an optimal size at 6 kb. No size selection was reagent cartridge and then onto the instrument along performed and only 600 ng of tagmented fragments with the flow cell. Automated cluster generation and were circularised. The circularised DNA was mechan- sequencing run were performed in a single 39-h run in ically sheared to small fragments with an optimal at a29 251-bp.

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Table 1 Fatty acid composition of strain GD1T and the type ribosomal RNAs were found by using RNAmmer strain of R. pyongyangensis (Lagesen et al. 2007) and BLASTn against the Fatty acid 1 2 GenBank database. Signal peptides and the number of transmembrane helices were predicted using Sig- Saturated nalP (Petersen et al. 2011) and TMHMM (Server) 12:0 – 2.47 respectively. ORFans were identified if their BLASTP 13:0 – 2.40 E-value was lower than 1e-03 for alignment lengths 14:0 1.8 13.87 greater than 80 amino acids. If alignment lengths were 15:0 3.0 – smaller than 80 amino acids, we used an E-value of 16:0 8.3 11.82 1e-05. Such parameter thresholds have already been 17:0 3.6 1.62 used in previous works to define ORFans. Here, we 18:0 3.0 3.50 compared the genome sequence of strain GD1T with Branched those of B. endophyticus strain 2102 (GenBank 12:0 iso – 1.48 accession number ALIM00000000), B. pumilus strain 13:0 iso – 9.79 ATCC 7061T (GenBank accession number 14:0 iso 8.9 13.62 ABRX00000000), L. sacchari strain 1-1 (GenBank 15:0 iso 35.6 4.78 accession number ASZU00000000) and S. kribbensis 16:0 iso 2.8 1.57 strain DSM 45090T (GenBank accession number 17:0 iso 14.9 1.05 ATZF00000000). Orthologues were identified using 13:0 anteiso – 11.95 the Proteinortho software (version 1.4) (Lechner et al. 15:0 anteiso 13.2 14.16 2011) using 30 % protein identity and 1e-05 E-value 17:0 anteiso 1.2 TR as thresholds. The average percentage of nucleotide Unsaturated sequence identity between corresponding orthologous 18:1 n9 2.8 – sets was determined using the Needleman–Wunsch algorithm global alignment technique. Artemis Strains: 1, GD1T;2,R. pyongyangensis MC 210T (data from Kim et al., 2015). - : not detected/reported; TR, traces (\1%) (Rutherford et al. 2000) was used for data management and DNA Plotter (Carver et al. 2009) was used for visualisation of genomic features. The Mauve align- Total information of 3.9 Gb was obtained from a ment tool was used for multiple genomic sequence 399 K/mm2 cluster density with a cluster passing alignment and visualisation (Darling et al. 2004). To quality control filters of 97.92 % (7,840,000 clusters). evaluate the genomic similarity between closely Within this run, the index representation for strain related species for which genomes have been GD1T was determined to 8.27 %. The 634,217 paired sequenced we determined dDDH that exhibits a high end reads were filtered according to the read qualities. correlation with DDH (Auch et al. 2010; Meier- These reads were trimmed and then assembled Kolthoff et al. 2013a). through CLCgenomicswb4 software.

Genome annotation and comparison with other genomes from other members of the family Results and discussion

Open Reading Frames (ORFs) were predicted using Strain GD1T was found to exhibit 98.01 % 16S rRNA Prodigal (Hyatt et al. 2010) with default parameters sequence identity with the type strain of R. pyongyan- but the predicted ORFs were excluded if they were gensis (GenBank accession number KJ729272), the spanning a sequencing gap region. The predicted currently closest phylogenetically related bacterial bacterial protein sequences were searched against the species with a validly published name (Fig. 1). This GenBank database (Benson et al. 2000) and the value is lower than the 98.7 % 16S rRNA gene Clusters of Orthologous Groups (COG) databases sequence threshold recommended by Stackebrandt using BLASTP. The tRNAScanSE tool (Lowe and and Ebers (2006) to delineate a new bacterial species Eddy 1997) was used to find tRNA genes, whereas without carrying out DNA–DNA hybridization, a 123 123

Table 2 Differential characteristics of strain GD1T from related members of the family Thermoactinomycetaceae Properties 1 2 3 4 5 6 7 8 9 10

Oxygen requirement Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic Gram stain Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Temperature for growth (°C) 22–37 22–42 20–37 30–60 28–65 30–65 35–65 28–65 22–45 45–60 formation ? ??? ?? ? ? ? na Starch hydrolysis ??– ?????–na Gelatin liquefaction – ? –– ?? – ?? ? Production of Alkaline phosphatase – na na na na na na na – na Acid phosphatase – na na na na na na na – na Catalase ??na ??na na na ? na Oxidase ??na ? –nanana? na Utilization of D-xylose – ? na – ? na – – – na D-fructose – – na ? –– ? na – ? D-glucose – ? na ????na – – D-mannose – ? na ??–v–– DNA G?C content (mol %) 40.1 42.1 42.1 45.5 55.6 49 48 47.9 37.8 47 Habitat Human gut Soil Soil Geothermal field Soil Soil and human Sugar cane Sediment Human blood Fermentation starter Strains: 1, GD1T;2,Risungbinella pyongyangensis MC 210T;3,Shimazuella kribbensis KCTC 9933T;4,Geothermomicrobium terrae YIM 77562T; 5, Lihuaxuella thermophila T T T

YIM 77831 (data in columns 2–5 are from Kim et al. 2015); 6, KCTC 3666 (Lacey and Cross 1989; Yoon et al. 2005); 7, Laceyella sacchari KCTC 9790 Leeuwenhoek van Antonie (Lacey 1971; Yoon et al. 2005); 8, Laceyella sediminis RHA1T (Chen et al. 2012); 9, Hazenella coriacea 23436T (Buss et al. 2013); 10, Thermoactinomyces daqus H-18T (Yao et al. 2014). w: weak; v: variable; na: not available Antonie van Leeuwenhoek

Fig. 3 Reference mass spectrum from R. massiliensis strain GD1T. Spectra from three individual colonies were compared and a reference spectrum was generated

Table 3 Nucleotide content and gene count levels of the Morphological, cultural and physiological genome characteristics Attribute Value % of totala Growth of strain GD1T was found to occur between 22 Size (bp) 3,440,191 100 and 37 °C, after 24–48 h of incubation. Early colonies G?C content (bp) 139,516 40.1 were 0.5 lm in diameter on blood-enriched Columbia Coding region (bp) 2,901,150 84.3 agar, and were 2 lm after 48–72 h. Growth was Total genes 3607 100.0 observed only aerobically. Gram staining showed RNA genes 67 1.9 Gram-positive branched . A motility test was Protein-coding genes 3540 98.1 negative. Colonies were observed to be round, grey, Genes with function prediction 2185 60.6 with rough texture, and adherent to the medium after Genes assigned to COGs 2447 67.8 24–48 h incubation. Growth was found to occur at pH Genes with peptide signals 143 4.0 6.0–8.0 and 0–5 % (w/v) NaCl. The optimal temper- Genes with transmembrane helices 925 25.6 ature, pH and NaCl concentration for growth were a The total is based on either the size of the genome in base determined to be 37 °C, pH 7.0 and 0–2 %(w/v) NaCl. pairs or the total number of protein coding genes in the Cells were found to have a mean width of 0.68 lm and annotated genome mean length of 3.03 lm (Fig. 2). The major fatty acids T of strain GD1 were identified as iso-C15:0 (35.6 %), iso-C17:0 (14.9 %) anteiso-C15:0 (13.2 %), iso-C14:0 threshold which has been reinforced by recent works (8.9 %), and C16:0 (8.3 %) (Table 1). (Meier-Kolthoff et al. 2013b; Kim et al. 2014). The Positive reactions were recorded for oxidase, catalase, 16S rRNA sequence of strain GD1T has been starch hydrolysis, leucine arylamidase, valine arylami- deposited in GenBank under number HF952921. dase, cystine arylamidase, trypsin, acid phosphatase,

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Fig. 4 Graphical circular map of the chromosome of R. massiliensis strain GD1T. From the outside in, the outer two circles shows open reading frames oriented in the forward (coloured by COG categories) and reverse (coloured by COG categories) direction, respectively. The third circle marks the rRNA gene operon (red) and tRNA genes (green). The fourth circle shows the G?C% content plot. The inner-most circle shows GC skew, purple indicating negative values and olive for positive values

alkaline phosphatase, naphthol-AS-BI-phosphohydro- allowing a further correct identification from a single lase, a-galactosidase, b-glucuronidase, a-glucosidase, colony with an identification score of 1.9 (Fig. 3). b-glucosidase, esculin hydrolysis and fermentation of D- maltose. Negative reactions were recorded for gelatin Genome properties hydrolysis, esterase (C4), esterase lipase (C8), b-galac- tosidase, lipase (C14), a-chymotrypsin, N acetyl-b- The genome of strain GD1T is 3,440,191 bp long with glucosaminidase, a-mannosidase, a-fucosidase, and a 40.1 % G?C content (Table 3; Fig. 4). Of the 3607 fermentation of L-arabinose, D-ribose, D-xylose, methyl- predicted genes, 3540 are protein-coding genes, and 3 bD-xylopyranoside, D-galactose, D-glucose, D-fructose, encode rRNAs. Three different rRNA genes (one 16S D-mannose, L-rhamnose, D-mannitol, N-acetylglu- rRNA, one 23S rRNA and one 5S rRNA) and 64 cosamine, amygdalin, arbutin, salicin, D-cellobiose, predicted tRNA genes were identified in the genome. D-lactose, D-melibiose, D-saccharose, D-trehalose, inulin, A total of 2447 genes (67.8 %) were assigned a D-melezitose, D-raffinose, starch, glycogen and D-lyxose. putative function. Three hundred and ninety genes By comparison with R. pyongyangensis MC210T, its were identified as ORFans (11.1 %). The remaining close phylogenetic neighbour, strain GD1T differs in genes were annotated as hypothetical proteins. The utilisation of D-xylose, D-fructose and D-mannose properties and the statistics of the genome are (Table 2). summarised in Table 3. The distribution of genes into Strain GD1T was found to be susceptible to penicillin COGs functional categories is presented in Table 4. G, amoxicillin, amoxicillin-clavulanic acid, ceftriax- one, imipenem, vancomycin, rifampicin, erythromycin, Comparison with other genomes from other gentamicin, ciprofloxacin and trimethoprim/sul- members of the family famethoxazole, but resistant to metronidazole. The reference mass spectrum recorded by MS The draft genome of strain GD1T has a larger size than MALDI TOF was then labeled as R. massiliensis, that of L. sacchari (3.32 Mb) but is smaller than that of 123 Antonie van Leeuwenhoek

Table 4 Number of genes Code Value % value Description associated with the 25 general COG functional J 166 4.69 Translation categories A 0 0 RNA processing and modification K 250 7.06 Transcription L 168 4.75 Replication, recombination and repair B 0 0 Chromatin structure and dynamics D 23 0.65 Cell cycle control, mitosis and meiosis Y 0 0 Nuclear structure V 60 1.69 Defense mechanisms T 93 2.63 Signal transduction mechanisms M 133 3.76 Cell wall/membrane biogenesis N 6 0.17 Cell motility Z 0 0 Cytoskeleton W 0 0 Extracellular structures U 33 0.93 Intracellular trafficking and secretion O 104 2.94 Posttranslational modification, protein turnover, chaperones C 127 3.59 Energy production and conversion G 171 4.83 Carbohydrate transport and metabolism E 281 7.94 Amino acid transport and metabolism F 76 2.15 Nucleotide transport and metabolism H 90 2.54 Coenzyme transport and metabolism I 107 3.02 Lipid transport and metabolism P 155 4.38 Inorganic ion transport and metabolism Q 66 1.86 Secondary metabolites biosynthesis, transport and catabolism a The total is based on the R 445 12.57 General function prediction only total number of protein S 265 7.49 Function unknown coding genes in the _ 1093 30.87 Not in COGs annotated genome

B. endophyticus, B. pumilus and S. kribbensis (5.11, 3.83 Unfortunately, R. pyongyangensis could not be included and 4.18 Mb, respectively) The G?C content of the in this comparison as its genome had not been sequenced. genome of strain GD1T is higher than those of B. endophyticus and S. kribbensis (40.1, 36.4 and 38.4 % respectively) but less than those of B. pumilus and L. Conclusion sacchari (41.7 and 48.9 % respectively). The gene content of the genome of strain GD1T is larger than On the basis of phenotypic, phylogenetic and genomic those of L. sacchari (3412) and smaller than those of B. analyses, strain GD1T (= DSM 46691 = CSUR P1082) endophyticus, B. pumilus and S. kribbensis (5238, 4162 is concluded to represent a novel species of the genus and 4860 respectively). However the distribution of Risungbinella, for which the name Risungbinella mas- genes into COG categories was similar in all genomes siliensis sp. nov. is proposed. The species is the fourth compared (Fig. 5). In addition, strain GD1T shares 3540, Thermoactinomycetaceae species isolated from humans 5179, 3900, 3271 and 4230 orthologous genes with B. and the first isolated from the human gut microbiota. endophyticus, B. pumilus, L. sacchari and S. kribbensis respectively. The average nucleotide sequence identity ranges from 69.69 to 64.11 % between the species Description of Risungbinella massiliensis sp. nov (Supplementary Table 3). Using dDDH, strain GD1T exhibits a similarity ranging from 22.6 to 32.5 % when Risungbinella massiliensis (mas.si.li.en0sis. L. masc. compared to other species (Supplementary Table 4). adj. massiliensis of Massilia, the ancient Greek and 123 Antonie van Leeuwenhoek

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