- Open Access
Non-contiguous finished genome sequence and description of Bacillus algeriensis sp. nov.
- Published: 15 June 2014
Abstract
Strain EB01T sp. nov. is the type strain of Bacillus algeriensis, a new species within the genus Bacillus. This strain, whose genome is described here, was isolated from sediment sample of the hypersaline lake Ezzemoul sabkha in northeastern Algeria. B. algeriensis is a facultative anaerobic Gram-positive bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,269,577 bp long genome contains 5,098 protein-coding and 95 RNA genes, including 12 rRNA genes.
Keywords
- Bacillus algeriensis
- hypersaline environments
- sediments
- genome
- taxono-genomics
Introduction
Bacillus massilioalgeriensis sp. nov. strain EB01T (= CSUR P857 = DSM 27334) is the type strain of B. massilioalgeriensis sp. nov. It is a new Gram-positive, facultatively anaerobic, motile, indole-negative, rod shaped bacterium with rounded ends. It was isolated from a sediment sample from the hypersaline lake Ezzemoul sabkha in the Oum-El-Bouaghi region in northeastern Algeria, which is an important wintering and resting site for several species of waterbirds, including the Greater Flamingo. This site is one of the Ramsar convention wetlands (http://www.ramsar.org). The genus Bacillus was created by Cohn about 142 years ago [1], and mainly comprises Gram-positive, rod-shaped, aerobic or facultatively anaerobic, spore-forming bacteria. The genus includes 279 species and 7 subspecies with validly published names [2]. Members of Bacillus genus are ubiquitous in nature, ranging from freshwater to marine sediments and from hot springs and desert sands to Arctic soils; many strains have been isolated from the gastrointestinal tracts of various insects and animals, from vegetation and from food [3]. Bacillus strains are biotechnologically priceless because of their high capacity to produce a wide range of antimicrobial compounds, enzymes and other metabolites that can be used in industry [4,5]. Some species of Bacillus are pathogenic, such as B. anthracis (responsible for causing anthrax) [6] and B. cereus (a major cause of food poisoning) [7]. Others are opportunists in immunocompromised patients, and may also be involved in various human infections, including pneumonia, endocarditis, ocular, cutaneous, bone or central nervous system infections and bacteremia [8].The current bacterial taxonomy is based on a combination of various phenotypic and genetic criteria [9,10]. However, the three essential genetic criteria that are used, comprising 16S rRNA gene based phylogeny [11], G+C content, and DNA-DNA hybridization [10,12] exhibit several drawbacks. As a result of the recent decrease in the cost of genomic sequencing, it has been proposed that whole genome sequencing information and MALDI-TOF spectrum [13] be combined with the main phenotypic characteristics as a polyphasic approach strategy (taxono-genomics) to describe new bacterial taxa [14–26].
Here we present a summary classification and a set of features for B. massilioalgeriensis sp. nov. strain EB01T together with the description of the complete genome sequence and annotation. These characteristics support the circumscription of the species B. massilioalgeriensis.
Classification and features
A consensus phylogenetic tree based on 16S rRNA gene sequence comparisons, highlighting the position of strain EB01T Bacillus massilioalgeriensis relative to other type strains within the Bacillus genus. GenBank accession numbers are displayed in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences made using the neighbor-joining method [38] within the MEGA 5 software [39]. Numbers above the nodes are percentages of bootstrap values from 1,000 replicates that support the node. Paenibacillus polymyxa was used as the outgroup. The scale bar represents 0.01 substitutions per nucleotide position.
Classification and general features of Bacillus massilioalgeriensis strain EB01T
MIGS ID | Property | Term | Evidence codea |
---|---|---|---|
Current classification | Domain Bacteria | TAS [27] | |
Phylum Firmicutes | |||
Class Bacilli | |||
Order Bacillales | |||
Family Bacillaceae | |||
Genus Bacillus | |||
Species Bacillus massilioalgeriensis | IDA | ||
Type strain EB01T | IDA | ||
Gram stain | Positive | IDA | |
Cell shape | Rod-shaped | IDA | |
Motility | Motile | IDA | |
Sporulation | Sporulating | IDA | |
Temperature range | Between 37°C and 55°C | IDA | |
Optimum temperature | 37°C | IDA | |
MIGS-6.3 | Salinity | Growth in LB medium + 0–2.5% NaCl | IDA |
MIGS-22 | Oxygen requirement | Facultative anaerobic | IDA |
Carbon source | Unknown | NAS | |
Energy source | Unknown | NAS | |
MIGS-6 | Habitat | Hypersaline sediment sample | IDA |
MIGS-15 | Biotic relationship | Free living | IDA |
Pathogenicity | Unknown | NAS | |
Biosafety level | 2 | NAS | |
MIGS-14 | Isolation | Sediment of Ezzemoul Sabkha Lake | IDA |
MIGS-4 | Geographic location | Algeria | IDA |
Sample collection | July 2012 | ||
MIGS-5 | time | ||
MIGS-4.1 | Latitude | 35.88167 | IDA |
MIGS-4.1 | Longitude | 6.503272 | IDA |
MIGS-4.3 | Depth | Unknown | NAS |
MIGS-4.4 | Altitude | 800 m | IDA |
Gram stain of B. massilioalgeriensis strain EB01T.
Transmission electron micrograph of B. massilioalgeriensis strain EB01T made using a Technai G2 Cryo (FEI) at an operating voltage of 200 kV. The scale bar represents 500 nm.
Strain EB01T exhibited catalase activity but oxidase activity was negative. Using the commercially available API 50CH system (BioMerieux) according to the manufacturer’s instructions, a weak positive reaction was observed for D-ribose, D-glucose, D-fructose, methyl α-D-glucopyranoside, N-acetylglucosamine, D-maltose, D-lactose, D-melibiose, D-saccharose, D-trehalose, D-tagatose, and hydrolysis of starch. Other tests were negative. Using the API ZYM system (BioMerieux), positive reactions were observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, α-chymotrypsin, β-glucuronidase, α-glucosidase, N-acetyl-glucosaminidase and a weak positive reaction was observed for acid phosphatase. The nitrate reduction and β-galactosidase reaction was also positive, but urease and indole production were negative. B. massilioalgeriensis was susceptible to amoxicillin, nitrofurantoin, erythromycin, doxycycline, rifampicin, vancomycin, gentamicin, imipenem, trimethoprim-sulfamethoxazole, ciprofloxacin, ceftriaxone and amoxicillin-clavulanic acid, but resistant to nalidixic acid.
Differential phenotypic characteristics between B. massilioalgeriensis sp. nov. strain EB01T and phylogenetically close Bacillus species†.
Characteristic | B. ma | B. su | B. fo | B. je | B. th | B. bo | B. ba | B. ne | B. kr |
---|---|---|---|---|---|---|---|---|---|
Cell-diameter(µm) | 0.7–1.1 | 0.5–0.8 | 1 | 0.8–1.1 | 0.5–0.7 | 0.5–0.9 | 0.7–1.2 | 1 | 1.4–2.0 |
Oxygen requirement | facultative anaerobic | facultative anaerobic | aerobic | facultative anaerobic | aerobic | na | facultative anaerobic | facultative anaerobic | aerobic |
Gram strain | + | − | + | V | V | + | + or V | + | + |
NaCl range (%,w/v) | 0–2.5 | 0–9 | 0–3 | 0–13 | 0–5 | 0–7 | na | 0–8 | 0–6 |
Motility | + | + | na | + | + | + | + | + | + |
Endospore formation | + | − | − | + | + | + | + | + | + |
Production of | |||||||||
Alkaline phosphatase | + | na | na | na | na | + | na | na | na |
Acid phosphatase | w | na | na | na | na | na | na | na | na |
Catalase | + | + | + | + | + | + | na | + | + |
Oxidase | − | − | + | − | − | + | na | na | − |
Nitrate reductase | + | + | + | + | + | − | + | − | − |
Urease | − | − | + | + | − | − | − | − | − |
α-galactosidase | − | na | na | na | na | na | na | na | na |
β-galactosidase | − | − | + | na | na | − | na | + | na |
β-glucuronidase | + | na | na | na | na | na | na | na | + |
N-acetyl-β-glucosaminidase | + | na | na | na | na | na | na | na | na |
Indole | − | − | na | − | − | − | − | − | na |
Esterase | + | na | na | na | na | na | na | na | + |
Esterase lipase | + | na | na | na | na | w | na | na | + |
Naphthyl-AS-BI-Phosphohydrolase | - | na | na | na | na | na | na | na | + |
Leucine arylamidase | + | na | na | na | na | + | na | na | na |
Cystine arylamidase | − | na | na | na | na | w | na | na | na |
Valine arylamidase | − | na | na | na | na | w | na | na | + |
Utilization of | |||||||||
D-mannose | − | − | + | − | − | + | + | + | − |
Amygdalin | − | na | + | − | − | + | w | − | na |
L-Arabinose | − | − | + | − | na | na | − | + | − |
Cellobiose | − | − | + | + | na | + | + | + | + |
Lactose | w | − | + | − | na | na | + | + | + |
D-xylose | − | + | + | − | na | na | − | + | + |
D-Glucose | w | + | + | + | + | na | + | + | + |
Mannitol | − | − | + | − | − | na | + | + | na |
Arabinose | − | − | + | − | − | na | − | − | na |
L-Xylose | − | + | + | − | na | na | − | na | + |
Glycerol | − | + | + | − | na | + | w | + | − |
D-Galactose | − | − | + | − | na | na | + | + | na |
Hydrolysis of | |||||||||
Starch | w | + | + | + | na | na | v | + | − |
Gelatin | − | + | + | + | w | − | + | − | + |
G+C content (mol%) | 42,22 | 43±1 | 43,1 | 41 | 43,8 | 41,1-42,2 | 39,6 | na | 43,3 |
Habitat | hyersaline sediment | deep subterranean thermal waters | alkaline ground water | fermented seafood | wastewater treatment culture system | soil | soil | spacecraft-assembly facility | soil |
Reference mass spectrum from B. massilioalgeriensis strain EB01T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.
Gel view comparing Bacillus massilioalgeriensis EB01T spectra with other members of the Bacillus genus (B. weihenstephanensis, B. vallismortis, B. thuringiensis, B. thioparans, B. subtilis subsp. subtilis, B. subterraneus, B. shackletonii, B. novalis, B. niacini, B. nealsonii, B. jeotgali, B. flexus, B. circulans, B. bataviensis and B. asahii). The Gel View displays the raw spectra of all loaded spectrum files as a pseudo-electrophoretic gel. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a grey scale scheme code. The grey scale bar on the right y-axis indicates the relation between the shade of grey a peak is displayed with and the peak intensity in arbitrary units.
Genome sequencing information
Genome project history
Project information
MIGS ID | Property | Term |
---|---|---|
MIGS-31 | Finishing quality | High-quality draft |
MIGS-28 | Libraries used | Nextera XT library |
MIGS-29 | Sequencing platform | Miseq-Illumina |
MIGS-31.2 | Sequencing coverage | 34× |
MIGS-30 | Assemblers | Velvet |
MIGS-32 | Gene calling method | Prodigal |
EMBL Date of Release | January 10, 2014 | |
EMBL ID | ERP003483 | |
MIGS-13 | Project relevance | Study of the Bacillus genus diversity in hypersaline lakes of northeastern Algeria |
Growth conditions and DNA isolation
Bacillus massilioalgeriensis sp. nov strain EB01T, was grown aerobically on 5% sheep blood enriched Columbia agar at 37°C. Three petri dishes were spread and resuspended in a 2 ml sterile Eppendorf tube containing 1ml of TE buffer with acid-washed glass beads (diameter ≤106 µm, Sigma, Saint-Quentin Fallavier, France). Three cycles of shaking were performed using a FastPrep BIO 101 apparatus (Qbiogene, Strasbourg, France) for 15 sec at level 6.5 (full speed). Then, the supernatant was placed in a new tube along with one hundred µl of 10% SDS and 50 µl of Proteinase K (Qiagen GmbH, Hilden, Germany) and incubated over night at 56°C. The digested mixture was used to perform DNA extraction using the classical phenol-chloroform method. The quality of the DNA was checked on an agarose gel (0.8%) stained with SYBR safe.
Genome sequencing
Genomic DNA of B. massilioalgeriensis sp. nov. strain EB01T was sequenced on the MiSeq platform (Illumina, Inc, San Diego CA 92121, USA) with a paired end and barcode strategy in order to be mixed with 7 others genomic projects constructed with the Nextera XT library kit (Illumina).
The gDNA was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA) to 34.4 ng/µL and dilution was performed to provide 1 ng of each small genome as input. The “tagmentation” step fragmented and tagged the DNA to generate an optimum insert size of 1.6 kb, validated on a high sensitivity labchip Calliper-Perkin Elmer (Caliper Life Sciences, Inc, Massachusetts, USA). Then limited cycle PCR amplification completed the tags adapters and introduced dual-index barcodes. After purification on Ampure beads (Lifetechnolgies, Carlsbad, CA, USA), the libraries were normalized on specific beads according to the Nextera XT protocol (Illumina). Normalized libraries are pooled into a single library for sequencing on the MiSeq. The pooled single strand library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and paired-end sequencing with dual index reads was performed in a single 39-hour run with a 2×250 bp read length. Within this pooled run, the index representation was determined to 7.1%. Total information of 2.4 G bases was obtained from a 320 K/mm2 density with 94.9% (5,757,000 clusters) of the clusters passing quality control (QC) filters. From the genome sequencing process, the 775,420 produced Illumina reads for B. massilioalgeriensis EB01T were filtered according to the read qualities and sizes using the fastq-mcf program (Ea-utils: command-line tools for processing biological sequencing data) [52]. 714,540 filtered read sequences were kept for genome assembly. The Velvet assembler was used with different kmer values (from 51 to 95) and the best assembly result with kmer value (n=91) producing 46 contigs with sizes from 872 bp to 409,112 bp, was retained for genome annotation.
Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [53] with default parameters. The predicted bacterial protein sequences were searched against the Clusters of Orthologous Groups (COG) database and the GenBank database [54] using BLASTP. Ribosomal RNAs were found by using RNAmmer 2.1 server [55,56] and BLASTn against the GenBank database, whereas the tRNAScanSE tool [57] was used to find tRNA genes. Transmembrane helices and lipoprotein signal peptides were predicted using phobius web server [58]. ORFans were identified if their BLASTP E-value was lower than 1e-03 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e-05. Artemis [59] was used for data management and DNA Plotter [60] was used for visualization of genomic features. To estimate the mean level of nucleotide sequence similarity at the genome level between B. massilioalgeriensis sp nov. strain EB01T and seven other Bacillus species, we use the Average Genomic Identity of Orthologous gene Sequences (AGIOS) in-house software. Briefly, this software combines the Proteinortho software [61] for pairwise comparison and detection of orthologous proteins between 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.
Genome properties
Graphical circular map of the chromosome. From outside to the center: Red and gray bars representing contigs, genes on the forward strand colored by COG categories (only genes assigned to COG), genes on the reverse strand colored by COG categories (only genes assigned to COG), RNA genes (tRNAs green, rRNAs red), GC content. The inner-most circle shows the GC skew, purple and olive indicating negative and positive values, respectively.
Nucleotide content and gene count levels of the genome
Attribute | Value | % of totala |
---|---|---|
Genome size (bp) | 5,269,577 | 100 |
DNA coding region (bp) | 4,342,253 | 82.4 |
DNA G+C content (bp) | 2,224,760 | 42.21 |
Total genes | 5,193 | 100 |
RNA genes | 95 | 1.82 |
Protein-coding genes | 5,098 | 98.17 |
Genes with function prediction | 3,217 | 63.1 |
Genes assigned to COGs | 3,041 | 59.65 |
Genes with peptide signals | 653 | 12.8 |
Genes with transmembrane helices | 1,297 | 25.44 |
Number of genes associated with the 25 general COG functional categories
Code | Value | % agea | Description |
---|---|---|---|
J | 176 | 3.45 | Translation, ribosomal structure and biogenesis |
A | 0 | 0 | RNA processing and modification |
K | 281 | 5.51 | Transcription |
L | 165 | 3.23 | Replication, recombination and repair |
B | 1 | 0.01 | Chromatin structure and dynamics |
D | 33 | 0.64 | Cell cycle control, mitosis and meiosis |
Y | 0 | 0 | Nuclear structure |
V | 63 | 1.23 | Defense mechanisms |
T | 144 | 2.82 | Signal transduction mechanisms |
M | 156 | 3.06 | Cell wall/membrane biogenesis |
N | 48 | 0.94 | Cell motility |
Z | 0 | 0 | Cytoskeleton |
W | 0 | 0 | Extracellular structures |
U | 50 | 0.98 | Intracellular trafficking and secretion |
O | 110 | 2.15 | Posttranslational modification, protein turnover, chaperones |
C | 206 | 4.04 | Energy production and conversion |
G | 277 | 5.43 | Carbohydrate transport and metabolism |
E | 370 | 7.25 | Amino acid transport and metabolism |
F | 82 | 1.60 | Nucleotide transport and metabolism |
H | 109 | 2.13 | Coenzyme transport and metabolism |
I | 142 | 2.78 | Lipid transport and metabolism |
P | 213 | 4.17 | Inorganic ion transport and metabolism |
Q | 86 | 1.68 | Secondary metabolites biosynthesis, transport and catabolism |
R | 521 | 10.21 | General function prediction only |
S | 327 | 6.41 | Function unknown |
- | 2057 | 40.34 | Not in COGs |
Comparison with other species Bacillus genomes
Genomic comparison of B. massilioalgeriensis sp. nov. strain EB01T with seven other Bacillus species†.
Species | Strain | Genome accession number | Genome size (Mb) | G+C content |
---|---|---|---|---|
Bacillus massilioalgeriensis | EB01T | ERP003483 | 5.26 | 42.22 |
Bacillus kribbensis | DSM 17871 | AUMQ00000000.1 | 5.05 | 43 |
Bacillus nealsonii | AAU1 | ASRU00000000.1 | 4.98 | 35.1 |
Bacillus bataviensis | LMG 21833 | AJLS00000000.1 | 5.37 | 39.6 |
Bacillus subtilis subsp. subtilis | 168 | NC_000964.3 | 4.22 | 43.5 |
Bacillus vallismortis | DV1-F-3 | AFSH00000000.1 | 3.88 | 43.8 |
Bacillus thuringiensis | BMB171 | NC_014171.1 | 5.64 | 35.2 |
Bacillus weihenstephanensis | KBAB4 | NC_010184.1 | 5.87 | 35.5 |
Genomic comparison of B. massilioalgeriensis sp. nov. strain EB01T with seven other Bacillus species†.
Species | B. th | B. ne | B. ba | B. subt | B. ma | B. kr | B. va | B. we |
---|---|---|---|---|---|---|---|---|
B. thuringiensis | 5,352 | 1,642 | 1,825 | 1,786 | 1,804 | 1,783 | 1,548 | 2,369 |
B. nealsonii | 67.32 | 4,789 | 1,746 | 1,679 | 1,778 | 1,751 | 1,450 | 1,702 |
B. bataviensis | 66.65 | 68.78 | 5,207 | 1,812 | 2,017 | 1,997 | 1,567 | 1,904 |
B. subtilis subsp. subtilis | 65.35 | 65.74 | 66.03 | 4,175 | 1,768 | 1,841 | 2,016 | 1,838 |
B. massilioalgeriensis | 64.77 | 66.61 | 69.33 | 65.44 | 5,098 | 1,985 | 1,541 | 1,863 |
B. kribbensis | 64.54 | 66.05 | 67.20 | 65.86 | 66.92 | 4,918 | 1,604 | 1,864 |
B. vallismortis | 64.56 | 65.05 | 65.54 | 91.06 | 64.92 | 65.30 | 4,097 | 1,592 |
B. weihenstephanensis | 89.95 | 67.27 | 66.70 | 65.46 | 64.87 | 64.56 | 64.70 | 5,653 |
Conclusion
On the basis of phenotypic (Table 2), phylogenetic and genomic analyses (taxonogenomics) (Table 6), we formally propose the creation of Bacillus massilioalgeriensis sp. nov. that contains the strain EB01T. This strain has been found in hypersaline lacustrine sediment sample collected from Algeria.
Description of Bacillus algeriensis sp. nov.
Bacillus algeriensis (al.ge.ri.en’sis. NL. masc.adj. algeriensis, of or pertaining to Algeria). Strain EB01T is a facultative anaerobic Gram-positive, endospore-forming, motile and rod shaped bacterium with rounded ends. Growth is achieved aerobically between 30 and 55°C (optimum 37°C), between 0% and 2.5% NaCl concentration and pH in the range of 6.5–9 (optimum at pH 7). Growth is also observed in microaerophilic atmosphere, however, weak growth was observed under anaerobic conditions. After 24h growth on 5% sheep blood-enriched Columbia agar (BioMerieux) at 37°C, bacterial colonies were smooth, light yellow with 2 mm in diameter. Cells have a length ranging from 2.4 µm to 4.9 µm (mean 3.6 µm) and a diameter ranging from 0.7 µm to 1.1 µm (mean 0.8 µm).
Catalase positive but oxidase negative. Using the commercially available API 50CH system (BioMerieux) according to the manufacturer’s instructions, a weak positive reaction was observed for D-ribose, D-glucose, D-fructose, methyl α-D-glucopyranoside, N-acetylglucosamine, D-maltose, D-lactose, D-melibiose, D-saccharose, D-trehalose, D-tagatose, and hydrolysis of starch. Other tests were negative. Using the API ZYM system (BioMerieux), positive reactions were observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, α chymotrypsin, β-glucuronidase, α-glucosidase, N-acetyl-glucosaminidase and a weak positive reaction was observed for acid phosphatase. The nitrate reduction and β-galactosidase reaction was also positive, but urease and indole production were negative. B. massilioalgeriensis was susceptible to amoxicillin, nitrofurantoin, erythromycin, doxycycline, rifampin, vancomycin, gentamycin, imipenem, trimethoprim-sulfamethoxazole, ciprofloxacin, ceftriaxone and amoxicillin/clavulanic acid, but resistant to nalidixic acid.
The G+C content of the genome is 42.22. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers HG315679 and EMBL database under accession number ERP003483, respectively. The type strain EB01T (= CSUR P857 = DSM 27334) was isolated from sediment sample of the hypersaline lake Ezzemoul sabkha of Oum-El-Bouaghi region in northeastern Algeria.
Declarations
Acknowledgements
The authors thank Linda Hadjadj for technical assistance and Xegen company for automating the genome annotation process.
Authors’ Affiliations
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