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Non-contiguous finished genome sequence and description of Paucisalibacillus algeriensis sp. nov.
Standards in Genomic Sciences volume 9, pages1352–1365 (2014)
Paucisalibacillus algeriensis strain EB02T is the type strain of Paucisalibacillus algeriensis sp. nov., a new species within the genus Paucisalibacillus. This strain, whose genome is described here, was isolated from soil sample from the hypersaline lake Ezzemoul Sabkha in northeastern Algeria. Paucisalibacillus algeriensis is a Gram-positive and strictly aerobic bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,006,766 bp long genome (1 chromosome but no plasmid) exhibits a low G+C content of 36% and contains 3,956 protein-coding and 82 RNA genes, including 9 rRNA genes.
Strain EB02T (= CSUR P858 = DSM 27335) is the type strain of Paucisalibacillus algeriensis sp. nov. It is a strictly aerobic Gram-positive rod, motile by means of peritrichous flagella, and spore-forming bacteria. It was isolated from a soil sample from the hypersaline lake Ezzemoul Sabkha of Oum-El-Bouaghi region in northeastern Algeria, which is the largest nesting area of Mediterranean flamingos. This lake is a Ramsar site (http://www.ramsar.org).
The genus Paucisalibacillusbelongs in the Bacillaceaefamily, and was created by Nunes in 2006 . To date, the genus contains only one validly published species, Paucisalibacillus globulusstrain B22T which was isolated from potting soil in Portugal . It has been described as a Gram positive rod-shaped bacterium, strictly aerobic, spore-forming and motile by means of two polar flagella at one end. It grows in the absence of NaCl, but a low NaCl concentration (1% w/v) improves growth .
The current bacterial taxonomy relies on a combination of various phenotypic, chemotaxonomic and genetic criteria [2–4]. The essential genetic criteria used are DNA-DNA hybridization, which is the ‘gold standard’ criterion to define bacterial species [3,5,6], G+C content and 16S rRNA gene sequence based phylogeny . However, these criteria have several drawbacks and their cutoffs can not be used for all bacterial genera . Presently, as the number of available bacterial genomes is increasing, while costs of whole genome sequencing are decreasing, it has been proposed that genomic information and MALDI-TOF spectra  be included with the main phenotypic characteristics of a strain, in a polyphasic approach (taxono-genomics) to the description of new bacterial taxa [8,10–23].
Here we present a summary classification and a set of features for Paucisalibacillus algeriensis sp. nov. strain EB02T together with the description of the complete genome sequence and annotation. These characteristics support the circumscription of the species Paucisalibacillus algeriensis.
Classification and features
Paucisalibacillus algeriensis strain EB02T was isolated accidentally in July 2012 during research work for the isolation of halophilic actinomycetes, and further characterized. The source of the isolate was a hypersaline soil sample from the Northwestern periphery of the hypersaline lake Ezzemoul Sabkha in the Oum-El-Bouaghi region of northeastern Algeria. This part of the lake is bounded by halophilic vegetation. Samples were taken aseptically at a depth of 10 cm and transferred to sterile containers, then transported in a cooler (4°C) to our lab in Algeria. 10 g of hyprersaline soil were suspended in 90 ml of sterile saline water (0.9% NaCl) and vigorously vortexed. Tenfold serial dilutions up to 10−5 of the soil suspension were plated in ISP (International Streptomyces Project) medium 2 (dextrose 4 g/l, malt extract 10 g/l, yeast extract 4 g/l, agar 20 g/l)  and the plates were incubated at 30°C for 21 days. Strain EB02T was obtained after 24 h of incubation. In order to obtain a pure culture, colonies were transferred after microscopic examination to Nutrient Agar (NA) medium (meat extract 1 g/l, peptone 5 g/l, yeast extract 2 g/l, sodium chloride 5 g/l, agar 15 g/l). Paucisalibacillus algeriensis sp. nov. strain EB02T (Table 1) was isolated by cultivation under aerobic conditions at 30°C. When compared to sequences available in GenBank database using the BLAST program through the National Center for Biotechnology Information (NCBI) server, the 16S rRNA gene sequence of Paucisalibacillus algeriensis strain EB02T (GenBank accession number HG315680) exhibited the highest identity (98.2%) with Paucisalibacillus globulustype strain DSM18846T (Figure 1), the phylogenetically closest validly published Paucisalibacillusspecies. This value was lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridizidation .
Six growth temperatures (25, 30, 37, 45, 50 and 55°C), ten pHs (5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11) and nine NaCl concentrations (0, 2.5, 5, 7.5, 10, 15, 20, 25, 30%) were tested. Growth occurred between 25°C and 50°C, however the optimal growth was observed between 30°C and 37°C, the strain was able to grow at between 0% and 5% NaCl concentration and at pHs in the range of 6.5–9 (optimum at pH 7). After 24 h of aerobic incubation under optimal growth conditions on sheep blood agar (BioMerieux), strain EB02T formed light beige, circular, slightly elevated colonies from 1mm to 2 mm in diameter. Growth of the strain was tested in anaerobic and microaerophilic atmospheres using GasPak EZ Anaerobe Pouch (Becton, Dickinson and Company) and CampyGen Compact (Oxoid) systems, respectively, and in an aerobic atmosphere, with or without 5% CO2. Growth was achieved under aerobic (with and without CO2) and microaerophilic conditions but no growth was observed under anaerobic conditions. Gram staining showed Gram positive rods (Figure 2). Cells grown on agar sporulate. A motility test was positive. The presence of peritrichous flagella and the size of cells were determined by negative staining transmission electron microscopy on a Technai G2 Cryo (FEI) at an operating voltage of 200 kV, the rods have a length ranging from 2.1 µm to 3.2 µm (mean 2.6 µm) and a diameter ranging from 0.4 µm to µm 0.6 (mean 0.5 µm) (Figure 3). Strain EB02T exhibited catalase activity but oxidase activity was negative. Using the commercially available API ZYM system (BioMerieux), positive reactions were observed for alkaline phosphatase, esterase (C4), trypsin, α-glucosidase, and a weak positive reaction was observed for esterase lipase (C8); the other tests were negative. Using the API 50CH system (BioMerieux) according to the manufacturer’s instructions, a weak positive reaction was observed for D-glucose, D-fructose, N-acetylglucosamine, D-saccharose, amygdalin, esculin and salicin. The remaining tests were negative. Indole production, β-galactosidase, urease, and hydrolysis of gelatin and starch were negative, but nitrate reduction reaction was positive. Paucisalibacillus algeriensis was resistant to nalidixic acid, but susceptible to amoxicillin, nitrofurantoin, erythromycin, doxycycline, rifampicin, vancomycin, gentamicin, imipenem, trimethoprim-sulfamethoxazole, ciprofloxacin, ceftriaxone and amoxicillin-clavulanic acid.
When compared to other Paucisalibacillus, Ornithinibacillus, Oceanobacillus and Virgibacillusspecies [1,37–43], Paucisalibacillus algeriensis sp. nov. strain EB02T exhibited the phenotypic differences detailed in (Table 2).
Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was performed as previously described [12,44,45]. Briefly, strain EB02T was cultivated on 5% sheep blood-enriched Columbia agar (BioMerieux) and incubated for 24 h at 30°C. Isolated bacterial colonies were picked, and then deposited as a thin film in 12 replicates on a MALDI-TOF steel target plate (Bruker Daltonics, Bremen, Germany). The plates were allowed to dry at room temperature. Each deposit was overlaid with 1.5 µl of matrix solution containing α-cyano-hydroxycinnamic acid (Sigma, Saint-Quentin Fallavier, France) saturated with 50% acetonitrile, 2.5% trifluoroacetic acid and high-performance liquid chromatography (HPLC)-grade water, and allowed to co-crystallize with the sample. Measurements were conducted using the Microflex LT spectrometer (Bruker Daltonics). Spectra were recorded in the linear positive ion mode over a mass range of 2 to 20 kDa. The acceleration voltage was 20 kV. Spectra were collected as a sum of 240 shots across a spot. The 12 EB02T spectra were imported into the MALDI BioTyper software (version 3.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against 6,335 bacterial spectra supplemented by the spectra from Ornithinibacillus californiensis DSM 16628T, Ornithinibacillus bavariensis DSM 15681T, Oceanobacillus iheyensis CIP 107618T which were the most closely related species on the basis of their 16S rRNA gene sequences, and Ornithinibacillus contaminans DSM 22953T, Oceanobacillus oncorhynchi subsp incaldanensis CIP 109235T, Oceanobacillus picturae CIP 108264T, Oceanobacillus profundus CIP 109535T, Oceanobacillus oncorhynchi subsp. oncorhynchi CIP 108867T, Oceanobacillus chironomi CIP 109536T, used as reference data in the BioTyper database. A score enabled the identification, or not, from the tested species: a score > 2 with a validated species enabled the identification at the species level, a score > 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification. For strain EB02T, the scores obtained ranged from 1.0 to 1.4 thus suggesting that our isolate was a new species. We added the spectrum from strain EB02T (Figure 4) to our database. Spectrum differences with those of Ornithinibacillus and Oceanobacillusrelated species are shown in (Figure 5).
Genome sequencing information
Genome project history The organism was selected for sequencing on the basis of its phylogenetic position and 16S rDNA sequence similarity to other members of the genus Paucisalibacillus, and is part of a study of the microbial diversity of the hypersaline lakes in northeastern Algeria. It was the 2nd genome of a Paucisalibacillusspecies and the first genome of Paucisalibacillus algeriensis sp. nov. The EMBL accession number is CBYO000000000 and consists of 23 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance .
Growth conditions and DNA isolation
Paucisalibacillus algeriensis sp. nov strain EB02T, was grown aerobically on 5% sheep blood enriched Columbia agar at 30°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.
Genomic DNA of Paucisalibacillus algeriensis sp. nov. strain EB02T was sequenced on the MiSeq instrument (Illumina, Inc, San Diego CA 92121, USA) with paired end and barcode strategies in order to be mixed with 7 others genomic projects constructed according 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 43.7 ng/µL and dilution was performed to require 1ng 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 Caliper-Perkin Elmer labchip (Caliper Life Sciences, Inc, Massachusetts, USA). Then limited cycle PCR amplification completed the tag adapters and introduce dual-index barcodes. After purification on Ampure beads (Life technolgies, Carlsbad, CA, USA), the libraries were then 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 2x250 bp read length. Within this pooled run, the index representation was determined to be 7.1%. Total information of 2.4 G bases was obtained from a 320 K/mm2 density with 94.9% (5,757,000) of the clusters passing quality control (QC) filters. From the genome sequencing process, the 1,375,572 produced Illumina reads for Paucisalibacillus algeriensis EB02T were filtered according to the read qualities and sizes using the fastq-mcf program (Ea-utils: command-line tools for processing biological sequencing data) . 1,296,442 filtered read sequences were kept for genome assembly. The SPAdes Genome assembler (http://bioinf.spbau.ru/spades) was used with different kmer values (from 67 to 99, interval 4) and the best assembly result, with kmer value (n=95) producing 23 contigs with sizes from 1214-bp to 717,245-bp and average size of 174,207-bp, was retained for genome annotation.
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters. The predicted bacterial protein sequences were searched against the Clusters of Orthologous Groups (COG) databases and the GenBank database  using BLASTP. Ribosomal RNAs were found by using RNAmmer 2.1 server [50,51] and BLASTn against the GenBank database, whereas the tRNAScan-SE tool  was used to find tRNA genes. Transmembrane helices and lipoprotein signal peptides were predicted using phobius web server . 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  was used for data management and DNA Plotter  was used for visualization of genomic features. To estimate the mean level of nucleotide sequence similarity at the genome level between Paucisalibacillus algeriensis sp nov. strain EB02T and Paucisalibacillus globulus, Ornithinibacillus scapharcae, Oceanobacillus iheyensis and Virgibacillus halodenitrificans, we used the Average Genomic Identity of Orthologous gene Sequences (AGIOS) home-made software. Briefly, this software uses the Proteinortho software  to detect orthologous proteins between genomes compared two by two, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm.
The genome is 4,006,766 bp long (1 chromosome but no plasmid) with 36% GC content (Figure 6 and Table 4). It is composed of 23 contigs. Of the 4,038 predicted genes, 3,956 were protein-coding genes, and 82 were RNAs (7 5S rRNA genes, 1 16S rRNA gene, 1 23S rRNA gene, and 73 tRNA genes). A total of 2,691 genes (68.02%) were assigned a putative function (by cogs or by NR blast), of which 179 were identified as ORFans (4.52%). The remaining genes were annotated as hypothetical proteins (821 genes, 20.75%). The distribution of genes into COGs functional categories is presented in Table 5. The properties and statistics of the genome are summarized in Tables 4 and 5.
f Paucisalibacillus algeriensis strain EB02T with those of Paucisalibacillus globulus strain B22T, Ornithinibacillus scapharcae strain TW25T, Oceanobacillus iheyensis strain HTE831T and Virgibacillus halodenitrificans strain 1806T (Table 6). The draft genome of Paucisalibacillus algeriensis (4.006Mb) is smaller in size than that of Paucisalibacillus globulus strain B22T (4.24 Mb) but larger than those of Oceanobacillus iheyensis strain HTE831T, Ornithinibacillus scapharcae strain TW25T and Virgibacillus halodenitrificans strain 1806T (3.63, 3.84 and 3.92 Mb, respectively). Paucisalibacillus algeriensis has a higher G+C content (36%) than those of Oceanobacillus iheyensis strain HTE831T and Paucisalibacillus globulus strain B22T (35.7% and 35.8%, respectively) but lower than those of Ornithinibacillus scapharcae strain TW25T and Virgibacillus halodenitrificans strain 1806T (36.7% and 37.4%, respectively). Paucisalibacillus algeriensis has more predicted protein coding genes (3,956) than Oceanobacillus iheyensis strain HTE831T and Virgibacillus halodenitrificans strain 1806T (3,500 and 3,886, respectively) but fewer genes than Ornithinibacillus scapharcae strain TW25T and Paucisalibacillus globulus strain B22T (3,986 and 4,191, respectively). In addition, Paucisalibacillus algeriensis shared 2,591, 2,502, 2,002, and 2,083 orthologous genes with Paucisalibacillus globulus, Ornithinibacillus scapharcae, Oceanobacillus iheyensis and Virgibacillus halodenitrificans respectively.
The average nucleotide sequence identity of orthologous genes ranges from 69.15 to 83.06% among all of the genomes which were used for the comparison, and from 69.49 to 83.06% between Paucisalibacillus algeriensis and the other genomes (Table 6,. Table 7.), presenting a high sequence identity of orthologous genes with Paucisalibacillusgenus (83.06%), thus confirming its new species status in the Paucisalibacillus genus.
On the basis of phenotypic (Table 2), phylogenetic and genomic analyses (taxonogenomics) (Table 6, Table 7.), we formally propose the creation of Paucisalibacillus algeriensis sp. nov. that contains the strain EB02T. This strain has been found in a hypersaline lacustrine soil sample collected from Algeria.
Description of Paucisalibacillus algeriensis sp. nov EB02T
Paucisalibacillus algeriensis (al.ge.ri.en’sis. N.L. masc.adj. algeriensis, of Algeria, where strain EB02T was isolated). Strain EB02T is a strictly aerobic Gram-positive rod, endospore-forming, motile by means of peritrichous flagella. Growth is achieved aerobically between 25°C and 50°C, but optimal growth was observed between 30°C-37°C. The strain was able to grow between 0% and 5% NaCl concentration and at pHs in the range of 6.5–9 (optimum at pH 7). Growth is also observed under a microaerophilic atmosphere, however, no growth was observed under anaerobic conditions. After 24h growth on 5% sheep blood-enriched Columbia agar (BioMerieux) at 30°C, bacterial colonies were light beige, circular, slightly elevated and from 1 mm to 2 mm in diameter. Cells have a length ranging from 2.1 µm to 3.2 µm (mean 2.6 µm) and a diameter ranging from 0.4 µm to µm 0.6 (mean 0.5 µm).
Catalase activity was positive but oxidase activity was negative. Using the commercially available API ZYM system (BioMerieux), positive reactions were observed for alkaline phosphatase, esterase (C4), trypsin, α-glucosidase, and weak positive reaction was observed for esterase lipase (C8). The other tests were negative. Using the API 50CH system (BioMerieux) according to the manufacturer’s instructions, a weak positive reaction was observed for, D-glucose, D-fructose, N-acetylglucosamine, D-saccharose, amygdalin, esculin and salicin. The remaining tests were negative. Indole production, β-galactosidase, urease, hydrolysis of gelatin and starch were negative, but the nitrate reduction reaction was positive. Paucisalibacillus algeriensis was resistant to nalidixic acid, but susceptible to amoxicillin, nitrofurantoin, erythromycin, doxycycline, rifampicin, vancomycin, gentamicin, imipenem, trimethoprim-sulfamethoxazole, ciprofloxacin, ceftriaxone and amoxicillin-clavulanic acid. The G+C content of the genome is 36%.
The 16S rRNA and genome sequences are deposited in GenBank under accession number HG315680 and EMBL database under accession number CBYO000000000, respectively. The type strain EB02T (= CSUR P858 = DSM 27335) was isolated from a soil sample from the margin of the hypersaline lake Ezzemoul Sabkha in the Oum-El-Bouaghi region of northeastern Algeria.
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The authors thank Linda Hadjadj for technical assistance and Xegen company for automating the genome annotation process.
These authors contributed equally to this work.