'Cellulomonas timonensis' sp. nov., taxonogenomics description of a new bacterial species isolated from human gut.

'Cellulomonas timonensis' sp. nov. strain sn7T is a new species within the Cellulomonas genus. We present the main phenotypic characteristics and provide a complete annotation of its genome sequence. This facultative anaerobic bacterium, isolated from the stool of 38-year-old obese Frenchman, is Gram-positive, has motile rods and is sporulating. The genome is 4 057 828 bp long with 72.42% G + C content. Of the 3732 predicted genes, 3667 were protein-coding genes and 65 were RNAs.

Introduction

The development of metagenomics has enabled a better exploration of the gut microbiota, thus bypassing the problem of noncultivable bacteria and providing an understanding of the relationship between altered gut microbiota and several pathologies such as obesity, inflammatory bowel disease and irritable bowel syndrome [1]. Nevertheless, bacterial culture remains essential in order to have a better representation of the viable population and would, in addition, allow for an extension of the known gut bacterial repertoire.

Recently our laboratory has developed a new concept known as microbial culturomics. This makes it possible to explore, as comprehensively as possible, the viable population of prokaryotes associated with the human gastrointestinal tract by varying culture media and physicochemical parameters [2]. As a result of this concept, several new bacteria, including new genera and species, have been reported in the human gut microbiota. As a result, culturomics has doubled the number of species isolated at least once from the human gut [3].

Current methods of defining a new bacterial species, which are based on genetic, phenotypic and chemotaxonomic criteria, are not reproducible and cannot be applied to all bacterial genera [4], [5]. Furthermore, the availability of genomic data for many bacterial species [6] has recently led to a new concept of bacterial description being proposed, including a proteomic description obtained y matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) [7], [8] alongside biochemical and genomic analyses of the new species [9].

The genus Cellulomonas was first described by Bergey et al. [10] and later amended by Clark and Stackebrandt et al. [11], [12]. The members of this genus are Gram-positive irregular rods with cellulolytic activity. They were cultivated in aerobic conditions, and most strains are also capable of anaerobic growth. They have l-ornithine in their peptidoglycan, contain menaquinone MK-9(H4) as the predominant respiratory quinone, have anteiso-C15:0 and C16:0 as the major fatty acids [11] and have a high genomic G + C content of 71 to 76 mol% [13]. To date, this genus consists of 28 species [9], [14]. Most of these species were originally isolated from environmental samples, and occasionally from rumen and activated sludge.

Here we present a summary classification and a set of features for the type strain C. timonensis sp. nov. strain sn7 (= CSUR P2058 = DSM 100699), a new bacterial species isolated by culturomics from the stool sample of an obese Frenchman, together with the description of the complete genomic sequence and its annotation.

Materials and methods

Organism information

A stool sample was collected from a 38-year-old Frenchman living in France who was included in a research protocol. The stool sample was frozen at −80°C after sampling at the La Timone hospital in Marseille. The patient provided written informed consent. Both this study and the consent procedure were approved by the ethics committee of the Federative Research Institute IFR48, Faculty of Medicine, Marseille, France (agreement 09-022).

Strain identification by MALDI-TOF MS and 16S rRNA sequencing

The stool sample was cultured on 5% sheep's blood–enriched Columbia agar (bioMérieux, Marcy l’Etoile, France) at 37°C in microaerophilic atmosphere generated by CampyGen (Oxoid, Dardilly, France). After 48 hours' incubation, the isolated colonies were deposited in duplicate on a MALDI-TOF MS MSP96 target plate (Bruker Daltonics, Leipzig, Germany), then covered with 1.5 μL of a matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid diluted in 50% acetonitrile, 2.5% trifluoroacetic acid, completed with high-performance liquid chromatography water). Proteomic analysis of our strain was carried out with MALDI-TOF MS as previously described [9] using a MicroFlex spectrometer (Bruker). Twelve distinct deposits were made for strain sn7T from 12 isolated colonies. Twelve spectra were thus obtained, imported into MALDI BioTyper software (version 2.0; Bruker) and analysed by standard pattern matching (with default parameter settings) against the main spectra of 7567 bacteria (Bruker database completed with the La Timone database, including species isolated by culturomics and in our routine laboratory). The comparison with the BioTyper database spectra enabled the identification and discrimination of the analysed species from those in the database as a result of the obtained score: a score of >2 with a validated species enabled identification at the species level, and a score of <1.7 did not enable any identification. If the colony was not identified, despite a clean spectrum, a sequencing of 16S rDNA was performed as previously described [15] to define taxonomic criteria. BLASTn searches were performed at the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi) to compare and identify the 16S rDNA sequence of our strain. A threshold of 98.7% similarity was determined to define a new species without performing DNA-DNA hybridization (DDH) [4].

Growth conditions

Different growth temperatures (28, 37, 45, 55°C) were tested under anaerobic and microaerophilic conditions using AnaeroGen and CampyGen respectively (Thermo Fisher Scientific, Courtaboeuf, France). The strain growth was also tested under aerobic conditions, in the presence of air and with or without 5% CO2. The tolerance of this strain sn7T to salt (0–5, 50–75 and 100 g/L NaCl) and pH (6, 7 and 8.5) was calculated.

Morphologic, biochemical and antibiotic susceptibility tests

Gram staining and motility were observed from fresh colonies between blades and slats using a DM1000 photonic microscope (Leica Microsystems, Nanterre, France) with a 40 × objective lens [16]. Spore formation was determined by thermal shock (80°C for 20 minutes) and observed under a microscope. Negative staining was carried out with detection formvar-coated grids placed on a drop of 40 μL of bacterial suspension and incubated at 37°C for 30 minutes, followed by a 10-second incubation in 1% ammonium molybdate. The grids were dried on blotting paper and then observed with a Tecnai G20 transmission electron microscope (FEI Company, Limeil-Brévannes, France). We studied the biochemical characteristics of this strain using API 20NE, API ZYM and API 50CH strips according to the manufacturer's instructions (bioMérieux). Oxidase and catalase reactions were determined using a BBL DrySlide (Becton, Le Pont de Claix, France) according to the manufacturer's instructions. The antimicrobial activity test was performed using the disc diffusion method (i2a, Montpellier, France) [17] on Mueller-Hinton agar in a petri dish (bioMérieux).

Fatty acid methyl ester analysis by gas chromatography/mass spectrometry

Cellular fatty acid methyl ester (FAME) analysis was performed by gas chromatography/mass spectrometry (GC/MS). Two samples were prepared with approximately 60 mg of bacterial biomass per tube collected from several culture plates. FAMEs were prepared as described by Sasser [18]. GC/MS analyses were carried out as previously described [19]. Briefly, FAMEs were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500–SQ 8 S; Perkin Elmer, Courtaboeuf, France). A spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1A (National Institute of Standards and Technology, Gaithersburg, MD, USA) and the FAME mass spectral database (Wiley, Chichester, UK).

Genome sequencing and assembly

Genomic DNA of C. timonensis was extracted as previously described [9] and sequenced on the MiSeq sequencer (Illumina, San Diego, CA, USA) using the mate-pair strategy. The genomic DNA was barcoded in order to be mixed with 11 other projects using the Nextera Mate Pair sample prep kit (Illumina). All genome sequencing steps were performed following the process previously reported by Lagier et al. [9]. Genome assembly was performed using a pipeline that enabled the creation of an assembly with different pieces of software (Velvet [20], Spades [21] and Soap Denovo [22]) on trimmed (MiSeq and Trimmomatic) [23] or untrimmed (only MiSeq software) data. For each of the six assemblies performed, GapCloser [22] was used to reduce gaps. Contamination with Phage Phix was then identified (BLASTn against Phage Phix174 DNA sequence) and eliminated. Finally, scaffolds <800 bp in size were removed. Scaffolds with a depth value of lower than 25% of the mean depth were also removed (identified as possible contaminants). The best assembly was selected by using different criteria (number of scaffolds, N50, number of N). For the studied strain, 250 bp gave the best assembly, with a depth coverage of 2 X 250 bp.

Genome annotation

Open reading frames (ORFs) were predicted using Prodigal [24] with default parameters, but the predicted ORFs were excluded if they spanned a sequencing gap region (containing N). The predicted bacterial protein sequences were searched against the Clusters of Orthologous Groups (COGs) database using BLASTP (E value of 1e-03, coverage 0.7 and identity 30%). If no hit was found, sequences were searched against the NR database using BLASTP (E value of 1e-03, coverage 0.7 and identity 30%). If the length of the sequence was <80 aa, we used an E value of 1e-05. The tRNAScanSE [25] tool was used to find transfer RNA genes, whereas ribosomal RNA genes were found using RNAmmer [26]. Lipoprotein signal peptides and the number of transmembrane helices were predicted using Phobius [27]. ORFans were identified if all the BLASTP runs performed did not give positive results (E value smaller than 1e-03 for ORFs with sequence size >80 aa or E value smaller than 1e-05 for ORFs with sequence length <80 aa). Such parameter thresholds have been used in previous studies to define ORFans. The annotation process was performed using DAGOBAH [28] including the Figenix [29] libraries, which provided the pipeline analysis.

16S rRNA phylogenetic tree

Sequences were recovered after a nucleotide BLAST against the 16S rRNA database of the All-Species Living Tree SILVA project (LTPs119). First, a filter to eliminate sequences smaller than 1450 bp was applied. (Sometimes this filter is decreased to retrieve more sequences.) Pass filter sequences were aligned using Muscle [30], and phylogenetic inferences were obtained using the approximate maximum-likelihood method within FastTree software [31]. Numbers at the nodes corresponding to local values were computed by the Shimodaira-Hasegawa test. A filter using PhyloPattern [32] was applied to the tree to remove duplicate species and bad taxonomic reference species. This pipeline was performed in DAGOBAH [28], which include the Figenix [29] libraries.

Genome comparison analysis

Species to be compared were automatically retrieved from the 16s RNA tree using PhyloPattern. For each selected species, the complete genome sequence, proteome sequence and ORFeome sequence were retrieved from the NCBI FTP site. If one specific strain did not have a complete and available genome, a complete genome of the same species was used. If ORFeomes and proteomes were not predicted, Prodigal was used with default parameters to predict them. All proteomes were analysed using proteinOrtho [33]; then, for each couple of genomes, a similarity score was computed. This score is the mean value of nucleotide similarity between all couples of orthologous genes between the two genomes studied (average genomic identity of orthologous gene sequences (AGIOS) tool). An annotation of all proteomes was also performed to define the distribution of functional classes of predicted genes according to COGs proteins (using the same method as that for genome annotation). The comparison process was performed by DAGOBAH [28], which includes the Figenix [29] libraries, which provided pipeline analysis, and PhyloPattern [32] for tree manipulation. To evaluate the genomic similarity among the Cellulomonas strains studied, we set two parameters: digital DDH, which exhibits a high correlation with DDH [34], [35], and AGIOS, which was designed to be independent of DDH.

Results

Strain identification and phylogenetic analyses

Strain sn7T was isolated in May 2015 by cultivation on 5% sheep's blood–enriched agar under microaerophilic conditions. This strain was not identified by MALDI-TOF MS, and its spectrum was added to our database (Fig. 1). Sequencing of the 16S rRNA gene demonstrated that this strain sn7T exhibited a nucleotide sequence similarity of 98.4% with Cellulomonas cellasea (GenBank accession no. KR922256), the phylogenetically closest bacterial species with a validly published name (Fig. 2). Its 16S rRNA sequence was deposited in the European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL-EBI) database under accession number LN870311. This value was lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers [4] to delineate a new species without carrying out DDH. Strain sn7T is thus a new species which has been named Cellulomonas timonensis (Table 1). The other closest species were C. chitinilytica (97.94%), C. biazotea (97.7%), C. fimi (97.5%) and C. xylanilytica (96.9%). The species C. timonensis, C. biazotea and C. fimi shared a single cluster, whereas C. xylanilytica is present in a distant clade in the phylogenetic tree. A comparison between the spectral differences of C. timonensis and other closest species is represented in a gel view (Fig. 3).

Fig. 1

Reference matrix-assisted desorption ionization–time of flight mass spectrometry analysis of Cellulomonas timonensis strain sn7T.

Fig. 2

Phylogenetic tree highlighting position of Cellulomonas timonensis strain sn7T relative to other type strains within genus Cellulomonas. GenBank accession numbers are indicated at right of species name. Sequences were aligned using CLUSTAL W, and phylogenetic inferences were obtained using maximum-likelihood method within MEGA software. Numbers at nodes are bootstrap values obtained by repeating analysis 500 times to generate majority consensus tree. Scale bar indicates 1% nucleotide sequence divergence.

Table 1

Classification and general features of Cellulomonas timonensis sn7T

Property	Term
Current classification	Domain: Bacteria
	Phylum: Actinobacteria
	Class: Actinobacteria
	Order: Micrococcales
	Family: Cellulomonadaceae
	Genus: Cellulomonas
	Species: Cellulomonas timonensis
	Type strain: sn7T
Gram stain	Positive
Cell shape	Rod
Motility	Motile
Sporulation	Sporulating
Temperature range	Mesophilic
Optimum temperature	37°C
Oxygen requirement	Facultative anaerobic
Salinity	0–5 g/L
Optimum salinity	1 g/L
pH	7–8.5
Optimum pH	7
Pathogenicity	Unknown
Habitat	Human gut
Isolation	Human faeces

Fig. 3

Gel view comparing Cellulomonas timonensis strain sn7T to other close species. Gel view displays raw spectra of loaded spectrum files arranged in pseudo–gel-like look. X-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity is expressed by greyscale scheme code. Colour bar and right y-axis indicate relation between colour of peak and its intensity in arbitrary units. Displayed species are indicated at left.

Phenotypic description

Growth of strain sn7T was observed between 28 and 37°C on 5% sheep's blood–enriched Colombia agar, and optimal growth was achieved at 37°C after 24 hours' incubation in aerobic conditions and 48 hours' incubation in anaerobic and microaerobic atmospheres. Cells were motile and sporulating. Colonies were irregular, with a diameter of 1.5 to 2 mm on blood-enriched Colombia agar after 48 hours. The results of pH testing showed that strain sn7T can survive under pH conditions ranging between 7 and 8.5, but has optimal growth at pH 7. It grows in salinity concentrations ranging from 0 to 5g/L NaCl. Gram staining (Fig. 4) showed Gram-positive rods. Using electron microscopy, the rods had a mean diameter of 0.3 μm and a length of 1.3 μm (Fig. 5).

Fig. 4

Gram staining of Cellulomonas timonensis strain sn7T.

Fig. 5

Transmission electron microscopy of Cellulomonas timonensis strain sn7T using Tecnai G20 device at operating voltage of 60 kV. Scale bar = 500 nm.

Catalase was positive for strain sn7T, and oxidase was negative. Using the API ZYM gallery, positive reactions were observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucosidase and N-acetyl-β-glucosaminidase. Cystine arylamidase, trypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase and α-fucosidase were negative. Using the API 20 NE system, a positive reaction was obtained for a nitrate reduction, indole formation, glucose fermentation, esculin hydrolysis and β-galactosidase, and assimilation for glucose, arabinose and mannose were observed. All other reactions were negative, including urease and gelatin hydrolysis. An API 50CH strip showed positive reactions for glycerol, l-arabinose, d-ribose, d-xylose, d-galactose, d-glucose, d-fructose d-mannose, arbutin, esculin ferric citrate, salicin, d-cellobiose, d-maltose, d-saccharose, d-trehalose, amidon and glycogen. Negative reactions were recorded for erythritol, d-arabinose, l-xylose, d-adonitol, methyl-βd-xylopyranoside, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl-αd-mannopyranoside, methyl-αd-glucopyranoside, N-acetyl-glucosamine, amygdalin d-lactose, d-melibiose, inulin, d-melezitose, d-raffinose, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. Table 2 shows a comparison of the principal phenotypic and biochemical features between C. timonensis and other species belonging to the Cellulomonas genus. Cells were susceptible to ceftriaxone, ciprofloxacin, clindamycin, doxycycline, erythromycin, gentamicin, penicillin, rifampicin, teicoplanin and vancomycin but were resistant to colistin, fosfomycin, oxacillin and trimethoprim/sulfamethoxazole.

Table 2

Differential characteristics of Cellulomonas timonensis strain SN7T, Cellulomonas cellasea DSM 20118T, Cellulomonas massiliensis strain JC225T, Cellulomonas fimi DSM 20113T, Cellulomonas chitinilytica X.bu-b, Cellulomonas soli Kc1T, Cellulomonas humilata ATCC 25174T, Cellulomonas xylanilytica XIL11 and Cellulomonas terrae DB5

Property	C. timonensis	C. cellasea	C. massiliensis	C. fimi	C. chitinilytica	C. soli	C. humilata	C. xylanilytica	C. terrae
Oxygen requirement	Facultative anaerobic	Facultative anaerobic	Aerobic	Facultative anaerobic	Facultative anaerobic	Facultative anaerobic	Aerobic/microaerophilic	Facultative anaerobic	Facultative anaerobic
Salt requirement	0–5 g/L	NA	5 g/L	NA	2 g/L	0–3 g/L	<4 g/L	NA	NA
Motility	+	−	+	+	−	+	−	−	−
Endospore formation	+	NA	−	−	NA	NA	−	−	NA
Indole	+	NA	−	NA	−	−	−	−	NA
Production of:
Alkaline phosphatase	+	NA	NA	NA	NA	NA	NA	NA	NA
Catalase	+	−	+	+	+	+	−	+	−
Oxidase	−	NA	+	NA	−	−	−	+	NA
Nitrate reductase	+	+	−	+	+	+	−	+	+
Urease	−	−	−	−	−	−	−	−	−
β-Galactosidase	+	+	+	−	+	+	NA	+	NA
N-Acetyl-glucosamine	−	NA	−	NA	NA	−	NA	+	NA
Acid from:
l-Arabinose	+	+	−	+	+	−	NA	NA	NA
Ribose	+	−	NA	+	−	−	−	−	−
Mannose	+	+	−	+	+	+	+	+	+
Mannitol	−	−	−	−	−	−	+	−	−
Sucrose	+	+	−	+	−	+	+	+	+
d-Glucose	+	+	−	+	−	+	+	+	+
d-Fructose	+	NA	NA	NA	NA	+	+	+	NA
d-Maltose	+	+	−	+	+	+	+	+	NA
d-Lactose	−	−	NA	+	−	−	NA	+	+
Habitat	Human gut	Soil	Human gut	Soil	Cattle farm compost	Soil	Soil	Elm tree	Soil

+, positive result; −, negative result; NA, data not available.

FAME analysis by GC/MS

The major fatty acid was the saturated and branched 12-methyl-tetradecanoic acid (74%). The detected compounds are mainly all saturated fatty acids. Very few unsaturated species are listed in Table 3.

Table 3

cellular fatty acid composition (%)

Fatty acid	IUPAC name	Mean relative %a
15:0 anteiso	12-methyl-Tetradecanoic acid	74.0 ± 0.2
17:0 anteiso	14-methyl-Hexadecanoic acid	8.4 ± 0.6
15:1n5 iso	13-methyl-Tetradec-9-enoic acid	5.0 ± 0.6
16:0	Hexadecanoic acid	3.9 ± 0.1
16:0 iso	14-methyl-Pentadecanoic acid	2.0 ± 0.1
15:0	Pentadecanoic acid	1.6 ± 0.1
15:0 iso	13-methyl-Tetradecanoic acid	1.4 ± 0.1
17:1 cyclo	11-methyl-Cyclohexylundecanoic acid	1.0 ± 0.1
18:1n5	13-Octadecenoic acid	TR
14:0	Tetradecanoic acid	TR
14:0 iso	12-methyl-Tridecanoic acid	TR
18:0	Octadecanoic acid	TR
18:2n6	9,12-Octadecadienoic acid	TR
5:0 anteiso	2-methyl-Butanoic acid	TR
17:0	Heptadecanoic acid	TR
17:0 iso	15-methyl-Hexadecanoic acid	TR
6:0 iso	4-methyl-Pentanoic acid	TR

IUPAC, International Union of Pure and Applied Chemistry; TR = trace amounts <1%.

Mean peak area percentage ± standard deviation.

Genome properties

The genome is 4 057 828 bp long with 72.42% G + C content (Table 4, Fig. 6), (accession no. FCOT00000000). It is composed of 13 scaffolds (composed of 13 contigs). Of the 3732 predicted genes, 3667 were protein-coding genes and 65 were RNAs (five genes were 5S rRNA, five genes 16S rRNA, five genes 23S rRNA and 50 genes tRNA). A total of 2727 genes (74.37%) were assigned a putative function (by COGs or by NR BLAST). A total of 176 genes (4.80%) were identified as ORFans. The remaining genes were annotated as hypothetical proteins (643 genes were >17.53%). Table 5 represents the distribution of C. timonensis genes into the different COGs categories.

Table 4

Nucleotide content and gene count levels of genome

Attribute	Value	% of totala
Size (bp)	4 057 828	100
G + C content (%)	2 938 504	72.41
Coding region (bp)	3 726 909	91.84
Total genes	3732	100
RNA genes	65	1.74
Protein-coding genes	3667	100
Genes with function prediction	2727	74.36
Genes assigned to COGs	2405	65.58
Genes with peptide signals	481	13.11
Genes with transmembrane helices	913	24.89
Crispr repeats	0	0
ORFan genes	176	4.79
Genes associated with PKS or NRPS	28	0.76
No. of antibiotic resistance genes	0	0

COGs, Clusters of Orthologous Groups database; NRPS, nonribosomal peptide synthase; PKS, polyketide synthase.

Total is based on either size of genome in base pairs or total number of protein-coding genes in annotated genome.

Fig. 6

Graphical circular map of chromosome of Cellulomonas timonensis strain sn7T. From outside to centre: genes on forward strand coloured by COGs categories (only genes assigned to COGs), genes on reverse strand coloured by COGs categories (only gene assigned to COGs), RNA genes (tRNAs green, rRNAs red), GC content and GC skew. COGs, Clusters of Orthologous Groups database.

Table 5

Number of genes associated with 25 general COGs functional categories

Code	Value	% of total	Description
J	184	5.017726	Translation
	1	0.027270248	RNA processing and modification
K	189	5.154077	Transcription
L	88	2.399782	Replication, recombination and repair
B	1	0.027270248	Chromatin structure and dynamics
D	27	0.7362967	Cell cycle control, mitosis and meiosis
Y	0	0	Nuclear structure
V	84	2.290701	Defense mechanisms
T	99	2.6997545	Signal transduction mechanisms
M	105	2.8633761	Cell wall/membrane biogenesis
N	52	1.4180529	Cell motility
Z	0	0	Cytoskeleton
W	19	0.5181347	Extracellular structures
U	35	0.95445865	Intracellular trafficking and secretion
O	102	2.7815652	Posttranslational modification, protein turnover, chaperones
X	12	0.32724297	Mobilome: prophages, transposons
C	156	4.2541585	Energy production and conversion
G	343	9.353695	Carbohydrate transport and metabolism
E	281	7.66294	Amino acid transport and metabolism
F	96	2.6179438	Nucleotide transport and metabolism
H	142	3.8723752	Coenzyme transport and metabolism
I	111	3.0269976	Lipid transport and metabolism
P	170	4.6359425	Inorganic ion transport and metabolism
Q	86	2.3452413	Secondary metabolites biosynthesis, transport and catabolism
R	248	6.7630215	General function prediction only
S	121	3.2997	Function unknown
—	1262	34.415054	Not in COGs

COGs, Clusters of Orthologous Groups database.

Genome comparison

We compared the genome of C. timonensis with other close species: Cellulomonas flavigena, Cellulomonas terrae, Cellulomonas xylanilytica, Cellulomonas chitinilytica, Cellulomonas fimi and Cellulomonas cellasea (Table 6). The draft genome sequence of C. timonensis is smaller than those of C. flavigena and C. fimi (4.06, 4.12 and 4.27 Mbp respectively), but larger than those of C. xylanilytica, C. cellasea, C. chitinilytica and C. terrae (1.78, 3.91, 1.17 and 1.84 Mbp respectively). The G + C content of C. timonensis is smaller than those of C. cellasea, C. flavigena and C. fimi (72.42, 74.55, 74.29 and 74.72% respectively), but larger than those of C. xylanilytica, C. chitinilytica and C. terrae (69.55, 68.99 and 69.55% respectively). The gene content of C. timonensis is smaller than those of C. flavigena and C. fimi (3667, 3678 and 3762 respectively), but larger than those of C. xylanilytica, C. cellasea, C. chitinilytica and C. terrae (3510, 3560, 2627 and 3606 respectively) (Table 6). We observed an identical distribution of genes into COGs categories in all compared genomes (Fig. 7). An analysis of orthologous genes shared among the different genomes revealed that C. timonensis shared 106, 353, 1623, 1742, 1487 and 77.31 orthologous genes with C. chitinilytica, C. terrae, C. flavigena, C. fimi, D. cellasea and C. xylanilytica respectively. Among species with standing in nomenclature, AGIOS values ranged from 64.51% to 87.97% among compared species, with the exception of C. timonensis. When compared to other species, the AGIOS values ranged from 66.61% with C. cellasea to 77.31% with C. xylanilytica (Table 7).

Table 6

Genome comparison of closely related species to Cellulomonas timonensis strain sn7T

Cellulomonas species	INSDC No.	Size (Mbp)	G + C %	Protein-coding genes
C. timonensis strain sn7	FCOT00000000	4.05	72.41	3667
C. xylanilytica strain XIL11	BBGX00000000.1	1.77	69.55	3510
C. cellasea strain DSM 20118T	AXNT00000000.1	3.91	74.55	3230
C. flavigena strain DSM 20109	CP001964.1	4.12	74.29	3678
C. chitinilytica strain X.bu-b	BBHG00000000.1	1.17	68.99	2627
C. terrae strain DB5	BBGZ00000000.1	1.83	69.55	3606
C. fimi strain DSM 20113T	CP002666.1	4.26	74.72	3725

INSDC, International Nucleotide Sequence Database Collaboration.

Fig. 7

Distribution of functional classes of predicted genes according to Clusters of Orthologous Groups database of proteins.

Table 7

Number of orthologous proteins shared between Cellulomonas genomes (upper right)

	C. chitinilytica	C. terrae	C. flavigena	C. fimi	C. cellasea	C. timonensis	C. xylanilytica
C. chitinilytica	2627	124	105	121	104	106	129
C. terrae	80.45	3606	354	391	324	353	308
C. flavigena	66.51	67.83	3678	1721	1477	1623	333
C. fimi	68.75	68.60	68.69	3762	1609	1742	390
C. cellasea	64.56	64.64	68.89	68.32	3560	1487	331
C. timonensis	77.00	76.99	67.41	67.92	66.61	3667	350
C. xylanilytica	80.64	87.97	67.23	69.29	64.51	77.31	3510

Average percentage similarity of nucleotides corresponding to orthologous proteins shared between genomes (lower left) and numbers of proteins per genome (bold).

Two parameters were used to evaluate genomic similarity among the studied strains: AGIOS (Table 7), which was designed to be independent of DDH, and digital DDH, which exhibits a high correlation with DDH [34], [35] (Table 8).

Table 8

Pairwise comparison of Cellulomonas timonensis sn7T with other Cellulomonas species using GGDC, formula 2 (DDH estimates based on identities/HSP length)

	C. timonensis	C. cellasea	C. chitinilytica	C. fimi	C. flavigena	C. terrae	C. xylanilytica
C. timonensis	100 (100–100%)	22.1 (19.8–24.6%)	28.4 (26–30.9%)	20.80 (18.5–23.2%)	20.60 (18.4–23%)	26.30 (24–28.8%)	26.8 (24.4–29.3%)
C. cellasea		100 (100–100%)	28 (25.7–30.5%)	21.9 (19.6–24.3%)	21.2 (19–23.7%)	26.1 (23.8–28.6%)	26.6 (24.3–29.1%)
C. chitinilytica			100 (100–100%)	30.8 (28.4–33.3%)	28.9 (26.5–31.4%)	30.6 (28.2–33.1%)	30.7 (28.3–33.2%)
C. fimi				100 (100–100%)	21.3 (19.1–23.8%)	27.7 (25.4–30.2%)	27.9 (25.6–30.4%)
C. flavigena					100 (100–100%)	26.4 (24–28.8%)	26.6 (24.3–29.1%)
C. terrae						100 (100–100%)	43.4 (40.9–45.9%)
C. xylanilytica							100 (100–100%)

DDH, DNA-DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; HSP, high-scoring segment pairs.

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Cellulomonas timonensis sp. nov., which contains the strain sn7T. This bacterial strain has been isolated from the faecal flora of a 38-year-old obese Frenchman.

Description of Cellulomonas timonensis sp. nov.

Cellulomonas timonensis (tim.o.nen'sis, L. masc. adj., timonensis, ‘of Timone,’ the name of the hospital where strain sn7T was first cultivated).

C. timonensis is a facultative anaerobic Gram-positive bacterium which is rod shaped with a mean diameter of 0.3 μm and a length of 1.3 μm. Optimal growth of strain sn7T occurs at 37°C in aerobic conditions. Colonies are white and smooth with an irregular diameter of 1.5 to 2 mm on 5% sheep's blood–enriched Colombia agar. Cells are sporulating and motile.

C. timonensis shows negative reactions for oxidase, cystine arylamidase, trypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase and α-fucosidase. Positive reactions were observed for catalase, esterase (C4), acid phosphatase, nitrate reduction, indole formation, glucose fermentation and esculin hydrolysis. Strain sn7T showed resistance to colistin, fosfomycin, oxacillin and trimethoprim/sulfamethoxazole. The fatty acids are mainly composed of 12-methyl-tetradecanoic acid (74%).

This strain exhibited a G + C content of 72.42% and a genome length of 4 057 828 bp. The 16S rRNA sequence and the whole genome shotgun sequence have been deposited in EMBL-EBI under accession numbers LN870311 and FCOT00000000, respectively. C. timonensis strain sn7T (=CSUR P2058 = DSM 100699) was isolated from a stool sample of an obese Frenchman.

Conflict of interest

None declared.