Non-contiguous finished genome sequence and description of Kurthia massiliensis sp. nov.
© The Author(s) 2012
Published: 19 December 2012
Kurthia massiliensis strain JC30T sp. nov. is the type strain of K. massiliensis 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. massiliensis is a Gram-positive aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,199,090 bp long genome contains 3,240 protein-coding genes and 86 RNA genes, including between 3 and 4 rRNA genes.
KeywordsKurthia massiliensis Firmicutes capsule Flagella
Kurthia massiliensis strain JC30T (CSUR 141T = DSM 24639T) is the type strain of K. massiliensis sp. nov. This bacterium is a Gram-positive, strictly aerobic rod that is capsulated, and motile by peritrichous flagella. This organism was originally isolated from the stool of a healthy Senegalese patient as part of a “culturomics” study aimed at cultivating all species within human feces, individually.
Currently, “the gold standard” for defining bacterial species is DNA-DNA hybridization . But this method is time-consuming and the inter-laboratory reproducibility is poor. Fortunately, the development of PCR and next-generation sequencing technologies have led to reliable and reproducible 16S rRNA comparison methods with generally agreed upon cutoff values that enable the taxonomic classification of new species for many bacterial genera . To describe new bacterial taxa, the use of a polyphasic approach was proposed  that includes their genome sequence, MALDI-TOF spectrum and main phenotypic characteristics (habitat, Gram-stain reaction, cultivation conditions, cell wall structure and metabolic characteristics).
The genus Kurthia was created in 1885 by Trevisan  in honor of Kurth who described the first species, Bacterium zopfii, isolated from the intestinal contents of chickens. As the stool samples had been stored at room temperature and the bacteria were strictly aerobic, it was assumed that the samples were contaminated by Kurthia, which multiplied during storage. The name Kurthia was first published in the seventh edition of Bergey’s Manual of Determinative Bacteriology  and was included in the Approved Lists of Bacterial Names . Currently, Kurthia includes 3 species: K. zopfii, K. gibsonii  and K. sibirica . The bacteria are members of the phylum Firmicutes, and the family Planococcaceae. There is no evidence of pathogenicity.
Here we present a summary classification and a set of features for K. massiliensis sp. nov. strain JC30T together with the description of the complete sequencing and annotation of its genome. These characteristics support the circumscription of the species K. massiliensis.
Classification and features
Classification and general features of Kurthia massiliensis strain JC30T according to the MIGS recommendations 
Species Kurthia massiliensis
Type strain JC30T
Motile by peritrichous flagella
Growth in BHI medium + 3% NaCl
Sample collection time
51 m above sea level
Different growth temperatures (25, 30, 37, 45, 50 and 55°C) were tested. Growth occurred between 25°C and 55°C, and optimal growth was observed between 25°C and 50°C. Growth of the strain was tested under aerobic atmosphere, in the presence of 5% CO2, and under anaerobic and microaerophilic atmospheres, which were created using GENbag anaer and GENbag microaer (bioMérieux), respectively. The strains were aerobic but also grew under microaerophilic conditions and in the presence of 5% CO2. Growth does not occur under anaerobic conditions. NaCl tolerance of strain JC30T was 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 0.5–3% NaCl. Growth in the range of pH 5.0–10.0 was tested using BBLTM Brain Heart Infusion (Becton Dickinson). pH tolerance revealed that growth could occur over a range of pH 6.0–9.0 with optimal growth between pH 7.0–9.0.
Differential phenotypic characteristics between strain JC30T and related species
potassium gluconate 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 [22,23]. Respiratory lipoquinones were separated into their different classes (menaquinones and ubiquinones) by thin layer chromatography on silica gel, using hexane:ter-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 with detection at 269 nm. The only respiratory quinone for strain JC30T was MK-7 (100%). 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 68.04% and C15:0 anteiso 16.92%. 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, phospholipid 1. The peptidoglycan of strain JC30T was isolated as described by Schleifer . Analysis was carried out as previously described [26,27] with the modification that TLC on cellulose was used rather than paper chromatography. Quantitative analysis of amino acids was performed following derivatization by gas chromatography and gas chromatography / mass spectrometry (320-MS Quadrupole GC/MS, Varian) . K. massiliensis showed the peptidoglycan type A4αL-Lys←D-Glu (type A11.33 according to reference ).
K. massiliensis was susceptible to penicillin G, amoxicillin, amoxicillin + clavulanic acid, imipenem, gentamycin, erythromycin, doxycycline, rifampicin, vancomycin, and nitrofurantoin. The organism was resistant to ceftriaxone, ciprofloxacin, sulfamethoxazole trimethoprim and metronidazole.
Genome sequencing information
Genome project history
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 12, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
K. massiliensis sp. nov. strain JC30T, CSUR P141T, DSM 24639T, was grown aerobically on 5% sheep blood-enriched Columbia agar at 37°C. Three petri dishes were spread and resuspended in 3×100 µl of G2 buffer. A first mechanical lysis was performed by glass powder on the Fastprep-24 device (Sample Preparation system) from MP Biomedicals, USA using 2×20 second cycles. DNA was then treated with lysozyme (4.17g/L, 30 minutes at 37°C) and extracted through the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration were measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 63.1/µl.
Genome sequencing and assembly
Shotgun and 3-kb paired-end sequencing strategies were used. The shotgun library was constructed with 500 ng of DNA with the GS Rapid library Prep kit (Roche). For 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 3.619 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 472 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 430 pg/µL. The library concentration equivalence was calculated as 1.69E+09 molecules/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 18.65 and 14.31% respectively. Approximately 340,000 beads for the shotgun sequencing and 790,000 beads for the 3kb paired end sequencing were loaded onto the GS Titanium PicoTiterPlate PTP Kit 70×75 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 294,263 passed filter wells were obtained and generated 81.3 Mb with a length average of 301 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 18 scaffolds and 72 large contigs (>1,500 bp).
Coding sequences (CDSs) were predicted using PRODIGAL with default parameters . The functional annotation of protein sequences was performed against the non-redundant GenBank database using BLASTP. Functional categories of these proteins were searched against the Clusters of Orthologous Groups (COG) database using COGNITOR . The prediction of RNAs genes, i.e., rRNAs, tRNAs and other RNAs was carried out using RNAmmer  and ARAGORN  algorithms. The transmembrane segments and peptide signals were identified using TMHMM  and SignalP tools .
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (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
To date, no genome of other strains or species belonging to the genus Kurthia were sequenced.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Kurthia massiliensis sp. nov., which contains the strain JC30T. This bacterium was found in Senegal.
Description of Kurthia massiliensis sp. nov.
Kurthia massiliensis (mas.si.li.en′sis. L. masc. adj. massiliensis of Massilia, the old Roman name for Marseille, where the type strain was isolated). Isolated from stool of a healthy Senegalese patient. K massiliensis are aerobic Gram-positive coccobacilli. On sheep blood agar after 24 h aerobic incubation at 37°C, colonies of strain JC30T are circular, greyish/yellowish, shiny, curved and smooth, 2–5 mm in diameter. Cells are motile by peritrichous flagella and encapsulated. Catalase activity is positive but oxidase activity is negative. Gelatine hydrolysis, maltose assimilation, potassium gluconate assimilation, malic acid assimilation, trisodium citrate assimilation are present. Esterase (C4), esterase lipase (C8), cystine arylaminidase, α-gluconidase activities are observed. Valine arylaminidase and alpha-chemotrypsin activities are weakly positive. The major fatty acids are iso C15:0 68.04% and anteiso C15:0 16.92%. Polar lipids found are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phospholipid 1. The peptidoglycan type is A4αL-Lys←D-Glu (type A11.33 according to ). Cells are susceptible to penicillin G, amoxicillin, amoxicillin + clavulanic acid, imipenem, gentamycin, erythromycin, doxycycline, rifampicin, vancomycin and nitrofurantoin. The G+C content of the genome is 39.26%. The type strain is JC30T (= CSUR P141T = DSM 24639T).
European Molecular Biology Laboratory
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