Genome sequence and description of Nesterenkonia massiliensis sp. nov. strain NP1T

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.


Introduction
Nesterenkonia massiliensis strain NP1 T (= CSUR P244 = DSM 26221) is the type strain of N. massiliensis sp. nov. This bacterium is a Grampositive, 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 [3], we recently proposed to use genomic properties in combination with phenotypic characteristics for the taxonomic classification of Bacteria . Herein, we present a summary classification and a set features for Nesterenkonia massiliensis sp. nov., strain NP1 T (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 [35]. This genus belongs to the family Micrococcaceae within the phylum Actinobacteria, and is most closely related to the genera Micrococcus, Arthrobacter and Kocuria [35]. The Nesterenkonia genus includes Grampositive, non spore-forming, aerobic, mesophilic bacteria that may be halotolerant or halophilic. Currently, the genus Nesterenkonia includes 12 species with validly published names [36]. 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][37][38][39][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 [41].

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 NP1 T (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 [51], 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 [52]. Ontology project [50]. 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), nonendospore-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 NP1 T 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, glu-cose, arabinose, mannose, mannitol, N-acetylglucosamine, 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 Dglucose, D-fructose, D-saccharose, ribose, mannose, mannitol, D-trehalose and L-rhamnose. No reaction was observed for esculin, salicin, Dcellobiose 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 NP1 T differed in cell shape, colony color, motility, optimal growth temperature, and mannitol fermentation ( Table 2).  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 NP1 T , 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 [1]. 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 [42].

Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [54] 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 [55] and the Clusters of Orthol-ogous Groups (COG) databases using BLASTP. The tRNAScanSE tool [56] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [57] and BLASTn against the GenBank database. Lipoprotein signal peptides and the number of transmembrane helices were predicted using SignalP [58] and TMHMM [59] 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 Evalue of 1e -5 . Such parameter thresholds have already been used in previous works to define ORFans. Artemis [60] and DNA Plotter [61] 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 [62]. 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 [34]. Briefly, this software combines the Proteinortho software [63] 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 NT1 T 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).

Genome properties
The genome of N. massiliensis strain NP1 T 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. Genes with transmembrane helices 599 22.49 a The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.    On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Nesterenkonia massiliensis sp. nov. that contains the strain NP1 T . 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 , 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 Grampositive, 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, βgalactosidase, glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate, phenylacetate assimilation and cytochrome oxidase. Fermentation of D-glucose, D-fructose, Dsaccharose, ribose, mannose, mannitol, Dtrehalose 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 NP1 T (= CSUR P244 = DSM 26221) was isolated from the fecal flora of a 32-year-old French female suffering from AIDS.