Non-contiguous finished genome sequence and description of Halopiger goleamassiliensis sp. nov.
© The Author(s) 2014
Published: 15 June 2014
Halopiger goleamassiliensis strain IIH3T sp. nov. is a novel, extremely halophilic archaeon within the genus Halopiger. This strain was isolated from an evaporitic sediment in El Golea Lake, Ghardaïa region (Algeria). The type strain is strain IIH3T. H. goleamassiliensis is moderately thermophilic, neutrophilic, non-motile and coccus-shaped. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,906,923 bp long genome contains 3,854 protein-encoding genes and 49 RNA genes (1 gene is 16S rRNA, 1 gene is 23S rRNA, 3 genes are 5S rRNA, and 44 are tRNA genes).
Halopiger goleamassiliensis sp. nov. strain IIH3T (=KC 430940 =CSUR P3036 = DSM on-going deposit) is the type strain of H. goleamassiliensis sp. nov. This organism is a Gram-negative, extremely halophilic, moderately thermophilic and strictly aerobic archaeon. It was isolated from evaporitic sediment in El Golea Lake, Ghardaïa region (Algeria) as part of a project studying archaeal diversity in hypersaline Lakes of Algeria.
The number of genera and species belonging to Halobacteria (Archaea, Euryarchaeota) has increased recently due to studies of several different hypersaline environments (thalassohaline and athalassohaline) combined with the use of different isolation media and culture conditions . At the time of writing, the family Halobacteriaceae, the single family described within the order Halobacteriales, accommodated 40 recognized genera . The genus Halopiger was proposed by Gutiérrez et al. (2007)  and contains only three species, Halopiger xanaduensis isolated from the Shangmatala Lake (China) , Halopiger aswanensis isolated from a hypersaline soil in Aswan (Egypt)  and Halopiger salifodinae recently isolated from a salt mine in Kuche county, Xinjiang province, China . So far, this genus is composed of strictly aerobic, Gram-negative, polymorphic and pigmented strains. We have recently used [6–18] a polyphasic approach for prokaryotic classification  that includes genomic data [20,21], MALDI-TOF spectra [22,23] and major phenotypic characteristics.
Using this approach, we report here a summary classification and a set of features for Halopiger goleamassiliensis sp.nov. strain IIH3T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the H. goleamassiliensis species.
Classification and features
Classification and general features of Halopiger goleamassiliensis according to the MIGS recommendations .
Evidence code a
Species Halopiger goleamassiliensis
Type strain IIH3T
Thermophile, between 40°C and 60°C
Halophile, 22.5%–25% (optimum)
Sugar or amino acids
Salt Lake sediment
Sediment of El Golea Lake
Differential phenotypic characteristics between strain IIH3T and related species
Cell diameter (µm)
NaCl range (%,w/v)
NaCl optimum (%,w/v)
Temperature range (°C)
Temperature optimum (°C)
Lipids from egg yolk
Utilization of carbohydrates and other compounds as sole carbon sources and acid production from these compounds were determined as described by Oren . Several sugars and amino acids can serve as sole carbon and energy sources (Table 2).
Antibiotic sensitivity tests were determined on SG medium agar plates with antibiotic discs. Strain IIH3T is susceptible to bacitracin (10 µg), novobiocin (30 µg), streptomycin (10 µg) and sulfamethoxazole (25 µg), but resistant to ampicillin (10 µg), cephalothin (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), penicillin G (10 µg), rifampicin (30 µg), tetracycline (30 µg), and vancomycin (30 µg).
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS is considered a reliable and rapid identification method for extremophilic prokaryotes [22,23] and it is used in the present study to characterize the strain IIH3T as previously described [6–18]. A pipette tip was used to pick one isolated archaeal colony from a culture agar plate, and to spread it as a thin film on a MTP 384 MALDI-TOF tar-get plate (Bruker Daltonics, Leipzig, Germany). The colonies from strain IIH3T and from other species of archaea were spotted in triplicate. After air-drying, 1.5 µl of matrix solution (a saturated solution of α-cyano-4-hydroxycinnaminic acid [CHCA] in 50% aqueous acetonitrile containing 2.5% trifluoroacetic acid) per spot was applied and allowed to dry for five minutes.
A score enabled the identification, or not, from the tested species: a score > 2.3 with a validly published 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 IIH3T, none of the obtained scores was > 1, thus suggesting that our isolate was not a member of a known species. We added the spectrum from strain IIH3T to our database for future reference. Figure 5 shows the MALDI-TOF MS spectrum differences between H. goleamassiliensis and other archaea.
Genome sequencing information
Genome project history
Paired-end 5 kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
EMBL Date of Release
June 18, 2018
Study of the archaeal diversity in hypersaline lakes of Algeria
Growth conditions and DNA isolation
H. goleamassiliensis sp.nov. strain IIH3T (= CSUR P3036 =DSM on-going deposit) was grown in SG medium at 55°C in aerobic condition. DNA was isolated and purified using the Genomic DNA purification kit, NucleoSpin Tissue procedure (MACHEREY-NAGEL) following the standard protocol as recommended by the manufacturer. The quality of the DNA was checked on an agarose gel (0.8%) stained with SYBR safe. The yield and the concentration were measured by the Quant-it Picogreen Kit (Invitrogen) on the Genios Tecan Fluorometer at 33.1 ng/µL.
Genome sequencing and assembly
A 5 kb paired-end sequencing strategy (Roche, Meylan, France) was used. This project was loaded on a 1/4 region on PTP Picotiterplate (Roche). Three µg of DNA was mechanically fragmented on the Covaris device (KBioScience-LGC Genomics, Teddington, UK) using miniTUBE-Red 5Kb. The DNA fragmentation was visualized through an Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 4.7 kb. The library was constructed according to the 454 GS FLX Titanium paired end-protocol. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was then loaded on a DNA labchip RNA pico 6000 on the BioAnalyzer. The pattern showed an optimum at 480 bp and the concentration was quantified on a Genios Tecan fluorometer at 642 pg/µL. The concentration equivalence of the library was calculated at 108 molecules/µL. The library was stored at −20°C until further use, and amplified in 2 emPCR reactions at 0.25 cpb, in 2 emPCR at 0.5 cpb and in 2 emPCR at 1 cpb with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yield of the 3 types of paired-end emPCR reactions was 3.68%, 8.05% and 10.69% respectively, in the quality range of 5 to 20% expected from the Roche procedure. These emPCR were pooled. Both libraries were loaded onto GS Titanium PicoTiterPlates (PTP Kit 70×75, Roche) and pyrosequenced with the GS Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche).
A total of 271,702 filter-passed wells were obtained and generated 84.39 Mb with an average length of 325 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp overlap. The final assembly contained 12 contigs (11 large contigs >1500 bp) arranged in 3 scaffolds and generated a genome size of 3.9 Mb, which corresponds to a coverage of 21.6× genome equivalent.
Open Reading Frames (ORFs) were predicted using prodigal with default parameters . ORFs spanning a sequencing gap region were excluded. Assessment of protein function was obtained by comparing the predicted protein sequences with sequences in the GenBank  and the Clusters of Orthologous Groups (COG) databases using BLASTP. RNAmmer  and tRNAscan-SE 1.21  were used for identifying the rRNAs and tRNAs, respectively. SignalP  and TMHMM  were used to predict signal peptides and transmembrane helices, respectively. For alignment lengths greater than 80 amino acids, ORFans were identified if their BLASTP E-value was lower than 1e-03. An E-value of 1e-05 was used if alignment lengths were smaller than 80 amino acids. DNA Plotter  was used for visualization of genomic features and Artemis  was used for data management. The mean level of nucleotide sequence similarity was estimated at the genome level between H. goleamassiliensis and 5 other members of the Halobacteriaceae family (Table 6), by BLASTN comparison of orthologous ORFs in pairwise genomes. Orthologous proteins were detected using the Proteinortho software using the following parameters: e-value 1e-05, 30% identity, 50% coverage and 50% of algebraic connectivity .
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA G+C content (bp)
DNA coding region (bp)
Genes with function prediction
Genes assigned to COGs
Genes with peptide signals
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Post-translational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Comparison with other genomes
Orthologous gene comparison and average nucleotide identity of H. goleamassiliensis with other compared genomes (upper right, numbers of orthologous genes; lower left, mean nucleotide identities of orthologous genes). Bold numbers indicate the numbers of genes or each genome.
Species (accession number)
Halopiger goleamassiliensis (PRJEB1780)
Natronomonas pharaonis (NC_007426)
Haloterrigena turkmenica (NC_013743)
Halalkalicoccus jeotgali (NC_014297)
Halopiger xanaduensis (NC_015666)
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Halopiger goleamassiliensis sp. nov. that contains the strain IIH3T. This archaeal strain has been found in Algeria.
Description of Halopiger goleamassiliensis sp. nov.
Halopiger goleamassiliensis (go.le’a. ma. si. li. en’sis. L. gen. masc. n. goleamassiliensis from the combination of El Golea, the Algerian region where the strain was isolated, and massiliensis, of Massilia, the Latin name of Marseille where the strain was sequenced). It has been isolated from an evaporitic sediment in El Golea Lake, Algeria.
Colonies were smooth, salmon-pigmented and small with 1 to 2 mm in diameter under optimal growth conditions. Strain is strictly aerobic, extremely halophilic and moderately thermophilic archaeon. Growth occurs at NaCl concentrations of 15–30%, at pH values in the range 7–11, and within the temperature range 40–60 °C. Optimal NaCl concentration, pH and temperature for growth are 22.5–25%, 8.0 and 55 °C, respectively. Magnesium is not required for growth. Cells are coccus-shaped (0.8–1.5 µm), Gram-negative, non-motile and lyse in distilled water. Cells are positive for catalase, oxidase and lysine decarboxylase production and negative for urease, arginine dihydrolase, ornithine decarboxylase, tryptophanase, phosphatase, β-galactosidase, D-mannitol, sacharose, starch, dextrose, and D-fructose fermentation. The following substrates are utilized as single carbon and energy sources for growth: pyruvate, D-glucose, D-mannose, D-ribose, D-xylose, maltose, sucrose, lactose, casamino acids, bacto-peptone, bacto-tryptone, and yeast extract. Tween 80, gelatin, and lipids from egg yolk are hydrolysed, whereas urea, starch, and casein are not. Methyl red, Voges-Proskauer, Simmons’ citrate tests, and H2S production are negative.
Cells are susceptible to bacitracin, novobiocin, streptomycin, and sulfamethoxazole but resistant to ampicillin, cephalothin, chloramphenicol, erythromycin, gentamicin, kanamycin, nalidixic acid, penicillin G, rifampicin, tetracycline, and vancomycin.
The G+C content of the DNA is 66.06%. The 16S rRNA and genome sequences are deposited in GenBank and EMBL under accession numbers KC430940 and CBMB010000001-CBMB010000011, respectively. The type strain IIH3T (=CSUR P3036 = DSM on-going deposit) was isolated from an evaporitic sediment in El Golea Lake, Algeria.
The authors thank the entire team of Christelle Desnues and more particularly Dr. Nikolay Popgeorgiev for his help with TEM and Sarah Temmam for her help with tree construction. The authors acknowledge the Xegen Company (www.xegen.fr) for automating the genomic annotation process.
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