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Genome sequence and description of Nesterenkonia massiliensis sp. nov. strain NP1T
Standards in Genomic Sciences volume 9, pages866–882 (2014)
Nesterenkonia massiliensis sp. nov., strain NP1T, is the type strain of Nesterenkonia massiliensis sp. nov., a new species within the genus Nesterenkonia. This strain, whose genome is described here, was isolated from the feces of a 32-year-old French woman suffering from AIDS and living in Marseille. Nesterenkonia massiliensis is a Gram-positive aerobic coccus. Here, we describe the features of this bacterium, together with the complete genome sequencing and annotation. The 2,726,371 bp long genome (one chromosome but no plasmid) contains 2,663 protein-coding and 51 RNA genes, including 1 rRNA operon.
Nesterenkonia massiliensis strain NP1T (= CSUR P244 = DSM 26221) is the type strain of N. massiliensis sp. nov. This bacterium is a Gram-positive, non-spore-forming, aerobic and motile coccus that was isolated from the fecal flora of AIDS-infected French female living in Marseille, France, as part of a “culturomics” study aiming at cultivating as many of the bacterial species in human feces as possible [1,2].
Taking advantage of the availability of more than 12,000 bacterial genome sequences , we recently proposed to use genomic properties in combination with phenotypic characteristics for the taxonomic classification of Bacteria [4–34].
Herein, we present a summary classification and a set features for Nesterenkonia massiliensis sp. nov., strain NP1T (CSUR= P244 = DSM 26221), including the description of its complete genome and annotation. These characteristics support the circumscription of the species Nesterenkonia massiliensis.
The genus Nesterenkonia was first described by Stackebrandt et al. in 1995 . This genus belongs to the family Micrococcaceae within the phylum Actinobacteria, and is most closely related to the genera Micrococcus, Arthrobacter and Kocuria . The Nesterenkonia genus includes Gram-positive, non spore-forming, aerobic, mesophilic bacteria that may be halotolerant or halophilic. Currently, the genus Nesterenkonia includes 12 species with validly published names . Members of the genus Nesterenkonia are ubiquitous bacteria, which have been isolated from various environments including hypersaline soil and lakes, soda lakes, sea food, and paper and cotton pulp mills [36–40]. However, prior to our study, Nesterenkonia species had not been reported in humans, with the exception of DNA sequences from Nesterenkonia sp. detected in the gut microbiota of patients with chronic kidney diseases .
Classification and features
A stool sample was collected from a Caucasian, AIDS-infected, 32-year-old French woman living in Marseille, France. The patient gave an informed and signed consent. This study and the assent procedure were approved by the Ethics Committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France under agreement number 09-022. The fecal sample was preserved at −80°C after collection. Strain NP1T (Table 1) was first isolated in March 2012 by cultivation on Columbia agar (BioMerieux, Marcy l’Etoile, France) under aerobic conditions after 14 days of preincubation of the stool sample with addition of 5ml of sheep rumen in blood bottle culture. The strain exhibited a 96.7% 16S rRNA nucleotide sequence identity with N. alba , the phylogenetically most closely related Nesterenkonia species with standing in nomenclature (Figure 1). This value was lower 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 .
Ontology project . If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.
Four different growth temperatures (25, 30, 37, 45°C) were tested. Growth was observed between 25 and 45°C on blood-enriched Columbia agar (BioMerieux), with optimal growth occurring at 37°C after 24 hours of incubation. Colonies were dark yellow and 1 mm in diameter. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMerieux), and under aerobic conditions, with or without 5% CO2. Optimal growth was obtained under aerobic conditions, but weak growth occurred in a microaerophilic atmosphere. No growth was observed under anaerobic conditions. Bacterial cells were Gram-positive (Figure 2), non-endospore-forming, and motile cocci. Cells grown on agar had a mean diameter and length of 0.67 µm and 1.4 µm, respectively (Figure 3).
Strain NP1T exhibited catalase but no oxidase activity. Using an API 20NE strip (BioMerieux), negative reactions were obtained for nitrate reduction, urease, indole production, glucose fermentation, arginine dihydrolase, β-galactosidase, glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate, phenyl-acetate assimilation and cytochrome oxidase. Substrate oxidation and assimilation were examined with an API 50CH carbohydrate fermentation strip (BioMerieux) at 37°C. Positive reactions were observed for D-glucose, D-fructose, D-saccharose, ribose, mannose, mannitol, D-trehalose and L-rhamnose. No reaction was observed for esculin, salicin, D-cellobiose and gentiobiose. N. massiliensis is susceptible to amoxicillin, imipenem, rifampin, ciprofloxacin, gentamicin, doxycycline and vancomycin but resistant to trimethoprim/sulfamethoxazole and metronidazole. When compared with representative species from the genus Nesterenkonia, N. massiliensis strain NP1T differed in cell shape, colony color, motility, optimal growth temperature, and mannitol fermentation (Table 2).
Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described  using a Microflex spectrometer (Brüker Daltonics, Leipzig, Germany). Twelve individual NP1T colonies were deposited on a MTP 384 MALDI-TOF target plate (Brüker). The twelve spectra were imported into the MALDI BioTyper software (version 2.0, Brüker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 4,706 bacteria, including 1 spectrum from N. lacusekhoensis, the only validly named Nesterenkonia species for which a spectrum was available in the BioTyper database. A score enabled the presumptive identification and discrimination of the tested species from those in a database: a score > 2 with a validated species enabled the identification at the species level; and a score < 1.7 did not enable any identification. For strain NP1T, no significant score was obtained, suggesting that our isolate was not a member of any known species (Figures 4 and 5).
Genome sequencing information
Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position, 16S rDNA similarity and phenotypic differences with other members of the genus Nesterenkonia, and is part of a “culturomics” study aiming at isolating individually all bacterial species within the human gut flora . It was the third genome of a Nesterenkonia species and the first genome of N. massiliensis sp. nov. A summary of the project information is shown in Table 3. The Genbank accession number is CBLL000000000 and consists of 141 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance .
Growth conditions and DNA isolation
N. massiliensis sp. nov strain NP1T, (= CSUR P244 = DSM 26221) was grown aerobically on sheep blood-enriched Columbia agar at 37°C. Two petri dishes were spread and the colonies resuspended in 6x100 µl of G2 buffer (EZI DNA Tissue Kit, Qiagen). A first mechanical lysis was performed with glass powder on the Fastprep-24 device (Sample Preparation system, MP Biomedicals, USA) using 2x20 second bursts. DNA was incubated with 2.5 µg/µL of lysozyme for 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 DNA concentration measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer was 94 ng/µl.
Genome sequencing and assembly
DNA (5 µg) was mechanically fragmented with a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3–4 kb. The DNA fragmentation was visualized through the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an average size of 3.5 kb. The library was constructed using a 454 GS-FLX Titanium paired-end rapid library protocol. Circularization and nebulization generated a pattern with an average size of 390 bp. After 20 cycles of PCR amplification, the double stranded paired-end library was quantified using the Quant-it Ribogreen kit (Invitrogen) using a Genios Tecan fluorometer at 820 pg/µL. The library concentration equivalence was calculated as 3.86 × 109 molecules/µL. The library was stored at −20°C until further use.
The library was clonally amplified with 1 cpb in two emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2. The yield of the emPCR was 13.67%, within the range of 5 to 20% recommended for the Roche procedure. Approximately 656,601 beads were loaded for a ¼ region on the GS Titanium PicoTiterPlate (PTP Kit 70x75, Roche) and sequenced with the GS Titanium Sequencing Kit XLR70. The run was performed overnight and analyzed on the cluster through the gsRunBrowser and Newbler assemblers (Roche). A total of 176,833 passed filter wells were obtained and generated 60.9 Mb with an average of length of 345 bp. The passed filter sequences were assembled on Newbler with 90% identity and 40 bp as overlap. The final assembly identified 18 scaffolds and 141 large contigs (>1,500 bp), and generated a genome size of 2.69Mb which corresponds to a coverage of 22.63 genome equivalents.
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters but the predicted ORFs were excluded if they spanned 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 tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer  and BLASTn against the GenBank database. Lipoprotein signal peptides and the number of transmembrane helices were predicted using SignalP  and TMHMM  respectively. ORFans were identified if their BLASTP E-value was lower than 1e−3 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e−5. Such parameter thresholds have already been used in previous works to define ORFans. Artemis  and DNA Plotter  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 . To estimate the mean level of nucleotide sequence similarity at the genome level, we used the Average Genomic Identity Of gene Sequences (AGIOS) home-made software . Briefly, this software combines the Proteinortho software  for detecting orthologous proteins in pairwise comparisons 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. As only one genome was available for the genus Nesterenkonia, we used genomes from closely related genera for the calculation of AGIOS values. N. massiliensis strain NT1T was compared to Nesterenkonia alba strain DSM 19423 (GenBank accession number ATXP00000000), Micrococcus luteus strain NCTC2665 (CP001628), Kocuria rhizophila strain DSM 2048 (AP009152) and Arthrobacter arilaitensis strain RE117 (FQ311875).
The genome of N. massiliensis strain NP1T is 2,726,371bp long (1 chromosome, but no plasmid) with a 62.47% G+C content (Table 4, Figure 6, Figure 7). Of the 2,714 predicted genes, 2,663 were protein-coding genes, and 51 were RNAs. Three rRNA genes (one 16S rRNA, one 23S rRNA and one 5S rRNA) and 48 predicted tRNA genes were identified in the genome. A total of 1,962 genes (73.68%) were assigned a putative function. One hundred and ninety-nine genes were identified as ORFans (7.47%). The remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 4. The distribution of genes into COGs functional categories is presented in Table 5 and a comparison is presented in Table 6.
We compared the genome of N. massiliensis strain NP1T to those of N. alba strain DSM 19423, M. luteus strain NCTC2665, K. rhizophila strain DSM 2048 and A. arilaitensis strain RE117. The draft genome of N. massiliensis had a larger size than those of N. alba and M. luteus (2.72, 2.59 and 2.7 Mb, respectively) but was smaller than that of A. arilaitensis (3.92 Mb). The G+C content of N. massiliensis was lower than those of N. alba, M. luteus and K. rhizophila (62.4, 63.8, 73 and 71.2%, respectively) but higher than that of A. arilaitensis (59.3%). The gene content of N. massiliensis was larger than those of N. alba, M. luteus and K. rhizophila (2,714, 2,403, 2,345 and 2,414 genes, respectively) but smaller than that of A. arilaitensis (3,771). In addition, N. massiliensis shared 1,142, 1,132, 1,087 and 1,208 orthologous genes with N. alba, M. luteus and K. rhizophila, respectively. N. massiliensis exhibited an AGIOS value of 75.61% with N. alba, and 68.57 to 70.5 with other members of the family Micrococcaceae.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Nesterenkonia massiliensis sp. nov. that contains the strain NP1T. The strain has been isolated from the fecal flora of an AIDS-infected patient living in Marseille, France. Several other bacterial species were also cultivated from different fecal samples through diversification of culture conditions [4–34], thus suggesting that the human fecal flora of humans remains only partially known.
Description of Nesterenkonia massiliensis sp. nov.
Nesterenkonia massiliensis (mas.si.li.en’sis. L. gen. fem. n. massiliensis of Massilia, the Roman name of Marseille, France, where the type strain was isolated).
Grows between 25 and 45°C on blood-enriched Columbia agar (BioMerieux). Optimal growth obtained at 37°C in aerobic atmosphere. Weak growth in microaerophilic atmosphere. No growth under anaerobic condition. Colonies are dark yellow and 1 mm in diameter. Cells are Gram-positive, non-endospore-forming, and motile cocci, with a mean diameter and length of 0.67 µm and 1.4 µm, respectively. Catalase positive, oxidase negative. Negative reactions obtained for nitrate reduction, urease, indole production, glucose fermentation, arginine dihydrolase, β-galactosidase, glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate, phenylacetate assimilation and cytochrome oxidase. Fermentation of D-glucose, D-fructose, D-saccharose, ribose, mannose, mannitol, D-trehalose and L-rhamnose. No reaction observed for esculin, salicin, D-cellobiose and gentiobiose. Cells are susceptible to amoxicillin, imipenem, rifampin, ciprofloxacin, gentamicin, doxycycline and vancomycin but resistant to trimethoprim/sulfamethoxazole and metronidazole.
The G+C content of the genome is 62.47%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JX424770 and CBLL00000000, respectively. The habitat of the microorganism is the human digestive tract. The type strain NP1T (= CSUR P244 = DSM 26221) was isolated from the fecal flora of a 32-year-old French female suffering from AIDS.
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The authors thank the Xegen Company (www.xegen.fr) for automating the genomic annotation process. This study was funded by the Mediterranée-Infection Foundation.
These 2 authors contributed equally