Noncontiguous finished genome sequence and description of Bacillus andreraoultii strain SIT1(T) sp. nov.

Bacillus andreraoultii strain SIT1(T) (= CSUR P1162 = DSM 29078) is the type strain of B. andreraoultii sp. nov. This bacterium was isolated from the stool of a 2-year-old Nigerian boy with a severe form of kwashiorkor. Bacillus andreraoultii is an aerobic, Gram-positive rod. We describe here the features of this bacterium, together with the complete genome sequencing and annotation. The 4 092 130 bp long genome contains 3718 protein-coding and 116 RNA genes.

Introduction

Bacillus andreraoultii strain SIT1T (= CSUR P1162 = DSM 29078) is the type strain of B. andreraoultii sp. nov. This bacterium was isolated from the stool of a 2-year-old Nigerian boy with kwashiorkor, a severe form of acute malnutrition, and is part of an effort called culturomics to cultivate all bacterial species from the human gut [1], [2]. This is a Gram-positive, aerobic or facultatively anaerobic, motile, spore-forming, indole-negative and rod-shaped bacillus.

The ruling taxonomic classification of prokaryotes is based on a combination of phenotypic and genotypic criteria [3], [4]. However, the three essential criteria that are used (16S rRNA gene-based phylogeny [5], G+C content and DNA-DNA hybridization (DDH)) [3], [6] exhibit several drawbacks. The number of sequenced bacterial genomes has rapidly increased due to the decrease in cost of sequencing (to date, almost 40 000 bacterial genomes have been sequenced). Therefore, the genomic data, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) spectrum [7] and phenotypic criteria have been proposed for the description of new bacterial species [8], [9].

The genus Bacillus was discovered in 1872 (Cohn, 1872) [10]. The genus is composed of 331 species with validly published names (Bacillus, http://www.doi.namesforlife.com/). Species of the genus Bacillus are ubiquitous bacteria isolated from various environments including soil, gastrointestinal tracts of various insects and animals, vegetation, fresh- and seawater and food [11]. In human beings, the Bacillus species may exist in opportunistic form in immunocompromised patients [12] or in pathogenic form, such as B. cereus (food poisoning) and B. anthracis (anthrax) [13]. Other species may also be found in various human infections, including pneumonia, endocarditis, and ocular, cutaneous, bone or central nervous system infections and bacteraemia [14].

The following is a summary classification and a set of features for Bacillus andreraoultii sp. nov. strain SIT1T together with the description of the complete genomic sequencing and annotation. These particularities support the circumscription of the species Bacillus andreraoultii.

Materials and methods

Sample collection, strain isolation and culture condition

A stool sample was obtained from a 2-year-old Nigerian boy with kwashiorkor, a severe form of acute malnutrition, who was admitted to the emergency room in the national hospital in Niamey, the capital city of Niger, in October 2013 [2]. The study was approved by the local ethics committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France, under agreement 09-022. The fecal specimen was preserved at 4°C after collection. Then the stool was sent to Marseille, where it was kept at −80°C until laboratory culture isolation. Strain SIT1T was isolated in March 2014 by cultivation on 5% sheep's blood–enriched Columbia agar (bioMérieux, Marcy l’Étoile, France) in an anaerobic atmosphere at 37°C after 10 days of stool specimen incubation in a culture bottle containing a blood-enriched Columbia agar liquid medium (bioMérieux). Growth of the strain was tested under anaerobic conditions using GENbag anaer system (bioMérieux), and under aerobic conditions, with or without 5% CO2. Different growth temperatures (25, 30, 37, 45, 55°C) were also tested.

MALDI-TOF and 16S sequencing

MALDI-TOF protein analysis was carried out as previously described [15] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). In brief, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate and spread it as a thin film on an MSP 96 MALDI-TOF target plate (Bruker). Twenty distinct deposits from 20 isolated colonies were tested for strain SIT1T. Each smear was overlaid with 2 μL of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoracetic acid) and allowed to dry for 5 minutes. Spectra were recorded in the positive linear mode for the mass range of 2000 to 20 000 Da (parameter settings: ion source 1 (ISI), 20 kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 240 shots with variable laser power. The time of acquisition was between 30 seconds and 1 minute per spot. The 20 SIT1T spectra were imported into MALDI BioTyper 3.0 software (Bruker) and analysed by standard pattern matching (with default parameter settings) against the main spectra of 7379 bacteria. The method of identification included the m/z from 3000 to 15 000 Da. For every spectrum, a maximum of 100 peaks were compared with spectra in the database. The resulting score enabled the identification of species whether tested or not: a score of ≥2 with a validly published species enabled identification at the species level, a score of ≥1.7 but <2 enabled identification at the genus level and a score of <1.7 did not enable any identification.

Identification of bacteria continued with a 16S rRNA standard PCR coupled with sequencing. That was performed using GeneAmp PCR System 2720 thermal cyclers (Applied Biosystems, Bedford, MA, USA) and ABI Prism 3130xl Genetic Analyser capillary sequencer (Applied Biosystems) respectively [16]. The 16S rRNA nucleotides sequence was corrected using Chromas Pro 1.34 software (Technelysium, Tewantin, Australia), and the BLASTn searches were performed in the online PubMed National Center for Biotechnology Information (NCBI) database (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi).

Morphologic, biochemical and antibiotic susceptibility tests

Bacillus andreraoultii strain SIT1T was observed, after negative colouration, using a Morgani 268D (Philips, Amsterdam, The Netherlands) transmission electron microscope at an operating voltage of 60 kV. The Gram colouration was performed using Color Gram 2 Kit (bioMérieux) and observed by using the DM1000 photonic microscope (Leica Microsystems, Wetzlar, Germany) with a 100× oil-immersion objective lens. The sporulation test was done doing a thermic shock (80°C during 30 minutes). To evaluate the motility of Bacillus andreraoultii, fresh colonies were observed between blades and slats using a DM1000 photonic microscope (Leica) with a 40× objective lens.

API ZYM, API 20 NE and API 50 CH (bioMérieux) gallery systems were used to perform biochemical assays. Oxidase (Becton Dickinson, Franklin Lakes, NJ, USA) and catalase assays (bioMérieux) were done separately. The antibiotic susceptibility was tested using SirScan Discs antibiotics (i2a, Montpellier, France).

Genome sequencing

Genomic DNA extraction of Bacillus andreraoultii strain SIT1T was performed according to the method previously described [17]. The DNA was resuspended in 205 μL TE buffer. The DNA concentration was 401.63 ng/μL as measured by a Qubit fluorometer using the high-sensitivity kit (Life Technologies, Carlsbad, CA, USA).

Genomic DNA of B. andreraoultii was sequenced on the MiSeq Technology (Illumina, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit (Illumina).

The gDNA was quantified by a Qubit assay with the high-sensitivity kit (Life Technologies) to 87.67 ng/μL. The mate pair library was prepared with 1 μg of genomic DNA using the Nextera mate pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate pair junction adapter. The pattern of fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) with a DNA 7500 lab chip. The DNA fragments ranged in size from 1 to 11 kb, with an optimal size of 4.851 kb. No size selection was performed, and 432.1 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared into small fragments with an optimal size of 684 bp on the Covaris device S2 in microtubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent), and the final concentration library was measured at 64.49 nmol/L.

The libraries were normalized at 2 nM and pooled. After denaturation and dilution at 15 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and a single 39-hour sequencing run were performed at a 2 × 251 bp read length.

All 8.2 Gb of information was obtained from a 947K/mm2 cluster density, with a cluster passing quality control filters with 99% (18 111 784 clusters). Within this run, the index representation for Bacillus andreraoultii was determined to 9.27%. The 1 513 908 paired reads were filtered according to the read qualities. The reads obtained from applications were trimmed, and an optimal assembly of 14 scaffolds and 58 contigs was obtained through the SOAPdenovo software, which generated a genome size of 4.09 Mb.

Genome annotation and comparison

Open reading frames (ORFs) were predicted using Prodigal [18] with default parameters, but the predicted ORFs were excluded if they spanned a sequencing gap region (contain N). The predicted bacterial protein sequences were searched against the Clusters of Orthologous Groups (COGs) database using BLASTP (e-value 1e-03, coverage of 0.7, and an identity percentage of 30%). If no hit was found, it search against the NR database using BLASTP with an e-value of 1e-03, coverage of 0.7 and an identity percentage of 30%. If sequence lengths were smaller than 80 amino acids, we used an e-value of 1e-05. The tRNAScanSE tool [19] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [20]. Lipoprotein signal peptides and the number of transmembrane helices were predicted using Phobius [21]. ORFans were identified if all the BLASTP performed did not retrieve positive results. Such parameter thresholds have already been used in previous works to define ORFans. Genomes were automatically retrieved from the 16s RNA tree using Phylopattern software [22]. For each selected genome, complete genome sequences, proteome genome sequences and Orfeome genome sequences were retrieved from the FTP site of NCBI. All proteomes were analysed with Proteinortho [23]. Then for each couple of genomes a similarity score was computed. This score is the mean value of nucleotide similarity between all couple of orthologues in the two genomes studied (average genomic identity of orthologous gene sequences, AGIOS) [9]. The resistome was analysed with the ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) database and BLASTp in GenBank [24]. The exhaustive bacteriocin database available in our laboratories (Bacteriocins from the URMITE database) (http://drissifatima.wix.com/bacteriocins) was performed by collecting all currently available sequences from the databases and from NCBI. Protein sequences from this database allowed putative bacteriocins from human gut microbiota to be identified using BLASTp methodology [25]. PHAST (PHAge search tool) was used to identify phage sequences [26].

An annotation of the entire proteome was performed to define the distribution of functional classes of predicted genes according to the clusters of orthologous groups of proteins using the same method as for the genome annotation.

Results

Strain identification and phylogenetic analyses

No significant MALDI-TOF score was obtained for strain SIT1T against the Bruker database, suggesting that our isolate was not a member of a known species. We added the spectrum from strain SIT1T to our database (Figure 1). Then a gel view was performed to show the spectral differences with other members of the genus Bacillus (Figure 2).

Fig. 1

Reference mass spectrum from Bacillus andreraoultii strain SIT1T. Spectra from 20 individual colonies were compared and reference spectrum generated.

Fig. 2

Gel view comparing Bacillus andreraoultii SIT1T to other Bacillus species. Gel view displays raw spectra of loaded spectrum files arranged in pseudo-gel-like look. x-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity is expressed in arbitrary units by greyscale scheme code, as indicated on right y-axis. Displayed species are indicated at left.

Using 16S rRNA phylogeny analyses, we demonstrated that strain SIT1T exhibited a 96% 16S rRNA sequence identity with Bacillus thermoamylovorans (GenBank accession no. HM030742), the phylogenetically closest bacterial species with standing in nomenclature (Figure 3). Its 16S rRNA sequence was deposited in GenBank under accession no. LK021120. This value was lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers [5] to delineate a new species without carrying out DNA-DNA hybridization. Thus, this bacterium was considered as a new species called Bacillus andreraoultii strain SIT1T belonging family Bacillaceae (Table 1).

Fig. 3

Phylogenetic tree highlighting position of Bacillus andreraoultii sp. nov. strain SIT1T (= CSUR P1162 = DSM 29078) relative to other type strains within Bacillus genus. Strains and their corresponding GenBank accession numbers for 16S rRNA genes are (type = T): B. infantis strain NRRL B-14911, NR121756; B. firmus strain 5695m-D2, AJ509007; B. subterraneus strain COOI3B, NR104749; B. niacini strain Et9/1, KJ722425; B. fumarioli strain R-14705, AJ581126; B. thermolactis strain R-33520, AM910339; B. thermoamylovorans strain N12-2, HM030742; B. circulans strain WSBC20059, Y13063; B. coagulans strain 36D1, DQ297926; B. oleronius strain ATCC700005, NR043325; Flaviflexus huanghaiensis strain H5, JN815236. Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using maximum likelihood method within MEGA6. Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 1000 times to generate majority consensus tree. Flaviflexus huanghaiensis strain H5 (JN815236) was used as outgroup. Scale bar = 2% nucleotide sequence divergence.

Table 1

Classification and general features of Bacillus andreraoultii strain SIT1T according to MIGS recommendations [27].

MIGS ID	Property	Term	Evidence codea
		Domain: Bacteria	TAS [28]
		Phylum: Firmicutes	TAS [29], [30], [31]
		Class: Bacilli	TAS [32], [33]
	Current classification	Order: Bacillales	TAS [34], [35]
		Family: Bacillaceae	TAS [35], [36]
		Genus: Bacillus	TAS [10], [35]
		Species: Bacillus andreraoultii	IDA
		Type strain SIT1T	IDA
	Gram stain	Positive	IDA
	Cell shape	Rod-shaped	IDA
	Motility	Motile	IDA
	Sporulation	Sporulating	IDA
	Temperature range	Mesophile	IDA
	Optimum temperature	37–45°C	IDA
MIGS-6.3	Salinity	0
MIGS-22	Oxygen requirement	Aerobic or facultative anaerobic	IDA
	Carbon source	Unknown	IDA
	Energy source	Unknown	IDA
MIGS-6	Habitat	Human gut	IDA
MIGS-15	Biotic relationship	Free-living	IDA
MIGS-14	Pathogenicity	Unknown
	Biosafety level	2
	Isolation	Human faeces
MIGS-4	Geographic location	Marseille, France	IDA
MIGS-5	Sample collection time	March 2013	IDA
MIGS-4.1	Latitude	43.296482	IDA
MIGS-4.1	Longitude	5.36978	IDA
MIGS-4.3	Depth	Surface	IDA
MIGS-4.4	Altitude	0 m above sea level	IDA

MIGS, minimum information about a genome sequence.

Evidence codes are as follows: IDA, inferred from direct assay; TAS, traceable author statement (i.e. a direct report exists in the literature); NAS, nontraceable author statement (i.e. not directly observed for the living, isolated sample, but based on a generally accepted property for the species or anecdotal evidence). These evidence codes are from the Gene Ontology project (http://www.geneontology.org/GO.evidence.shtml). If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or by an expert or reputable institution mentioned in the acknowledgements.

Phenotypic and biochemical characteristics

B. andreraoultii growth occurred at all temperatures tested; however, optimal growth was observed between 37 and 45°C after 24 hours of incubation. Colonies were 0.1 to 0.3 μm diameter on blood-enriched Columbia agar. Growth was achieved aerobically and weak growth anaerobically. Gram staining showed rod-shaped, Gram-positive bacilli (Figure 4). Cells were grown on agar sporulate. A motility test was positive. Cells grown on agar are smooth and greyish after 24 hours of incubation, and they have an average width and length of 0.5 μm and 3 μm, respectively, and exhibited flagella (Figure 5).

Fig. 4

Gram staining of B. andreraoultii strain SIT1T.

Fig. 5

Transmission electron microscopy of B. andreraoultii strain SIT1T using Morgani 268D (Philips) at operating voltage of 60kV. Scale bar = 500 nm.

Strain SIT1T showed catalase activity but was negative for oxidase. Using an API ZYM strip, positive reactions were observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase. Negative reactions were observed for lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, β-glucuronidase, N-acetyl- β-glucosaminidase, α-mannosidase and α-fucosidase. Using an API 20 NE strip, the nitrate reductase–hydrolysis reaction (β-glucosidase, esculine), and β-galactosidase and assimilation reaction (potassium gluconate) were also positive. Negative reactions were found for urease, indole, arginine dihydrolase and fermentation (glucose). Using an API 50 CH strip, positive reactions were recorded for l-arabinose, d-ribose, d-xylose, d-glucose, d-fructose, d-mannose, N-acetylglucosamine, arbutine, esculine, salicine, d-celiobiose, d-maltose, d-saccharose, d-trehalose and amidon. Negative reactions were recorded for glycerol, erythritol, d-arabinose, l-xylose, d-adonitol, methyl-βd-xylopyranoside, d-galactose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl-αd-mannopyranoside, methyl-αd-glucopyranoside, amygdalin, d-lactose, d-melibiose, inulin, d-melezitose, d-raffinose, glycogen, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate.

Bacillus andreraoultii SIT1 was resistant to trimethroprim-sulphamethoxazole, macrolide (erythromycin), vancomycin and the third generation of cephalosporin (ceftriaxone) but was susceptible to fosfomycin, imipenem, penicillin, amoxicillin, gentamicin, ciprofloxacin, doxycycline and rifampicin. Five species with validly published names in the Bacillus genus were selected to make a phenotypic comparison with B. andreraoultii (Table 2).

Table 2

Differential characteristics of Bacillus andreraoultii SIT1T; B. thermoamylovorans strain LMG 18084T; B. thermolactis strain R-6488T; B. circulans strain LMG 13261T; B. subterraneus strain COO13BT; and B. niacin 2923T

Property	B. andreraoultii	B. thermoamylovorans	B. thermolactis	B.circulans	B. subterraneus	B. niacini
Cell diameter (μm)	0.1–0.3	0.5–4	1–4	1–3	0.5–12	3–5
Mean length (μm)	3	5	10	4.2	25	5.6
Oxygen requirement	F/anaerobic	F/anaerobic	F/anaerobic	F/anaerobic	F/anaerobic	Aerobic
Gram stain	+	+	+	−	−	+
Motility	+	+	−	+	+	−
Flagella	+	NA	+	+	+	−
Endospore formation	+	+	+	+	−	+
Production of:
Alkaline phosphatase	+	NA	NA	NA	NA	NA
Acid phosphatase	+	NA	NA	NA	NA	NA
Catalase	+	+	+	NA	+	NA
Oxidase	−	+	+	−	−	+
Nitrate reductase	+	+	+	v	+	+
Indole	−	−	−	−	−	−
Urease	−	−	−	−	−	−
α-Galactosidase	+	NA	NA	NA	NA	NA
β-Galactosidase	+	NA	NA	NA	−	NA
β-Glucuronidase	−	NA	NA	NA	−	NA
α-Glucosidase	+	NA	NA	NA	+	NA
β-Glucosidase	+	NA	NA	NA	+	NA
Esterase	+	NA	NA	NA	NA	NA
Esterase lipase	+	NA	NA	NA	NA	NA
Naphthol-AS-BI-phosphohydrolase	+	NA	NA	NA	NA	NA
N-acetyl-β-glucosaminidase	−	NA	NA	NA	NA	NA
Pyrazinamidase	NA	NA	NA	NA	NA	NA
α-Mannosidase	−	NA	NA	NA	NA	−
α-Fucosidase	−	NA	NA	NA	NA	NA
Leucine arylamidase	−	NA	NA	NA	NA	NA
Valine arylamidase	−	NA	NA	NA	NA	NA
Cystine arylamidase	−	NA	NA	NA	NA	NA
α-Chemotrypsin	−	NA	NA	NA	NA	NA
Trypsin	−	NA	NA	NA	NA	NA
Utilization of:
5-Keto-gluconate	−	−	−	v	NA	NA
d-Xylose	+	+	+	+	+	−
d-Fructose	+	+	+	+	+	+
d-Glucose	+	+	+	+	+	+
d-Mannose	+	+	−	+	−	NA
Habitat	Human gut	Wine, grass . . .	Raw milk	Bee larvae	Subterranean water	Soil

+, positive result; −, negative result; v, variable result; NA, data not available.

Genomic characteristics and genome comparison

Bacillus andreraoultii SIT1T was selected for sequencing on the basis of its phenotypic differences, phylogenetic position and 16S rRNA sequence similarity to other members of the Bacillus genus. It was part of a culturomics study aimed at isolating all bacterial species from human digestive flora in patients with kwashiorkor, an acute form of malnutrition. It is the first sequenced genome from B. andreraoultii sp. nov. The European Molecular Biology Laboratory (EMBL) accession number of B. andreraoultii genome is CCFJ00000000 and consists of 14 scaffolds and 58 contigs (Figure 6). Table 3 shows the project information and its association with minimum information about a genome sequence (MIGS) version 2.0 compliance [27]. The genome is 4 092 130 bp long with 35.42% G+C content. On the 3843 predicted genes, 3718 were protein-coding genes and 116 were RNAs genes. The remaining genes were annotated as hypothetical proteins (712 genes, >19.15%). The properties and statistics of the genome are summarized in Table 4. The distribution of genes into functional COGs categories is presented in Table 5. The draft genome sequence of Bacillus andreraoultii was smaller than those of Bacillus halodurans C-125, Bacillus pseudofirmus OF4 and Lysinibacillus sphaericus C3-41 (4.09, 4.20, 4.25 and 4.82 MB respectively) but larger than those of Solibacillus silvestris StLB046, Bacillus pumilus SAFR-032 and Bacillus coagulans 2-6 WK1 (3.98, 3.70 and 3.07 MB, respectively). The G+C content of Bacillus andreraoultii was smaller than those of Bacillus pumilus SAFR-032, Bacillus coagulans 2-6, Bacillus halodurans C-125, Bacillus pseudofirmus OF4 and Lysinibacillus sphaericus C3-41 (35.42, 41.29, 47.29, 43.69, 39.86 and 37.13% respectively). The gene content of Bacillus andreraoultii was smaller than those of Bacillus halodurans C-125, Bacillus pseudofirmus OF4 and Lysinibacillus sphaericus C3-41 (3718, 4052, 4335 and 4771, respectively) but larger than those of Bacillus pumilus SAFR-032 and Bacillus coagulans 2-6 (3681 and 2971 respectively) (Table 6). The distribution of genes into COGs categories in the genomes from all seven compared Bacillus species, Anoxybacillus flavithermus, Lysinibacillus sphaericus and solibacillus silvestris (Figure 7).

Fig. 6

Graphical circular map of genome. From outside to center: Contigs (red/grey), COGs category of genes on forward strand (three circles), genes on forward strand (blue circle), genes on reverse strand (red circle), COGs category on reverse strand (three circles), GC content.

Table 3

Project information

MIGS ID	Property	Term
MIGS-31	Finishing quality	High-quality draft
MIGS-28	Libraries used	Mate pair
MIGS-29	Sequencing platform	Illumina MiSeq
MIGS-31.2	Fold coverage	185×
MIGS-30	Assemblers	SOAPdenovo
MIGS-32	Gene calling method	Prodigal
	GenBank date of release is 8-07-2015
	NCBI project ID	PRJEB6477
	EMBL accession	CCFJ00000000
MIGS-13	Project relevance	Study of human gut microbiome

EMBL, European Molecular Biology Laboratory; MIGS, minimum information about a genome sequence; NCBI, National Center for Biotechnology Information.

Table 4

Nucleotide content and gene count levels of genome

Attribute	Value	% of totala
Size (bp)	4 092 130	100
G+C content (bp)	1 434 521	35.42
Coding region (bp)	3 248 339	79.38
Total genes	3834	100
RNA genes	116	3.0
Protein-coding genes	3718	100
Genes with function prediction	2505	67.37
Genes assigned to COGs	2420	65.08
Genes with peptide signals	308	8.26
Genes with transmembrane helices	918	24.69

COGs, Clusters of Orthologous Groups database.

Total is based on either the size of genome in base pairs or total number of protein-coding genes in annotated genome.

Table 5

Number of genes associated with 25 general COGs functional categories

Code	Value	% Value	Description
J	163	4.3840775	Translation
A	0	0	RNA processing and modification
K	192	5.164067	Transcription
L	233	6.26681	Replication, recombination and repair
B	1	0.02689618	Chromatin structure and dynamics
D	31	0.8337816	Cell cycle control, mitosis and meiosis
Y	0	0	Nuclear structure
V	62	1.6675632	Defense mechanisms
T	118	3.1737492	Signal transduction mechanisms
M	107	2.8778913	Cell wall/membrane biogenesis
N	65	1.7482517	Cell motility
Z	0	0	Cytoskeleton
W	0	0	Extracellular structures
U	41	1.1027434	Intracellular trafficking and secretion
O	105	2.824099	Posttranslational modification, protein turnover, chaperones
C	145	3.899946	Energy production and conversion
G	172	4.626143	Carbohydrate transport and metabolism
E	255	6.858526	Amino acid transport and metabolism
F	71	1.9096289	Nucleotide transport and metabolism
H	95	2.5551372	Coenzyme transport and metabolism
I	116	3.119957	Lipid transport and metabolism
P	181	4.8682084	Inorganic ion transport and metabolism
Q	47	1.2641205	Secondary metabolites biosynthesis, transport and catabolism
R	344	9.252286	General function prediction only
S	245	6.589564	Function unknown
—	1298	34.911243	Not in COGs

COGs, Clusters of Orthologous Groups database.

Table 6

Numbers of orthologous proteins shared between genomes (upper right) and AGIOS values obtained (lower left)

	Bacillus halodurans	Bacillus pseudofirmus	Bacillus coagulans	Lysinibacillus sphaericus	Bacillus pumilus	Bacillusandreraoultii
B. halodurans	4052a	1819	1293	1360	1603	1429
B. pseudofirmus	68.7%	4335a	1300	1388	1577	1452
B. coagulans	63.5%	63.2%	2971a	1166	1366	1362
L. sphaericus	63.2%	63.9%	62.4%	4771a	1409	1317
B. pumilus	64.8%	65.4%	64.4%	64%	3681a	1472
B. andreraoultii	64.4%	65.2%	64.1%	65.6%	65.6%	3718a

AGIOS, average genomic identity of orthologous gene sequences.

Numbers of proteins per genome. Percentage 0.660 = 6.

Fig. 7

Distribution of functional classes of predicted genes in genomes from Bacillus andreraoultii, Anoxybacillus flavithermus, Bacillus atrophaeus, Bacillus clausii, Bacillus coagulans, Bacillus halodurans, Bacillus pseudofirmus, Bacillus pumilus, Lysinibacillus sphaericus and Solibacillus silvestris chromosomes according to clusters of orthologous groups of proteins.

Bacillus andreraoultii contained a bacteriocin (colicin) consisting of 175 amino acids harboured with the bacteriocins of Paenibacillus and Lactobacillus (Figure 8) and sharing 62% of the homology of Bacillus vireti. The results did not indicate the presence of nonribosomal peptide synthetases and polyketide synthases and phage. We performed the analysis of the resistome of Bacillus andreraoultii SIT1T antibiotic classes of the macrolide–lincosamide–streptogramin B (MLSB) antibiotics, such as ATP-binding transporters (ABC) lsaB and msrD, major facilitator transporters mefA, transferases (vatA) and two-component system vancomycin resistance vanS/vanR, norA (Table 7).

Fig. 8

Molecular phylogenetic analysis by maximum likelihood method of representatives of genus Bacillus andreraoultii SIT1T inferred from 16S rRNA gene sequence. Tree with highest log likelihood (−2930.4905) is shown. Percentage of trees in which associated taxa clustered together is shown next to branches. Initial trees for heuristic search were obtained automatically by applying neighbour-joining and BioNJ algorithms to matrix of pairwise distances estimated using JTT model, then selecting topology with superior log likelihood value. Tree is drawn to scale, with branch lengths measured in number of substitutions per site. Analysis involved 14 amino acid sequences. There 155 positions in final data set. Evolutionary analyses were conducted in MEGA5.

Table 7

List of genes associated with antibiotic resistance in Bacillus andreraoultii SIT-1

Gene function	ORF	Gene name	GC	Size (aa)	Function	Best BLAST hit, GenBank	% Coverage	% Identity
MLSB	328	lsaB	32.7	494	ABC transporter	Bacilli	100	94
Glycopeptide	395	van S	34.7	378	Sensor histidine kinase VanS	Bacillus sp. J37	99	82
Glycopeptide	396	vanR	36.6	232	Vancomycin response regulator VanR	Clostridium clariflavum	100	96
MLSB	431	vatA	35	213	Chloramphenicol acetyltransferase	Paucisalibacillus globulus	100	99
MLSB	572	msrd	38.7	487	ABC transporters	Clostridium kluyveri NBRC 12016	100	100
MLSB	573	mefA	37.2	408	Macrolide-efflux protein	Clostridium kluyveri	100	100
MLS	583	msrd	35.8	177	ABC-F type ribosomal protection protein	Bacteroides	100	91

ORF, open reading frame; MLSB, macrolide–lincosamide–streptogramin B.

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses (taxonogenomics), we formally propose the creation of Bacillus andreraoultii sp. nov. that contains the strain SIT1T. This bacterium was isolated from the stool of a 2-year-old Nigerian boy with kwashiorkor, a severe form of acute malnutrition.

Description of Bacillus andreraoultii sp. nov.

The Bacillus andreraoultii name come from André Raoult, who was a military doctor who worked with malnutrition and kwashiorkor in Senegal and who described its specific features [37].

Strain SIT1T is aerobic, Gram positive, endospore forming, motile and rod shaped. Growth was achieved aerobically between 25 and 55°C (optimum 37 to 45°C). After 24 hours of growth on 5% sheep's blood–enriched Columbia agar at 37°C, bacterial colonies were smooth and greyish with a diameter of 0.1 to 0.3 mm. The cells had a mean width and length of 0.5 μm and 3 μm, respectively, and exhibited flagella. They were catalase positive and oxidase negative.

Bacillus andreraoultii SIT1T was resistant to trimethroprim-sulphamethoxazole, macrolide (erythromycin), vancomycin and the third generation of cephalosporin (ceftriaxone). It contained a bacteriocin.

The genome is 4 092 130 bp long, and the G+C content is 35.42%. The 16S rRNA gene sequence and whole-genome shotgun sequence of B. andreraoultii strain SIT1T are deposited in GenBank under accession nos. LK021120 and CCFJ00000000, respectively. The type strain SIT1T (= CSUR P1162 = DSM 29078) was isolated from the stool of a 2-year-old Nigerien boy with kwashiorkor, a severe form of acute malnutrition.

Conflict of Interest

None declared.