Genome sequence of ”Leucobacter massiliensis” sp. nov. isolated from human pharynx after travel to the 2014 Hajj. T. Leangapichart, P. Gautret, T. T. Nguyen, N. Armstrong, J.-M. Rolain To cite this version: T. Leangapichart, P. Gautret, T. T. Nguyen, N. Armstrong, J.-M. Rolain. Genome sequence of ”Leucobacter massiliensis” sp. nov. isolated from human pharynx after travel to the 2014 Hajj.. New Microbes and New Infections, Wiley Online Library 2018, 21, pp.42-48. 10.1016/j.nmni.2017.10.007. hal-01795906 HAL Id: hal-01795906 https://hal-amu.archives-ouvertes.fr/hal-01795906 Submitted on 22 May 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. NEW SPECIES Genome sequence of “Leucobacter massiliensis” sp. nov. isolated from human pharynx after travel to the 2014 Hajj T. Leangapichart, P. Gautret, T. T. Nguyen, N. Armstrong and J.-M. Rolain Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE) CNRS-IRD UMR 6236, Méditerranée Infection, Faculté de Médecine et de Pharmacie, Aix-Marseille-Université, Marseille, France Abstract “Leucobacter massiliensis” strain 122RC15T sp. nov. is a new species within the genus Leucobacter. The genome of this strain is described here. It was isolated from the pharynx of a 76-year-old Algerian female after travelling from the 2014 Hajj. “Leucobacter massiliensis” is a Gram- positive, aerobic bacillus. Here we describe the features including complete genome and annotation of this strain. The 3 136 406-bp long genome contains 2797 protein-coding genes and 49 RNA genes. © 2017 The Authors. Published by Elsevier Ltd. Keywords: Culturomics, genome, Hajj, Leucobacter massiliensis, new species, taxonogenomics Original Submission: 26 June 2017; Revised Submission: 9 October 2017; Accepted: 17 October 2017 Article published online: 26 October 2017 Materials and methods Corresponding author: J.-M. Rolain, Faculté de Médecine et de Pharmacie, Aix-Marseille-Université, Marseille, France E-mail: [email protected] Strain identification A pharyngeal sample was obtained from a healthy pilgrim after travelling to the 2014 Hajj. The pharyngeal sample was pro- cessed as previously described [1]. This strain was isolated by Introduction cultivation on Chocolate agar PolyViteX (bioMérieux, Marcy l’Étoile, France) under a 5% CO2 atmosphere at 37°C after 1 fi “Leucobacter massiliensis” strain 122RC15T (= DSM 29913= day of incubation. For bacterial identi cation, matrix-assisted – fl CSUR P1430) is the type strain of L. massiliensis sp. nov. This laser desorption ionization time-of- ight mass spectrometry bacterium was isolated from the pharynx of a 76-year-old (MALDI-TOF-MS) protein analysis was carried out as previ- woman originating from Algeria and living in Marseille after ously described [13]. The 122RC15 spectra were imported into travelling from the 2014 Hajj [1]. This pilgrim had respiratory MALDI BIOTYPER 3.0 software (Bruker Daltonics, Leipzig, tract symptoms including cough, sore throat and loss of voice Germany) and analysed by standard pattern matching (with but without antibiotic usage during the Hajj. The genus Leuco- default parameter settings) against 7765 spectra of bacteria. bacter was first designed by Takeuchi et al. [2] and formerly From the resulting scores, the tested strain may or may not be fi ’ included 25 validly described species. Members of Leucobacter identi ed compared with the instrument s database; a score of fi have been isolated from several sources including waste-water 2 with a validly published species enabled identi cation at the fi [3–7], soil environments [8,9], air [10], foods [11] and nema- species; a score 1.7 and <2 allows identi cation at the genus fi todes [12]. However, there are no known human pathogens level; and a score <1.7 does not enable any identi cation. among the genus Leucobacter. fi “L. massiliensis” is an aerobic, Gram-positive bacillus. With Phylogenetic analysis and classi cations current knowledge, this strain 122RC15 is the first species of The 16S rRNA gene was sequenced as previously described Leucobacter genus isolated from human pharynx. Here, we describe [14]. The 16S rRNA gene sequence was blasted to the NCBI fi the main characteristics of strain 122RC15 with a phenotypic and database for species identi cation. The strain is considered as a phylogenetic analysis and the complete genome description. new species if the percentage of similarity is <98.7%. For New Microbe and New Infect 2018; 21: 42–48 © 2017 The Authors. Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) https://doi.org/10.1016/j.nmni.2017.10.007 NMNI Leangapichart et al. Genomes of “Leucobacter massiliensis” in a pilgrim 43 phylogenetic classification, homologous sequences from type trimmed. The assembly of the three runs leaded to 3 392 578 strains found on the List of Prokaryotic Names with Standing in paired end reads. SPADES assembler was used and assembled in Nomenclature (LPSN) website (http://www.bacterio.net/) were to 24 scaffolds. downloaded from the NCBI database. The phylogenetic tree was constructed using a neighbour-joining method and the Genome annotation and comparisons maximum composite likelihood substitution model with 1000 Open reading frames were predicted using PRODIGAL (http:// bootstraps on MEGA6 (Molecular Evolutionary Genetics prodigal.ornl.gov/) with default parameters. The predicted Analysis) software [15]. open reading frames were excluded if they spanned a sequencing gap region. The predicted protein sequences were Phenotypic properties and biochemical searched against the GenBank [17] and Clusters of Ortholo- characterization gous Groups database (COGs) databases using BLASTP. The Growth of the strain was tested under aerobic conditions with tRNAScanSE tool [18] and RNAmmer [19] were used to find or without 5% CO2 including anaerobic and microaerophilic tRNA genes and ribosomal RNAs, respectively. Signal peptides conditions using GENbag anaer and GENbag microaer systems, and transmembrane helices were predicted by SIGNALP [20] and respectively (bioMérieux) with different temperatures (25°C, TMHMM [21]. Furthermore, mobile genetic elements were 30°C, 37°C, 42°C and 45°C). Optimal salt concentration was predicted using PHAST [22] and RAST server [23]. The resis- determined by growing the strain at 0%, 0.5%, 2%, 7% and 10% tome analysis was identified with the ARG-ANNOT database NaCl. Gram staining, motility and sporulation were observed [24]. Analysis of the presence of polyketide synthases/non- using a light microscope (DM1000; Leica Microsystems, Nan- ribosomal peptide synthetases and bacteriocins was identified terre, France). Electron microscopy of “L. massiliensis” was as previously described [25]. Genomic islands were predicted performed using a TechnaiG2 Cryo (FEI Company, Limeil- by ISLANDVIEWER [26]. The genome sequences of available spe- Brevannes, France) at an operating voltage of 200 keV. cies in the genus Leucobacter were downloaded from NCBI. Biochemical assays were characterized using API ZYM and Average nucleotide identity of draft genomes within the Leu- API 50 CH (bioMérieux) gallery systems. Cellular fatty acid cobacter genus was calculated by pairwise comparisons using methyl ester analysis was performed by gas chromatography/ JSPECIESWS [27]. Moreover, proteome comparisons were per- mass spectrometry as previously described [16]. formed by CMG-BIOTOOLS [28] with minimum query coverage of 50% and minimum identity of 50%. Genome sequencing and assembly Genomic DNA of “L. massiliensis” was sequenced using the MiSeq Technology (Illumina, San Diego, CA, USA) with the pair Results end and mate-pair strategies. First, automated cluster genera- tion and sequencing runs were performed in a single 39-h run at Phylogenetic analysis a 2 × 250-bp read length. Total information of 7.3 Gb was After isolation of strain 122RC15, identification was performed 2 obtained from an 800 K/mm cluster density, with a cluster by MALDI-TOF analysis. However, MALDI-TOF-MS was un- fi fi passing quality control lters of 92.2% (14 034 000 passing lter able to identify the strain 122RC15 because its spectrum was clusters). Within this run, the index representation for not part of the database. BLAST results for 16S rRNA gene se- “ ” L. massiliensis was 10.25%. The 1 438 190 paired reads were quences showed 98.3% similarity with Leucobacter kyeonggiensis trimmed. Moreover, two mate-pair libraries were performed in strain F3-P9 (GenBank Accession no. NR132679.1), which was fi a single 39-h run in a 2 × 151-bp read length. The rst library confirmed by phylogenetic tree construction (Fig. 1). Hence, was loaded twice in the same run where total information of we propose the creation of a new species, which putatively 2 6.1 Gb was obtained from a 653 K/mm cluster density with a classifies it as a member of the Leucobacter genus within the fi cluster passing quality control lters of 96.1% (12 031 000 Microbacteriaceae family in the Actinobacteria phylum (Table 1). fi passing lter paired reads). Within this run, the index repre- The 16S rRNA sequence was deposited in NCBI under Gen- “ ” sentation for L. massiliensis was 4.29%. The 978 060 paired Bank Accession no. LN849775. reads were trimmed then assembled. The second library was loaded once and 6.5 Gb of total information was obtained from Phenotypic analysis 2 a 696 K/mm cluster density with a cluster passing quality Strain 122RC15 was a Gram-positive rod, non-motile, non- control filters of 95.6% (12 863 000 passing filter paired reads).