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Genome sequence and description of Corynebacterium ihumii sp. nov.

Standards in Genomic Sciences volume 9, pages 1128–1143 (2014)Cite this article

Abstract

Corynebacterium ihumii strain GD7T sp. nov. is proposed as the type strain of a new species, which belongs to the family Corynebacteriaceae of the class Actinobacteria. This strain was isolated from the fecal flora of a 62 year-old male patient, as a part of the culturomics study. _Corynebacterium ihumii is a Gram positive, facultativly anaerobic, nonsporulating bacillus. Here, we describe the features of this organism, together with the high quality draft genome sequence, annotation and the comparison with other member of the genus Corynebacteria. C. ihumii genome is 2,232,265 bp long (one chromosome but no plasmid) containing 2,125 protein-coding and 53 RNA genes, including 4 rRNA genes. The whole-genome shotgun sequence of _Corynebacterium ihumii strain GD7T sp. nov has been deposited in EMBL under accession number GCA_000403725.

Introduction

Corynebacterium ihumii strain GD7T sp. nov. (= CSUR P902, = DSM 45751) is the type strain of Corynebacterium ihumii strain GD7T sp. nov. This bacterium is a Gram-positive, facultativly anaerobic, non spore-forming, non-motile bacillus that was isolated from the stool of a 62 year-old French male who was admitted to the intensive care unit in the Timone Hospital, Marseille, France, for respiratory distress. This strain was isolated as a part of “culturomics” project whose scope is to cultivate all species within human feces [1,2].

The current classification of prokaryotes is based on a combination of phenotypic and genotypic characteristics [3,4] that include 16S rRNA gene phylogeny and nucleotide sequence similarity, G + C content and DNA-DNA hybridization (DDH). Despite being considered as a “gold standard” these genotypic tools exhibit several drawbacks that are overcome by newer sequencing methods [5,6]. Because of the rapidly declining cost of sequencing, the number of sequenced bacterial genomes rapidly increased (almost 7,000 to date [7]). Hence, we recently proposed to incorporate genomic information among criteria used for the description of new bacterial species [8–29].

Corynebacteria are Gram-positive bacteria that belong to the phylum Actinobacteria and have a high G+C content. They are found in diverse ecological niches such as soil, clinical specimens, cheese smear, vegetables, sewage etc. The genus Corynebacterium was created by Lehmann and Neumann in 1896 [30] which currently comprises 112 distinct species and 11 subspecies [31]. Many Corynebacterium species are involved in human and animal diseases and include C. diphtheriae [32], C. jeikeium, C. urealyticum, C. striatum, C. pseudotuberculosis, and C. ulcerans [33]. Others have industrial applications for amino acid production like C. glutamicum [34].

Here, we present a summary classification and a set of features for Corynebacterium ihumii strain GD7T sp. nov. (=CSUR P902, =DSM 45751) together with the description of the genome sequencing and annotation.

Classification and Features

A stool sample was collected from a 62 year-old male admitted to the intensive care unit of the Timone Hospital in Marseille, France. The patient gave a written informed consent for the study. The study was approved by the Ethics Committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France, under agreement number 09-022. The fecal specimen was preserved at −80°C after collection. Strain GD7T (Table 1) was isolated in January 2012 by cultivation on PVX agar (BioMerieux, Marcy l’Etoile, France) in aerobic condition with 5% CO2 at 37°C, after 21 days of incubation.

Table 1. Classification and general features
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To understand the phylogenetic relationships of C. ihumii GD7T, we constructed a 16S rRNA-based neighbor joining tree with 90 Corynebacterium species (Figure 1). The 16S rRNA sequence similarity among Corynebacterium species ranged from 82.9 to 99.60%. Strain GD7T exhibited a highest 16S rRNA sequence similarity of 99.1% with C. pilbarense. This value, although higher 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 [4], is in the range of values observed within the genus Corynebacterium.

Figure 1.
figure 1

Phylogenetic tree highlighting the position of Corynebacterium ihumii strain GD7T relative to other type strains within the Corynebacterium genus. GenBank accession numbers are indicated for each strain. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the neighbor-joining method within the MEGA software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1,000 times to generate a majority consensus tree. Mycobacterium tuberculosis was used as an outgroup. The scale bar represents a 2% nucleotide sequence divergence.

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Various growth temperatures (25, 30, 37, 45 and 56°C) were tested. Growth occurred between 30 and 45°C on blood-enriched Columbia agar (BioMérieux), with the optimal growth being obtained at 37°C. Growth of the strain was tested under anaerobic and microaerophilic conditions using the GENbag Anaer and GENbag microaer systems, respectively (BioMérieux), and under aerobic conditions, with or without 5% CO2. Optimal growth was achieved aerobically, but cell growth was also observed under microaerophilic and anaerobic conditions. The motility test was negative and the cells were nonsporulating. Colonies were white and granular with a diameter of 0.5 mm on blood-enriched Columbia agar (BioMérieux). Gram staining showed short Gram-positive rods (Figure 2). By electron microscopy, cells grown on agar had a mean length and diameter of 1.26 µm (range 1.1 – 1.4) and 0.7 µm (range 0.6–0.85), respectively (Figure 3). Strain GD7T was catalase positive and oxidase negative. Using the API ZYM system (BioMérieux), positive reactions were observed for alkaline phosphatase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. Negative reactions were observed for esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N actetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using the API CORYNE system (BioMérieux), positive reactions were observed for pyrazinamidase, alkaline phosphatase, and glucose and ribose fermentation. Negative reactions were observed for reduction of nitrates, pyrolidonyl arylamidase; β-glucuronidase, β-galactosidase, α-glucosidase N-acetyl-β-glucosaminidase, β-glucosidase, urease, gelatin hydrolysis, fermentation of xylose, mannitol, maltose, lactose, saccharose and glycogen. Using an API 50CH strip (BioMérieux), positive reactions were observed for fermentation of L-arabinose, D-ribose, D-xylose, methyl-βD xylopranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, methyl-αD-xylopranoside, methyl-αD-glucopranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-maltose, D-lactose, D-mellibiose, D-saccharose, D-trehalose, inulin, D-raffinose, amidon, glycogen and D-lyxose. Negative reactions were observed for fermentation of glycerol, erythritol, D-arabinose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, esculin ferric citrate, D-melezitose, D-xylitol, gentiobiose, D-turanose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-ketogluconate. Table 2 summarizes the differential phenotypic characteristics of C. ihumii, C. pilbarense, C. coylae, C. glaucum, and C. mucifaciens. C. ihumii strain GD7T was susceptible to amoxicillin, amoxicillin-clavulanic acid, ceftriaxone, imipenem, doxycycline, vancomycin, erythromycin, rifampicin, trimethoprim/sulfamethoxazole and ciprofloxacine whereas it was resistant to metronidazole.

Figure 2.
figure 2

Gram staining of C. ihumii strain GD7T

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Figure 3.
figure 3

Transmission electron microscopy of C. ihumii strain GD7T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 1 µm.

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Table 2. Differential characteristics of C. ihumii sp. nov. strain GD7T, C. pilbarense, C. coylae, C. glaucum and C. mucifaciens.
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Table 2. Project information
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Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was peformed as previously described [46] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). The spectra from twelve isolated distinct GD7T colonies were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 4,706 bacteria, including spectra from validated Corynebacterium species, that were part of the reference data contained in the BioTyper database. The presumptive identification and discrimination of the tested species from those in the database was interpreted as follows: a score > 2 with a validly published species enabled the identification at the species level; a score > 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification. For strain GD7T, no significant score was obtained, suggesting that GD7 isolate was not a member of any known species or genus (Figures 4 and 5).

Figure 4.
figure 4

Reference mass spectrum from C. ihumii strain GD7T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

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Figure 5.
figure 5

Gel view comparing C. ihumii sp. nov. strain GD7T (= CSUR P902 = DSM 45751) to other members of the Corynebacterium genus. The gel view displays the raw spectra of all loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a Gray scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units.

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Genome sequencing information

Genome project history

As part of a ‘culturomics’ study of the human digestive flora, this organism was isolated and selected for sequencing on the basis of its phenotypic differences, phylogenetic position and 16S rRNA and rpoB sequence similarity to other members of the genus Corynebacterium [1,2]. It is the first sequenced genome of C. ihumii sp. nov. The GenBank Bioproject number is PRJEB646 and consists of 41 large contigs in 5 scaffolds. Table 3 shows the project information and its association with MIGS version 2.0 compliance [47].

Growth conditions and DNA isolation

C. ihumii sp. nov. strain GD7T strain was cultivated in Columbia broth (BioMérieux) at 37°C. Chromosomal DNA was extracted from 50mL of culture, following centrifugation at 4°C at 2000 xg for 20 min. Cell pellets were resuspended in 1 mL Tris/EDTA/NaCl [10mM Tris/HCl (pH7.0), 10 mM EDTA (pH8.0), and 300 mM NaCl] and re-centrifuged under the same conditions. The pellets were then re-suspended in 200µL TE buffer and proteinase K and kept overnight at 37°C for cell lysis. DNA purification with phenol/chloroform/isoamylalcohol (25:24:1) was followed by an overnight precipitation with ethanol at −200C. Then, the DNA was resuspended in 200 µL TE buffer. DNA concentration was 18.3ng/µl as measured using the Genios Tecan fluorometer and the Quant-it Picogreen kit (Invitrogen).

Genome sequencing and assembly

The 454 GS-FLX Titanium paired-end protocol (Roche, Meylan, France) was used for the library construction of C. ihumii strain GD7T which was then pyrosequenced. Briefly, 3.7µg of purified chromosomal DNA was mechanically fragmented on the Covaris device (KBioScience-LGC Genomics, Middlesex, UK) through miniTUBE-Red with an enrichment size at 5kb. The DNA fragmentation was visualized through the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 2.5 kb. Circularization and nebulization were performed on 100ng of the fragmented DNA and generated an optimal pattern of 443 bp. This was followed by 17 PCR amplification cycles followed by double size selection. The single stranded paired-end library was then quantified using Quant-it Ribogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 207 pg/µL. The library concentration equivalence was calculated as 8.57E+08 molecules/µL. The library was stored at −20°C until further use. The shotgun library was clonally amplified with 0.5cpb and 1cpb in 2 emPCR reactions for each condition, using the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yield of the shotgun emPCR reactions was 5.27 and 7.56% respectively for the two kinds of paired-end emPCR reactions according to the quality expected (range of 5 to 20%) from the Roche procedure. The library was loaded on the 1/4 region of a GS Titanium PicoTiterPlate (PTP Kit 70x75, Roche) and pyrosequenced with the GS Titanium Sequencing Kit XLR70 and the GS FLX Titanium sequencer (Roche). The run was performed overnight and analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 186,723 passed filter wells were obtained and generated 69.4Mb with a length average of 371 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40bp as overlap. The assembly lead to 5 scaffolds and 41 large contigs (>1500bp) and generated a genome size of 2,232,265 bp which corresponds to a coverage of 30.84× genome equivalent.

Genome annotation

Open Reading Frames (ORFs) prediction was performed using Prodigal [48] with default parameters. The predicted ORFs were excluded if they spanned a sequencing gap region. Functional assessment of protein sequences was carried out by comparing them with sequences in the GenBank [49] and Clusters of Orthologus Groups (COG) databases using BLASTP. tRNAs, rRNAs, signal peptides and transmembrane helices were identified using tRNAscan-SE 1.21 [50], RNAmmer [51], SignalP [52] and TMHMM [53], respectively. ORFans were identified if their BLASTP E-value was lower than 1e−3 for alignment lengths greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e−5 [54]. PHAST was used to identify, annotate and graphically display prophage sequences within bacterial genomes or plasmids [55]. Artemis [56] was used for data management whereas DNA Plotter [57] was used for visualization of genomic features. In-house perl and bash scripts were used to automate these routine tasks.

To estimate the mean level of nucleotide sequence similarity at the genome level between C. ihumii and another 42 members of the genus Corynebacterium, we used the Average Genomic Identity of Orthologous gene Sequences (AGIOS) home-made pipeline. Briefly, this pipeline combines the Proteinortho software (with the following parameters: e-value 1e−5, 30% of identity, 50% coverage and algebraic connectivity of 50%) [58] for detecting orthologous proteins between genomes compared pairwise, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm.

Genome properties

The genome of C. ihumii sp. nov. strain GD7T is 2,232,265 bp long (1 chromosome in 5 scaffolds, no plasmid) with a 65.1% GC content (Table 4, Figure 6). Of the 2,182 predicted genes, 2,125 were protein-coding genes and 57 were RNAs (53 tRNA and 4 rRNA genes). A total of 1,562 genes (71.58%) were assigned a putative function. Four hundred and twenty-two genes (19.8%) were annotated as hypothetical proteins, and 126 genes ORFans (5.9%). The distribution of genes into COGs functional categories is presented in Table 5. The properties and statistics of the genome are summarized in Tables 4 and 5. A quick search with PHAST revealed that C. ihumii harbors an incomplete bacteriophage.

Figure 6.
figure 6

Graphical circular map of the chromosome. From the outside in, the outer two circles show open reading frames oriented in the forward and reverse directions (colored by COG categories), 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 the GC skew, purple and olive indicating negative and positive values, respectively.

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Table 4. Nucleotide content and gene count levels of the genome
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Table 4. Number of genes associated with the 25 general COG functional categories
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Comparative genomics

Presently there are more than 75 genomic sequences (finished or draft) available for Corynebacterium species in GenBank. Here, we have compared C. ihumii sp. nov. strain GD7T with 41 finished or draft genome sequences from 25 Corynebacterium species. Table 6 shows a comparison of genome size, GC%, coding-density, and numbers of proteins for the compared Corynebacterium genomes. C. ihumii had a smaller genome than all other compared genomes except that of C. urealyticum strain DSM 7111. AGIOS values identities ranged from 65.23 to 80.59% among Corynebacterium species, and from 97.97 to 99.99% within Corynebacterium species (Supplementary Table). By comparison with other species, C. ihumii exhibited AGIOS values ranging from 67.15% with C. pseudotuberculosis to 76.30% with C. lipophiloflavum, thus confirming its new species status.

Table 6. Main characteristics of Corynebacterium genomes compared to that of C. ihumii strain GD7T.
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Figure 7 shows the comparison of gene distribution into COG categories of C. ihumii with C. glutamicum strain ATCC 13032, C. efficiens YS 314, C. jeikeium K411, C. aurimucosum ATCC 700975, C. kroppenstedtii DSM 44385, C. resistens DSM 45100, C. variabile DSM 44702, C. diphtheriae BH8, C. pseudotuberculosis 1002, C. ulcerans 0102, C. halotolerans YIM 70093 and C. callunae DSM 20147. The overall COG distribution is similar, except C. variabile for category L genes.

Figure 7.
figure 7

Distribution of functional classes of predicted genes of Corynebacterium ihumii strain GD7T (colored in thick red line) along with other Corynebacterium genomes according to the clusters of orthologous groups of proteins.

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Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Corynebacterium ihumii sp. nov. which contains strain GD7T (= CSUR P902 = DSM 45751). This bacterium was isolated from the fecal flora of a 62 year-old male admitted in intensive care unit for respiratory distress.

Description of Corynebacterium ihumii strain GD7T sp. nov.

Colonies are white and granular with a 0.5 mm diameter on blood-enriched Columbia agar. Cells are rod-shaped with a mean length and diameter of 1.26 µm (range 1.1 – 1.4) and 0.7 µm (range 0.6–0.85), respectively. Growth is observed between 30 and 45°C, with optimal growth obtained at 37°C on blood-enriched Columbia agar. Optimal growth is achieved aerobically, but cell growth is also observed under microaerophilic and anaerobic conditions. Cells stain Gram-positive, are nonmotile and nonsporulating. Catalase is positive, oxidase is negative. Using the API ZYM system, positive reactions are observed for alkaline phosphatase, leucine arylamidase, valine arylamidase, cystin arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. Negative reactions are observed for esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N actetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using the API CORYNE system, positive reactions are observed for pyrazinamidase, alkaline phosphatase, and glucose and ribose fermentation. Negative reactions are observed for reduction of nitrates, pyrolidonyl arylamidase; β-glucuronidase, β-galactosidase, α-glucosidase N-acetyl-β-glucosaminidase, β-glucosidase, urease, gelatin hydrolysis, fermentation of xylose, mannitol, maltose, lactose, saccharose and glycogen. Using the API 50CH system, positive reactions are observed for fermentation of L-arabinose, D-ribose, D-xylose, methyl-βD xylopranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, methyl-αD-xylopranoside, methyl-αD-glucopranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-maltose, D-lactose, D-mellibiose, D-saccharose, D-trehalose, inulin, D-raffinose, amidon, glycogen and D-lyxose. Negative reactions are observed for fermentation of glycerol, erythritol, D-arabinose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, esculin ferric citrate, D-melezitose, D-xylitol, gentiobiose, D-turanose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-ketogluconate. Cells are susceptible to amoxicillin, amoxicillin-clavulanic acid, ceftriaxone, imipenem, doxycycline, vancomycin, erythromycin, rifampicin, trimethoprim/sulfamethoxazole and ciprofloxacine but was resistant to metronidazole. The G+C content of the genome is 65.1%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JX424769 and CAVS000000000, respectively.

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Acknowledgements

The authors thank the Xegen Company for automating the genomic annotation process. This study was funded by the Mediterranee-Infection foundation.

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Authors and Affiliations

  1. URMITE, UM63, CNRS7278, IRD198, Inserm1095, IHU Méditerranée-Infection, Aix-Marseille Université, Faculté de médecine, France

    Roshan Padmanabhan, Grégory Dubourg, Jean-Christophe Lagier, Carine Couderc, Caroline Michelle, Didier Raoult & Pierre-Edouard Fournier

  2. Special Unit Agents, King Fahd Medical Research Center, King Abdul Aziz University, Jeddah, Saudi Arabia

    Didier Raoult

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Correspondence to Pierre-Edouard Fournier.

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Padmanabhan, R., Dubourg, G., Lagier, JC. et al. Genome sequence and description of Corynebacterium ihumii sp. nov.. Stand in Genomic Sci 9, 1128–1143 (2014). https://doi.org/10.4056/sigs.5149006

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Keywords

Environmental Microbiome

ISSN: 2524-6372