Non-contiguous finished genome sequence and description of Kurthia senegalensis sp. nov.
© The Author(s) 2014
Published: 15 June 2014
Kurthia senegalensis strain JC8ET sp. nov. is the type strain of K. senegalensis sp. nov., a new species within the genus Kurthia. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. K. senegalensis is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,975,103 bp long genome contains 2,889 protein-coding genes and 83 RNA genes, including between 4 and 6 rRNA genes.
KeywordsKurthia senegalensis Firmicutes capsule ]flagella culturomics
Kurthia senegalensis strain JC8ET (CSUR P138T = DSM 24641T) is the type strain of K. senegalensis sp. nov. This bacterium is a Gram-positive strictly aerobic rod, capsulated, motile by peritrichous flagella and was isolated from the stool of a healthy Senegalese patient as part of a “culturomics” study aiming at cultivating individually all species within human feces .
Presently, “the gold standard method” to define a bacterial species is DNA-DNA hybridization (DDH) . But this method is time consuming and the inter-laboratory reproducibility is poor. So, with the development of PCR and sequencing methods, 16S rRNA gene sequence comparison is often the preferred approach for recognizing a new taxon when a gene sequence similarity less than 97% is found . To make descriptions more complete, phenotypic criteria (morphology, biochemical tests, growth conditions, chemotaxonomy) have to be included to characterize a prokaryote strain . Fortunately, sequencing whole prokaryote genomes is now possible for more laboratories, and descriptions of sequencing protocols should be included in all species descriptions. Such activity would supplant the need for most other methods used during genome annotation, and new bioinformatics methods based on genome-to-genome comparison have been proposed to replace the DDH approach .
Here we present a summary classification and a set of features for K. senegalensis sp. nov. strain JC8ET together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species K. senegalensis.
Kurth described Bacterium zopfii, isolated from the intestinal contents of chickens, which became later the first species of the genus Kurthia, K. zopfii. The genus Kurthia was created in 1885 by Trevisan  in honor of Kurth. The name Kurthia was first published in the seventh edition of Bergey’s Manual of Determinative Bacteriology . Currently, the genus includes 4 species: K. zopfii, K. gibsonii , K. sibirica  and K. massiliensis . The bacteria are included in the Firmicutes phylum, in the Planococcaceae family.
Classification and features
Classification and general features of Kurthia senegalensis strain JC8ET 
Species Kurthia senegalensis
Type strain JC8ET
Motile by peritrichous flagella
Growth in BHI medium + 2% NaCl
Sample collection time
51 m above sea level
Six different growth temperatures (25, 30, 37, 45, 50 and 55°C) were tested. Growth occurred between 25°C and 50°C, and optimal growth was observed between 30°C and 50°C. Growth of the strain was tested under an aerobic atmosphere, in the presence of 5% CO2, and also in anaerobic and microaerophilic atmospheres which were created using GENbag anaer and GENbag microaer (bioMérieux), respectively. The strains were aerobic and also grew under microaerophilic conditions and in the presence of 5% CO2 but did not grow in an anaerobic atmosphere. The NaCl concentrations allowing growth of strain JC8ET, were determined on DifcoTMBrain Heart Infusion Agar plates (Becton Dickinson). The powder was supplemented with NaCl (Euromedex) to obtain the tested concentrations (0.5, 1, 2, 3, 5 10, 15%, w/v). Growth occurred between 0.5–5% NaCl but the optimum growth was between 0.5–2% NaCl.
Growth in the range of pH 5.0–10.0 was tested using BBLTM Brain Heart Infusion (Becton Dickinson). Final pH was adjusted with HCl or NaOH solution. Growth occurred between pH 5–9.
Diagnostic traits differentiating five Kurthia species.
potassium gluconate assimilation
capric acid assimilation
trisodium citrate assimilation
esterase lipase (C8)
Analysis of respiratory quinones by HPLC was carried out by the Identification Service and Dr Brian Tindall, DSMZ, Braunschweig, Germany. Respiratory lipoquinones were extracted from 100 mg of freeze dried cell material as described by Tindall [26,27]. Respiratory lipoquinones were separated into their different classes (menaquinones and ubiquinones) by thin layer chromatography on silica gel, using hexane:tert-butylmethylether (9:1 v/v) as solvent. UV absorbing bands corresponding to menaquinones or ubiquinones were removed from the plate and further analyzed by HPLC at 269 nm. The respiratory quinones were MK-7 (100%) for strain JC8ET. Preparation and determination of cellular fatty acids were carried out by following the procedures given for the Sherlock Microbial identification System (MIDI). The major fatty acids were C15:0 iso 50.75% and C15:0 anteiso 24.05%. Polar lipids were extracted from 100 mg of freeze dried cell material using a chloroform:methanol:0.3% aqueous NaCl mixture 1:2:0.8 (v/v/v) (modified after ). The extraction solvent was stirred overnight and the cell debris pelleted by centrifugation. Polar lipids were recovered into the chloroform phase by adjusting the chloroform:methanol:0.3% aqueous NaCl mixture to a ratio of 1:1:0.9 (v/v/v). Polar lipids were separated as previously described . The polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipids 1 and 2, unidentified aminophospholipid and glycolipid. The peptidoglycan of the bacteria was isolated as described by Schleifer . The determination was carried out as previously described [30,31] with the modification that TLC on cellulose was applied instead of paper chromatography. Quantitative analysis of amino acids was performed after derivatization by gas chromatography and gas chromatography / mass spectrometry (320-MS Quadrupole GC/MS, Varian) . K. senegalensis showed the peptidoglycan type A4αL-Lys←D-Glu (type A11.33 according to ref ).
K. senegalensis was susceptible to penicillin G, amoxicillin, amoxicillin plus clavulanic acid, imipenem, gentamycin, erythromycin, doxycycline, rifampicin, vancomycin, nitrofurantoin. It was resistant to ceftriaxone, ciprofloxacin, sulfamethoxazole trimethoprim and metronidazole.
Genome sequencing information
Genome project history
Project information 
One paired end 3-kb library and one Shotgun library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
EMBL Date of Release
February 28, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
K. senegalensis sp. nov. strain JC8ET, CSUR P138T, DSM 24641T, was grown aerobically on 5% sheep blood-enriched Columbia agar at 37°C. 3 petri dishes were spread and resuspended in 3×100µl of G2 buffer. A first mechanical lysis was performed with glass powder on the Fastprep-24 device (Sample Preparation system) from MP Biomedicals, USA using 2×20 second bursts. DNA was then treated with lysozyme (30 minutes at 37°C) and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 86 ng/µl.
Genome sequencing and assembly
Shotgun and 3-kb paired-end sequencing strategies were performed. The shotgun library was constructed with 500 ng of DNA with the GS Rapid library Prep kit (Roche). For the paired-end sequencing, 5 µg of DNA was mechanically fragmented on a Hydroshear device (Digilab) with an enrichment size at 3–4 kb. The DNA fragmentation was visualized using the 2100 BioAnalyzer (Agilent) on a DNA labchip 7500 with an optimal size of 3.679 kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimal size of 497 bp. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired-end library was then quantified using the Genios fluorometer (Tecan) at 888 pg/µL. The library concentration equivalence was calculated as 3.28 × 109molecules/µL. The library was stored at −20°C until further use.
The shotgun and paired-end libraries were clonally-amplified with 3 cpb and 1cpb in 3 and 4 emPCR reactions respectively on the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 14.72 and 20% respectively. 340,000 beads for the shotgun application and 790,000 beads for the 3kb paired end were loaded on the GS Titanium PicoTiterPlate PTP Kit 70x75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 307,968 passed filter wells were obtained and generated 69.7 Mb with a length average of 223 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 17 scaffolds and 42 large contigs (>1,500 bp).
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters but the predicted ORFs were excluded if they were spanning a sequencing GAP region. The predicted bacterial protein sequences were searched against the GenBank database  and the Clusters of Orthologous Groups (COG) databases  using BLASTP. The tRNAscan-SE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer .
Transmembrane domains and signal peptides were predicted using TMHMM  and SignalP , respectively. ORFans were identified if their BLASTp E-value was lower than 1e-03 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e-05. Such parameter thresholds have been used in previous works to define ORFans.
To estimate the mean level of nucleotide sequence similarity at the genome level between K. senegalensis and K. massiliensis (GenBank accession number CAEU01000000), the only available Kurthia genome to date, we compared the ORFs only using comparison sequences found in the server RAST  at a query coverage of ≥60% and a minimum nucleotide length of 100 bp.
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA G+C content (bp)
DNA coding region (bp)
Genes with function prediction
Genes assigned to COGs
Genes with peptide signals
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Comparison with other Kurthia genomes
Genome characteristics of Kurthia representatives.
K. senegalensis strain JC8ET
K. massiliensis JC30T
Genome size (bp)
DNA G+C content (%)
Prophage genome properties
Prophage Finder  and PHAST  were used to identify potential prophages in K. senegalensis strain JC8ET genome. The genome contains at least one genetic element of around 36.3 kb (with a GC content of 38.9%), which we named KS1, on contig 21. The overall G + C content of the KS1 genome (38.9%) is comparable with the overall G + C content of K. senegalensis genome (38.21%), allowing KS1 to be maintained and regulated inside the host .
A total of 49 open reading frames (ORFs) larger than 98 nucleotides were recovered from KS1, and most of them (24) encode proteins sharing a high identity with proteins found in Bacillales genus phages. The majority of the putative genes (43) have the same orientation and six are located on the complementary strand. Preliminary annotation of KS1 was performed and the majority of the putative genes (31) encode hypothetical proteins. The 19 ORFs with an attributed function encode proteins involved in DNA packaging, head and tail morphogenesis structure, cell lysis and lysogeny control, DNA replication, recombination, and modification.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Kurthia senegalensis sp. nov. that contains the strain JC8ET. This strain originated in Senegal.
Description of Kurthia senegalensis sp. nov.
Kurthia senegalensis (se.ne.gal.e’n.sis, L. gen. masc. n. senegalensis pertaining to Senegal, the country where the type strain was isolated). Isolated from stool of a healthy Senegalese patient. K senegalensis are aerobic Gram-positive coccobacilli. Surface colonies were observed on sheep blood agar after 24 h aerobic incubation at 37°C. The colonies of the strain JC8ET were circular, greyish/yellowish, shiny, curved and smooth, 2–5 mm in diameter. Cells are motile by peritrichous flagella and capsulated. Catalase activity is positive but oxidase activity is negative. Gelatine hydrolysis, N-acetyl-glucosamine assimilation, potassium gluconate assimilation, capric acid assimilation and malic acid assimilation are present. Alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylaminidase, trypsin, α-chemotrypsin and acid phosphatase activities are observed. The major fatty acids are C15:0 iso 50.75% and C15:0 anteiso 24.05%. The polar lipids present are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipids 1 and 2, unidentified aminophospholipid and glycolipid. The peptidoglycan type is A4αL-Lys←D-Glu (type A11.33). Cells are susceptible to penicillin G, amoxicillin, amoxicillin plus clavulanic acid, imipenem, gentamycin, erythromycin, doxycycline, rifampicin, vancomycin and nitrofurantoin. The genome is 2,975,103 bp long with a 38.21% G+C content. A 36.3 kb prophage, KS1, was identified. The type strain is JC8ET (= CSUR P138T = DSM 24641T). The 16S rRNA gene sequence was deposited in GenBank with the accession number JF824796. The whole genome shotgun sequence of K. senegalensis strain JC8ET was deposited in GenBank/DDBJ/EMBL under accession number CAEW01000000.
European Molecular Biology Laboratory
DNA Data Bank of Japan
The authors thank Julien Paganini at Xegen Company for automating the genomic annotation process. This study was funded by the Mediterranée-Infection Foundation.
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