Standards in Genomic Sciences (2013) 8:264-278 DOI:10.4056/sigs.3496989 Non contiguous-finished genome sequence and description of Bacillus massiliosenegalensis sp. nov. Dhamodharan Ramasamy1, Jean-Christophe Lagier1, Aurore Gorlas1, Didier Raoult1 and Pierre-Edouard Fournier1* 1Aix-Marseille Université, URMITE, Faculté de médecine, Marseille, France Corresponding author: Pierre-Edouard Fournier ([email protected]) Keywords: Bacillus massiliosenegalensis, genome, culturomics, taxono-genomics Bacillus massiliosenegalensis strain JC6T sp. nov. is the type strain of Bacillus massiliosenegalensis sp. nov., a new species within the genus Bacillus. This strain was isolat- ed from the fecal flora of a healthy Senegalese patient. B. massiliosenegalensis is an aerobic Gram-positive rod-shaped bacterium. Here we describe the features of this organism, togeth- er with the complete genome sequence and annotation. The 4,981,278-bp long genome comprises a 4,957,301-bp chromosome and a 23,977-bp plasmid. The chromosome contains 4,925 protein-coding and 72 RNA genes, including 4 rRNA genes. The plasmid contains 29 protein-coding genes. Introduction Bacillus massiliosenegalensis strain JC6T (= CSUR strain JC6T together with the description of the P151 = DSM 25957) is the type strain of B. complete genomic sequencing and annotation. massiliosenegalensis sp. nov., a new species within These characteristics support the creation of the the genus Bacillus. This bacterium is a Gram- species B. massiliosenegalensis. positive, aerobic, catalase-positive and indole- The genus Bacillus (Cohn 1872) was created in negative bacillus that was isolated from the stool 1872 [26] and currently consists of mainly Gram- of a healthy Senegalese patient as part of a study positive, motile, and spore-forming bacilli. Cur- aimed at individually cultivating all human enteric rently, 173 Bacillus species and 4 subspecies are bacterial species [1,2]. validly published [27]. Members of the genus Ba- Currently, bacterial taxonomy relies on a combi- cillus are ubiquitous bacteria, mostly isolated from nation of various genetic and phenotypic criteria. environmental sources. However, several species However, the three main genetic criteria that are are associated with humans, either as pathogens used, including 16S rRNA gene-based phylogeny or commensals [28]. and nucleotide similarity [3,4], DNA-DNA hybridi- zation [5] and G+C content suffer significant Classification and features drawbacks and their cutoffs are not applicable to A stool sample was collected from a healthy 16- all genera and species. Over recent years, the in- year-old male Senegalese volunteer patient living troduction of high-throughput genome sequencing in Dielmo (rural village in the Guinean-Sudanian and proteomic analyses [6] provided a source of zone in Senegal), who was included in a research exhaustive information about characterized bac- protocol. Written assent was obtained from this terial isolates. Such data may now be included individual. No written consent was needed from among the criteria used for taxonomic identifica- his guardians for this study because he was older tion. We recently proposed to use a polyphasic than 15 years old (in accordance with the previ- approach to describe new bacterial taxa that is ous project approved by the Ministry of Health of based on their genome sequence, MALDI-TOF Senegal and the assembled village population and spectrum and main phenotypic characteristics [7- as published elsewhere [29].) 25]. Both this study and the assent procedure were Here we present a summary classification and a approved by the National Ethics Committee of set of features for B. massiliosenegalensis sp. nov. The Genomic Standards Consortium Ramasamy et al. Senegal (CNERS) and the Ethics Committee of the Senegalemassilia anaerobia, Peptoniphilus obesi, Institut Fédératif de Recherche IFR48, Faculty of Peptoniphilus senegalensis, Enterobacter Medicine, Marseille, France (agreement numbers massiliensis, Alistipes obesi, Peptoniphilus 09-022 and 11-017). Several other new bacterial grossensis, Brevibacillus massiliensis [7-25]. species were isolated from this specimen using The fecal specimen was conserved at -80°C after various culture conditions, including the recently collection. Strain JC6T was (Table 1) was isolated described Alistipes senegalensis, Alistipes in January 2011 by cultivation on 5% sheep blood- timonensis, Anaerococcus senegalensis, Bacillus enriched Brain Heart infusion (BHI) agar (Becton timonensis, Clostridium senegalense, Peptoniphilus Dickinson, Heidelberg, Germany). The strain ex- timonensis and Paenibacillus senegalensis, hibited a 97.3% nucleotide sequence similarity Herbaspirillum massiliense, Kurthia massiliensis, with B. siralis (Pettersson et al. 2000), the Brevibacterium senegalense, Aeromicrobium phylogenetically closest Bacillus species (Figure massilense, Cellulomonas massiliensis, 1). Table 1. Classification and general features of Bacillus massiliosenegalensis strain JC6T according to the MIGS recommendations [30] MIGS ID Property Term Evidence codea Domain Bacteria TAS [31] Phylum Firmicutes TAS [32-35] Class Bacilli TAS [36,37] Current classification Order Bacillales TAS [32,38] Family Bacillaceae TAS [32,39] Genus Bacillus TAS [32,40,41] Species Bacillus massiliosenegalensis IDA Type strain JC6T IDA Gram stain Positive IDA Cell shape Rod IDA Motility Motile IDA Sporulation Sporulating IDA Temperature range Mesophile IDA Optimum temperature 30°C IDA MIGS-6.3 Salinity Growth in BHI medium + 5% NaCl IDA MIGS-22 Oxygen requirement Aerobic IDA Carbon source Unknown NAS Energy source Unknown NAS MIGS-6 Habitat Human gut IDA MIGS-15 Biotic relationship Free living IDA Pathogenicity Unknown Biosafety level 2 MIGS-14 Isolation Human feces NAS MIGS-4 Geographic location Senegal IDA MIGS-5 Sample collection time September 2010 IDA MIGS-4.1 Latitude 13.7167 IDA MIGS-4.1 Longitude -16.4167 IDA MIGS-4.3 Depth Surface IDA MIGS-4.4 Altitude 51 m above sea level IDA Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report ex- ists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolat- ed sample, but based on a generally accepted property for the species, or anecdotal evidence). These evi- dence codes are from the Gene Ontology project [42]. If the evidence is IDA, then the property was di- rectly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. http://standardsingenomics.org 265 Bacillus massiliosenegalensis sp. nov. 56 Bacillus massiliosenegalensis (JF824800) 75 Bacillus siralis (NR 028709) 73 Bacillus firmus (HM030743) Bacillus koreensis (FJ889614) 78 100 Bacillus beringensis (FJ889612) Bacillus oceanisediminis (JQ799086) 25 96 Bacillus firmus (D16268) Bacillus pumilus (AY876289) 89 Bacillus amyloliquefaciens (JX094152) 100 49 Bacillus subtilis (AJ276351) 53 Bacillus licheniformis (JX281696) 34 Bacillus atrophaeus (AB021181) Bacillus megaterium (D16273) 99 Bacillus coahuilensis (EF014452) 37 Bacillus weihenstephanesis (AB021199) 79 Bacillus thuringiensis (D16281) 100 85 Bacillus cereus (AE016877) Bacillus timonensis (JF824810) 34 Geobacillus thermoglucosidasius (FN428685) Paenibacillus lautus (AB073188) Brevibacillus brevis (AB271756) Clostridium butyricum (AJ458420) 0.02 Figure 1. Phylogenetic tree showing the position of Bacillus massiliosenegalensis strain JC6T relative to other type strains within the Bacillus genus. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method within the MEGA software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 500 times to generate a majority consensus tree. Clostridium butyricum was used as outgroup. The scale bar represents a 2% nucleotide sequence divergence. Different growth temperatures (25, 30, 37, 45°C) Strain JC6T exhibited catalase activity but not oxi- were tested. Growth occurred between 25°C and dase activity. Using the API 50CH system, we ob- 45°C, and the optimal growth was observed at served positive reactions for aesculin, D-cellobiose, 30°C. Colonies were translucent and 2 mm in diam- D-glucose, D-maltose, N-acetyl-glucosamine and D- eter on blood-enriched Columbia agar. Growth of trehalose. Using the API ZYM system, a positive re- the strain was tested under anaerobic and -glucosidase and weak microaerophilic conditions using GENbag anaer reactions were observed for alkaline phosphatase, and GENbag microaer systems respectively esteraseaction was lipase, observed valine forarylamidase α and trypsin. Us- (BioMérieux) in the presence of air with or without ing the API 20E system, a positive reaction was ob- 5% CO2. Growth was achieved in aerobic condition served for nitrate reduction and negative reactions (with or without CO2), and weak growth was ob- were observed for indole production and urease. B. served in microaerophilic and anaerobic condi- massiliosenegalensis is susceptible to amoxicillin, tions. Gram staining showed a rod-shaped Gram- ceftriaxone, imipenem, trime- positive bacterium (Figure 2). The motility test was thoprim/sulfamethoxazole, gentamicin, ciprofloxa- positive by means of peritrichous flagella. Cells cin, rifampicin and vancomycin, but resistant to have a mean diameter of 0.65 µm and a mean metronidazole and erythromycin. The differential length of 3.076 µm