Standards in Genomic Sciences (2013) 9:93-105 DOI:10.4056/sigs.4388124 Non-contiguous finished genome sequence and description of Bacillus massiliogorillae sp. nov. Mamadou Bhoye Keita1, Seydina M. Diene1, Catherine Robert1, Didier Raoult1,2, Pierre- Edouard Fournier1, and Fadi Bittar1* 1URMITE, Aix-Marseille Université, Faculté de Médecine, Marseille, France 2King Fahad Medical Research Center, King Abdul Aziz University, Jeddah, Saudi Arabia *Correspondence: Fadi Bittar ([email protected]) Keywords: Bacillus massiliogorillae, genome, culturomics, taxonogenomics Strain G2T sp. nov. is the type strain of B. massiliogorillae, a proposed new species within the genus Bacillus. This strain, whose genome is described here, was isolated in France from the fecal sample of a wild western lowland gorilla from Cameroon. B. massiliogorillae is a facul- tative anaerobic, Gram-variable, rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,431,633 bp long genome (1 chromosome but no plasmid) contains 5,179 protein-coding and 98 RNA genes, including 91 tRNA genes. Introduction Strain G2T (= CSUR P206 = DSM 26159) is the type Here we present a summary classification and a strain of B. massiliogorillae sp. nov. This bacterium set of features for B. massiliogorillae sp. nov. strain is a Gram-variable, facultatively anaerobic, indole- G2T together with the description of the complete negative bacillus having rounded-ends. It was iso- genome sequence and annotation. These charac- lated from the stool sample of Gorilla gorilla goril- teristics support the circumscription of the spe- la as part of a “culturomics” study aiming at culti- cies B. massiliogorillae [9]. vating bacterial species within gorilla feces. The genus Bacillus (Cohn 1872) was created about Classification and features 140 years ago [1]. To date this genus, comprised In July 2011, a fecal sample was collected from a mostly of Gram-positive, motile, and spore- wild western lowland gorilla near Messok, a vil- forming bacteria, includes 276 species with validly lage in the south-eastern part of the DJA FAUNAL published names [2]. Members of the genus Bacil- Park (Cameroon). The collection of the stool sam- lus are ubiquitous bacteria isolated from various ple was approved by the Ministry of Scientific Re- environments including soil, fresh and sea water, search and Innovation of Cameroon. No experi- food, and occasionally from humans and animals mentation was conducted on this gorilla. The fecal in which they are either pathogens, such as B. specimen was preserved at -80°C after collection anthracis (the causative agent of anthrax) [3] and and sent to Marseille. Strain G2T (Table 1) was iso- B. cereus (associated mainly with food poisoning) lated in January 2012 by cultivation on Brucella [4], or saprophytes [5]. Bacillus species may also agar medium (Oxoid, Dardilly, France). This strain rarely be involved in a variety of human infec- exhibited a 97.3% 16S rRNA nucleotide sequence tions, including pneumonia, bacteremia, meningi- similarity with Bacillus simplex, the tis, endocarditis, endophthalmitis, osteomyelitis phylogenetically closest validly published Bacillus and skin/soft tissue infection [5]. However, in species (Figure 1). This value was lower than the great apes, few data are available about the pres- 98.7% 16S rRNA gene sequence threshold rec- ence of the genus Bacillus. Recent reports have ommended by Stackebrandtia and Beers to delin- described the isolation of atypical B. anthracis (B. eate a new species without carrying out DNA-DNA anthracis-like bacteria) in wild chimpanzees and hybridization [23]. gorillas from Africa [6-8]. The Genomic Standards Consortium Bacillus massiliogorillae sp. nov. Table 1. Classification and general features of Bacillus massiliogorillae strain G2T MIGS ID Property Term Evidence codea Domain Bacteria TAS [10] Phylum Firmicutes TAS [11-13] Class Bacilli TAS [14,15] Order Bacillales TAS [16,17] Current classification Family Bacillaceae TAS [16,18] Genus Bacillus TAS [16,19,20] Species Bacillus massiliogorillae IDA Type strain G2T IDA Gram stain Variable IDA Cell shape Rod IDA Motility Motile IDA Sporulation Sporulating IDA Temperature range Mesophile IDA Optimum temperature 37°C IDA MIGS-6.3 Salinity Growth in BHI medium + 2% NaCl IDA MIGS-22 Oxygen requirement Facultative anaerobic IDA Carbon source Unknown NAS Energy source Unknown NAS MIGS-6 Habitat Gorilla gut IDA MIGS-15 Biotic relationship Free living IDA MIGS-14 Pathogenicity Unknown NAS Biosafety level 2 NAS Isolation Gorilla feces NAS MIGS-4 Geographic location Cameroon IDA MIGS-5 Sample collection time July 2011 IDA MIGS-4.1 Latitude Unknown NAS MIGS-4.1 Longitude Unknown NAS MIGS-4.3 Depth Unknown NAS MIGS-4.4 Altitude Unknown NAS 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 [21]. If the evidence is IDA, then the property was direct- ly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. 94 Standards in Genomic Sciences Keita et al. 39 Bacillus infernus (NR027227) 18 Bacillus methanolicus (NR 040985) 15 Bacillus fumarioli (FJ973527) 23 Bacillus jeotgali (JX094165) Bacillus lentus (NR040792) Bacillus smithii (NR036987) 62 56 54 Bacillus badius (EU717967) Bacillus firmus (JX428993) 52 40 Bacillus benzoevorans (Y14693) 78 Bacillus nealsoniis (NR044546) 100 Bacillus circulans (JF833093) 74 Bacillus massiliogorillae (JX650055) 97 Bacillus simplex (HQ327114) 87 Bacillus psychrosaccharolyticus (JX429005) Bacillus flexus (JN033557) 100 Bacillus megaterium (FJ685764) Bacillus thuringiensis (HF545324) Clostridium botulinum (AM412317) Figure 1. Phylogenetic tree highlighting the position of Bacillus massiliogorillae strain G2T relative to other type strains within the Bacillus genus. GenBank accession numbers are indicated in parentheses. Se- quences were aligned using CLUSTAL X (V2), and phylogenetic inferences obtained using the maximum- likelihood method within the MEGA 5 software [22]. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1,000 times to generate a majority consensus tree. Clostridium botulinum was used as outgroup. The scale bar represents a 2% nucleotide sequence divergence. Different growth temperatures (25, 30, 37, 45°C) Strain G2T exhibited catalase activity but not oxi- were tested. Growth occurred at all tested tem- dase activity. Using the API 50CH system peratures, and the optimal growth was observed (BioMerieux), a positive reaction was observed for at 37°C. Colonies were 2-5 mm in diameter on Co- D-glucose, D-fructose, D-ribose, N- lumbia agar, grey opaque in color. Growth of the acetylglucosamine, amygdalin, arbutin, aesculin, strain was tested under anaerobic and salicin, cellobiose, maltose, D-lactose, D-trehalose, microaerophilic conditions using GENbag anaer D-saccharose, and hydrolysis of starch. Using the and GENbag microaer systems, respectively API ZYM system, positive reactions were observed (BioMérieux), and in aerobic conditions, with or for esterase (C4), esterase lipase (C8), phospha- without 5% CO2. Growth was achieved under aer- - glucosidase and N-acetyl- - obic (with and without CO2), microaerophilic and glucosaminidase. The urease reaction was also anaerobic conditions. Gram staining showed Gram positive,tase acid, but nitrateα reduction and indole produβc- variable bacilli (Figure 2). A motility test was posi- tion were negative. B. massiliogorillae is suscepti- tive. Cells grown on agar sporulate and the rods ble to amoxicillin, nitrofurantoin, erythromycin, have a length ranging from 3.2 to 7.5 µm (mean doxycycline, rifampin, vancomycin, gentamycin 5.4 µm) and a diameter ranging from 0.8 to 1.2 µm and imipenem but resistant to trimethoprim- (mean 1 µm) as determined by negative staining sulfamethoxazole, ciprofloxacin, ceftriaxon and transmission electron microscopy (Figure 3). amoxicillin-clavulanic acid. http://standardsingenomics.org 95 Bacillus massiliogorillae sp. nov. Figure 2. Gram staining of B. massiliogorillae strain G2T Figure 3. Transmission electron microscopy of B. massiliogorillae strain G2T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 1 µm. 96 Standards in Genomic Sciences Keita et al. Table 2. Differential phenotypic characteristics between B. massiliogorillae sp. nov. strain G2T and phylogenetically close Bacillus species. Characteristic B. massiliogorillae sp. nov. B. simplex B. psychrosaccharolyticus B. circulans Cell diameter (µm) 0.87-1.2 0.7-0.9 0.9-1 0.5-0.8 Oxygen requirement aerobic aerobic facultative anaerobic aerobic Gram stain var var var var Salt requirement < 5% <7% <10% <7% Motility + v + + Endospore formation + + + + Production of Alkaline phosphatase + na na na Acid phosphatase + na na na Catalase + + + + Oxidase - - na na Nitrate reductase - na + na Urease + na na w α-galactosidase - na na na β-galactosidase - na + + β -glucuronidase - na na na α -glucosidase + na na na N-acetyl- β- glucosaminidase + na na na Indole - na na na Esterase + na na na Esterase lipase + na na na Naphthyl-AS-BI- phosphohydrolase - na na na Phenylalanine arylamidase - na na na Leucine