- Open Access
Non contiguous-finished genome sequence and description of Clostridium jeddahense sp. nov.
- Published: 15 June 2014
Abstract
Clostridium jeddahense strain JCDT (= CSUR P693 = DSM 27834) is the type strain of C. jeddahense sp. nov. This strain, whose genome is described here, was isolated from the fecal flora of an obese 24 year-old Saudian male (BMI=52 kg/m2). Clostridium jeddahense strain JCDT is an obligate Gram-positive bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,613,503 bp long genome (1 chromosome, no plasmid) exhibits a G+C content of 51.95% and contains 3,462 protein-coding and 53 RNA genes, including 4 rRNA genes.
Keywords
- Clostridium jeddahense
- genome
- culturomics
- taxonogenomics
Introduction
Clostridium jeddahense strain JCDT (=CSUR P693 = DSM 27834), is the type strain of Clostridium jeddahense sp. nov. This bacterium is a Gram-positive, anaerobic, spore-forming indole, positive bacillus that was isolated from the stool of an obese 24 year-old Saudian individual, as a part of a culturomics study as previously reported.
The usual parameters used to delineate a bacterial species include 16S rDNA sequence identity and phylogeny [1,2], genomic G + C content diversity, and DNA-DNA hybridization (DDH) [3,4]. Nevertheless, some limitations appeared notably because the cutoff values vary dramatically between species and genera [5]. The introduction of high-throughput sequencing techniques made genomic data for many bacterial species available [6]. We recently proposed a new method (taxono-genomics), which includes genomic data in a polyphasic approach to describe new bacterial species [6]. This strategy combines phenotypic characteristics, including MALDI-TOF MS spectrum, and genomic analysis [7–37].
Here, we present a summary classification and a set of features for C. jeddahense sp. nov. strain JCDT (=CSUR P693 = DSM 27834), together with the description of the complete genome sequencing and annotation. These characteristics support the circumscription of the species C. jeddahense.
The genus Clostridium was created in 1880 [38] and consists of obligate anaerobic rod-shaped bacilli able to produce endospores [38]. More than 200 species have been described to date (http://www.bacterio.cict.fr/c/clostridium.html). Members of the genus Clostridium are mostly environmental bacteria or associated with the commensal digestive flora of mammals. However, several are major human pathogens, including C. botulinum, C. difficile and C. tetani [38].
Classification and features
A consensus phylogenetic tree highlighting the position of Clostridium jeddahense strain JCDT relative to other type strains within the Clostridum genus. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using the maximum-likelihood method in the MEGA software package. Numbers at the nodes are the percentages of bootstrap values from 500 replicates that support the node. Clostridium ramosum was used as outgroup. The scale bar represents a 2% nucleotide sequence divergence.
Classification and general features of Clostridium jeddahense strain JCDT according to the MIGS recommendations [39]
MIGS ID | Property | Term | Evidence codea |
---|---|---|---|
Current classification | Domain Bacteria | TAS [40] | |
Phylum Firmicutes | |||
Class Clostridia | |||
Order Clostridiales | |||
Family Clostridiaceae | |||
Genus Clostridium | |||
Species Clostridium jeddahense | IDA | ||
Type strain JCDT | IDA | ||
Gram stain | Positive | IDA | |
Cell shape | Rod | IDA | |
Motility | Motile | IDA | |
Sporulation | Sporulating | IDA | |
Temperature range | Mesophile | IDA | |
Optimum temperature | 37°C | IDA | |
MIGS-6.3 | Salinity | Unknown | IDA |
MIGS-22 | Oxygen requirement | Anaerobic | IDA |
Carbon source | Unknown | IDA | |
Energy source | Unknown | IDA | |
MIGS-6 | Habitat | Human gut | IDA |
MIGS-15 | Biotic relationship | Free living | IDA |
Pathogenicity | Unknown | ||
Biosafety level | 2 | ||
MIGS-14 | Isolation | Human feces | |
MIGS-4 | Geographic location | Jeddah, Saudi Arabia | IDA |
MIGS-5 | Sample collection time | July 2013 | IDA |
MIGS-4.1 | Latitude | 21.422487 | IDA |
MIGS-4.1 | Longitude | 39.856184 | IDA |
MIGS-4.3 | Depth | Surface | IDA |
MIGS-4.4 | Altitude | 0 m above sea level | IDA |
Gram stain of Clostridium jeddahense strain JCDT
Transmission electron micrograph of C. jeddahense strain JCDT, taken using a Morgani 268D (Philips) at an operating voltage of 60kV.The scale bar represents 500 nm.
Properties | C. jeddahense | C. sporosphaeroides | C. cellulolyticum | C. leptum | C. senegalense | C. dakarense | C. beijerinckii | C. difficile |
---|---|---|---|---|---|---|---|---|
Cell diameter (µm) | 1.0 | 0.5–0.6 | 1.5 | 0.6–0.8 | 1.1 | 1.2 | 1.7 | 3.0 |
Oxygen requirement | Strictly anaerobic | Strictly anaerobic | Strictly anaerobic | Strictly anaerobic | Strictly anaerobic | Strictly anaerobic | Strictly anaerobic | Strictly anaerobic |
Gram stain | Positive | Positive | Positive | Positive | Positive | Positive | Variable | Variable |
Motility | Motile | Non Motile | Motile | Non Motile | Motile | Motile | Motile | Motile |
Endospore formation | + | + | + | + | + | + | + | + |
Indole | + | − | − | Na | − | + | Na | Na |
Production of | ||||||||
Alkaline phosphatase | + | Na | Na | Na | − | + | Na | Na |
Catalase | − | Na | − | Na | − | − | − | Na |
Oxidase | − | Na | Na | Na | − | − | Na | Na |
Nitrate reductase | − | − | − | Na | − | − | − | − |
Urease | − | − | − | Na | − | − | − | Na |
β-galactosidase | − | Na | Na | Na | − | − | Na | Na |
N-acetyl-glucosamine | − | Na | Na | Na | + | Na | Na | |
Acid from | ||||||||
L-Arabinose | − | − | Na | Na | Na | − | + | − |
Ribose | − | − | Na | W | Na | − | − | − |
Mannose | − | − | Na | − | Na | − | + | + |
Mannitol | − | − | Na | − | Na | − | + | + |
Sucrose | − | -Na | Na | W | Na | − | + | + |
D-glucose | − | Na | Na | Na | Na | + | + | Na |
D-fructose | − | Na | Na | Na | Na | − | + | + |
D-maltose | − | Na | Na | Na | Na | + | + | − |
D-lactose | − | Na | Na | Na | Na | − | + | − |
G+C content (%) | 52 | 41 | 51 | 26.8 | 27.98 | 28 | 28 | |
Habitat | Human gut | Environment | Compost | Human gut | Human gut | Human gut | Human gut | Human gut |
C. jeddahense is susceptible to amoxicillin, amoxicillin-clavulanate, imipenem, metronidazole, doxycycline, rifampicin, vancomycin but resistant to ceftriaxone, ciprofloxacin and trimethoprim-sulfamethoxazole. The comparisons with other Clostridium species are summarized in Table 2.
Reference mass spectrum from C. jeddahense strain JCDT. Spectra from 12 individual colonies were compared and a reference spectrum was generated.
Gel view comparing C. jeddahense strain JCDT to other Clostridium species. The gel view displays the raw spectra of loaded spectrum files arranged as a pseudo-electrophoretic gel. 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 grey scale scheme code. The grey scale bar on the right y-axis indicates the relation between the shade of grey a peak is displayed with and the peak intensity in arbitrary units. Species names are shown on the left.
Genome sequencing information
Genome project history
Project information
MIGS ID | Property | Term |
---|---|---|
MIGS-31 | Finishing quality | High-quality draft |
MIGS-28 | Libraries used | Paired end and Mate pair |
MIGS-29 | Sequencing platform | MySeq Illumina |
MIGS-31.2 | Fold coverage | 94.91× |
MIGS-30 | Assemblers | Newbler |
MIGS-32 | Gene calling method | PRODIGAL |
Genbank Date of Release | February 12, 2014 | |
Genbank project ID | CBYL00000000 | |
MIGS-13 | Project relevance | Study of the human gut microbiome |
Growth conditions and DNA isolation
C. jeddahense sp. nov., strain JCDT (= CSUR P693 = DSM 27834) was grown on 5% sheep blood-enriched Columbia agar (BioMerieux) at 37°C in anaerobic atmosphere. Bacteria grown on three Petri dishes were harvested and resuspended in 4x100µL of TE buffer. Then, 200 µL of this suspension was diluted in 1ml TE buffer for lysis treatment that included a 30-minute incubation with 2.5 µg/µL lysozyme at 37°C, followed by an overnight incubation with 20 µg/µL proteinase K at 37°C. Extracted DNA was then purified using 3 successive phenol-chloroform extractions and ethanol precipitation at −20°C overnight. After centrifugation, the DNA was resuspended in 160 µL TE buffer.
Genome sequencing and assembly
Genomic DNA of Clostridium jeddahense was sequenced on a MiSeq sequencer (Illumina, Inc, San Diego CA 92121, USA) with 2 applications: paired end and mate pair. The paired end and the mate pair strategies were barcoded in order to be mixed respectively with 14 other genomic projects constructed according the Nextera XT library kit (Illumina) and 11 others projects with the nextera Mate pair kit (Illumina).
The gDNA was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 11.1 ng/µL and dilution was performed such that 1ng of each strain’s gDNA was used to construct the paired end library. The “tagmentation” step fragmented and tagged the DNA.Then limited cycle PCR amplification completed the tag adapters and introduced dual-index barcodes. After purification on Ampure beads (Life Technolgies, Carlsbad, CA, USA), the libraries were normalized on specific beads according to the Nextera XT protocol (Illumina). Normalized libraries are pooled into a single library for sequencing on the MiSeq. The pooled single strand library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and paired-end sequencing with dual index reads was performed in a single 39-hour run at a 2x250 bp read length. Within this pooled run, the index representation was determined to be 7.3%. Total information of 5.3 Gbases was obtained from a 574 K/mm2 density with 95.4% (11,188,000 clusters) of the clusters passing quality control (QC) filters. From the genome sequencing process, the 753,292 produced Illumina reads for Clostridium jeddahense were filtered according to the read qualities.
The mate pair library was constructed from 1 µg of genomic DNA using the Nextera Mate Pair Illumina guide. The genomic DNA sample is simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) with a DNA7500 labchip. The DNA fragments range in size from 1 kb up to 11 kb with a mean size of 7kb. No size selection was performed and 600 ng tagmented fragments were circularized. The larger circularized DNA molecules were physically sheared to smaller sized fragments with a mean size of 625 bp on the Covaris device S2 in microtubes (Woburn, MA, USA).The library’s profile and the quantitation were visualized on a High Sensitivity Bioanalyzer LabChip. The libraries were normalized to 2 nM and pooled. After a denaturation step and dilution at 10 pM the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing run was performed in a single 39-hour run at a 2x250 bp read length.
Total information of 3.9 Gb was obtained from a 399 K/mm2 density with 97.9% (7,840,000 clusters) of the clusters passing quality control (QC) filters. Within this pooled run, the index representation for Clostridium jeddahense was determined to be 6.54%.
From this genome sequencing process, the 501,426 produced Illumina reads for Clostridium jeddahense were filtered according to the read qualities.
Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [56] with default parameters. However, the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank [57] and Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAs and rRNAs were predicted using the tRNAScan-SE [58] and RNAmmer [59] tools, respectively. Signal peptides and numbers of transmembrane helices were predicted using SignalP [60] and TMHMM [61], respectively. Mobile genetic elements were predicted using PHAST [62] and RAST [63]. 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. Such parameter thresholds have already been used in previous work to define ORFans. Artemis [64] and DNA Plotter [65] were used for data management and visualization of genomic features, respectively. The Mauve alignment tool (version 2.3.1) was used for multiple genomic sequence alignment [66].
To estimate the mean level of nucleotide sequence similarity at the genome level between C. jeddahense and 7 other members of the genus Clostridium, we used the Average Genomic Identity Of gene Sequences (AGIOS) home-made software [6]. Briefly, this software combines the Proteinortho software [67] for detecting orthologous proteins between pairs of genomes, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm. C. jeddahense strain JCDT was compared to C. senegalense strain JC122, C. dakarense strain FF1, Clostridium beijerinckii strain NCIMB 8052, C. difficile strain B1, Clostridium cellulolyticum strain H10, Clostridium leptum strain DSM 753, and Clostridium sporosphaeroides strain DSM 1294 (see Table 6B).
Genome properties
Graphical circular map of the chromosome. From the outside in: open reading frames oriented in the forward (colored by COG categories) direction, open reading frames oriented in the reverse (colored by COG categories) direction, RNA operon (red), and tRNAs (green), GC content plot, and GC skew (purple: negative values, olive: positive values).
Nucleotide content and gene count levels of the genome
Attribute | Value | % of totala |
---|---|---|
Genome size (bp) | 3,613,503 | |
DNA G+C content (bp) | 1,877,214 | 51.95 |
DNA Coding region (bp) | 3,152,277 | 87.23 |
Number of replicons | 1 | |
Extra chromosomal element | 0 | |
Total genes | 3,515 | 100 |
RNA genes | 53 | 1.51 |
Protein-coding genes | 3,462 | 98.49 |
Genes with function prediction | 2,193 | 87.19 |
Genes assigned to COGs | 2,515 | 71.55 |
Genes with peptide signals | 135 | 3.84 |
Genes with transmembrane helices | 887 | 25.23 |
Number of genes associated with the 25 general COG functional categories
Code | Value | % agea | Description |
---|---|---|---|
J | 154 | 4.45 | Translation |
A | 0 | 0 | RNA processing and modification |
K | 296 | 8.55 | Transcription |
L | 138 | 3.98 | Replication, recombination and repair |
B | 1 | 0.03 | Chromatin structure and dynamics |
D | 24 | 0.69 | Cell cycle control, mitosis and meiosis |
Y | 0 | 0 | Nuclear structure |
V | 73 | 2.11 | Defense mechanisms |
T | 156 | 4.5 | Signal transduction mechanisms |
M | 116 | 3.35 | Cell wall/membrane biogenesis |
N | 62 | 1.79 | Cell motility |
Z | 0 | 0 | Cytoskeleton |
W | 0 | 0 | Extracellular structures |
U | 48 | 1.38 | Intracellular trafficking and secretion |
O | 66 | 1.9 | Posttranslational modification, protein turnover, chaperones |
C | 154 | 4.45 | Energy production and conversion |
G | 237 | 6.84 | Carbohydrate transport and metabolism |
E | 328 | 9.47 | Amino acid transport and metabolism |
F | 56 | 1.61 | Nucleotide transport and metabolism |
H | 92 | 2.66 | Coenzyme transport and metabolism |
I | 85 | 2.45 | Lipid transport and metabolism |
P | 164 | 4.74 | Inorganic ion transport and metabolism |
Q | 53 | 1.53 | Secondary metabolites biosynthesis, transport and catabolism |
R | 346 | 10 | General function prediction only |
S | 195 | 5.63 | Function unknown |
- | 947 | 27.35 | Not in COGs |
Genome comparison with other Clostridium genomes
Genomic comparison of C. jeddahense with 7 other Clostridium species†.
Species | Strain | Genome accession number | Genome size (Mb) | G+C content |
---|---|---|---|---|
C. jeddahense | JCDT | CBYL00000000 | 3.61 | 51.95 |
C. sporosphaeroides | DSM 1294 | ARTA01000000 | 3.17 | 53.5 |
C. cellulolyticum | H10 | NC_011898 | 4.07 | 37.4 |
C. dakarense | DSM 27086 | CBTZ010000000 | 3.73 | 27.98 |
C. difficile | B1 | NC_017179 | 4.46 | 28.4 |
C. leptum | DSM 753 | ABCB02000000 | 3.27 | 50.2 |
C. senegalense | DSM 25507 | CAEV01000001 | 3.89 | 26.8 |
C. beijerincki | NCIMB 8052 | NC_009617 | 6.0 | 29.0 |
Genomic comparison of C. jeddahense with 7 other Clostridium species†
C. jeddahense | C. sporosphaeroides | C. cellulolyticum | C. dakarense | C. difficile | C. leptum | C. senegalense | C. beijerincki | |
---|---|---|---|---|---|---|---|---|
C. jeddahense | 3,462 | 1,573 | 876 | 816 | 847 | 1,030 | 770 | 1,044 |
C. sporosphaeroides | 91.97 | 2,951 | 854 | 776 | 819 | 1,016 | 745 | 1,015 |
C. cellulolyticum | 61.60 | 60.62 | 3,923 | 806 | 851 | 754 | 814 | 946 |
C. dakarense | 57.30 | 56.34 | 65.70 | 5,020 | 1,271 | 665 | 1,110 | 1,142 |
C. difficile | 57.56 | 56.80 | 65.65 | 77.74 | 3,390 | 714 | 1,098 | 1,171 |
C. leptum | 67.98 | 68.07 | 61.94 | 58.55 | 58.84 | 3,591 | 651 | 780 |
C. senegalense | 57.52 | 56.73 | 65.71 | 70.18 | 69.41 | 58.72 | 3,704 | 1,125 |
C. beijerincki | 58.63 | 57.95 | 65.93 | 68.98 | 68.48 | 59.58 | 71.37 | 3,818 |
The draft genome of C. jeddahense (3.61 Mb) is larger than C. sporosphaeroides and C. leptum (3.17 and 3.27 Mb respectively) but smaller than C. beijerincki, C. cellulolyticum, C. difficile, C. senegalense and C. dakarense (6.0, 4.07, 4.46, 3.89, 3.73 Mb respectively). It exhibits a higher G+C content than all other compared genome except C. sporosphaeroides (53.5%). C. jeddahense has a higher gene content (3,462) than C. sporosphaeroides, C. difficile (2,951 and 3,390 respectively) but smaller than C. leptum, C. beijerincki, C. cellulolyticum, C. senegalense and C. dakarense (3,591, 3,818, 3,923, 3,704, 5,020 respectively). C. jeddahense shared 1,573, 876, 816, 847, 1,030, 770 and 1,044 orthologous genes with C. sporosphaeroides, C. cellulolyticum, C. dakarense, C. difficile, C. leptum, C. senegalense and C. beijerincki respectively.
Distribution of predicted genes of C. jeddahense and 7 other Clostridium species into COG categories. C.jdm= C. jeddahense, C.spo= C. sporosphaeroides, C. lep= C. leptum, C.bej = C. beijerinckii, C. cel = C. cellulolyticum, C. diff = C. difficile, C. sen = C. senegalense, C. dak = C. dakarense.
Conclusion
On the basis of phenotypic, phylogenetic and genomic analyses (taxono-genomics), we formally propose the creation of Clostridium jeddahense sp. nov. that contains strain JCDT. This strain was isolated from the fecal flora of an obese 24 year-old Saudian individual living in Jeddah.
Description of C. jeddahense sp. nov.
Clostridium jeddahense (jed.dah..en’.se L.gen. neutr. n. combination of Jeddah, the city in Saudi Arabia where the specimen was obtained from an obese Saudian patient sample.) Transparent colonies were 0.2 to 0.3 mm in diameter on blood-enriched agar. C. jeddahense is a Gram-positive, obligate anaerobic, endospore-forming bacterium with a mean diameter of 1 µm. Optimal growth on axenic medium was observed at 37°C.
C. jeddahense is catalase negative and oxidase negative. Alkaline phosphatase, arginine arylamidase, proline arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase and serine arylamidase activities were positive. Arginine dihydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, α-arabinosidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, α-fucosidase, reduction of nitrate, leucyl glycine arylamidase, fermentation of mannose and raffinose, urease, β-galactosidase-6-phosphatase, β-glucuronidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase and tyrosine arylamidase activities were negative. Asaccharolytic. Positive for indole. Cells are susceptible to amoxicillin, amoxicillin-clavulanate, imipenem, metronidazole, doxycycline, rifampicin, vancomycin but resistant to ceftriaxone, ciprofloxacin and trimethoprim-sulfamethoxazole.
The G+C content of the genome is 51.95%. The 16S rDNA and genome sequences are deposited in GenBank under accession numbers HG726040 and CBYL00000000, respectively. The type strain is JCDT (= CSUR P693 = DSM 27834).
Declarations
Acknowledgements
This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, under grant No. (1-141/1433 HiCi). The authors, therefore, acknowledge technical and financial support of KAU. The authors thank the Xegen Company (www.xegen.fr) for automating the genomic annotation process.
Authors’ Affiliations
References
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