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Draft genome sequence of Thermoactinomyces sp. strain AS95 isolated from a Sebkha in Thamelaht, Algeria
Standards in Genomic Sciences volume 11, Article number: 68 (2016)
The members of the genus Thermoactinomyces are known for their protein degradative capacities. Thermoactinomyces sp. strain AS95 is a Gram-positive filamentous bacterium, isolated from moderately saline water in the Thamelaht region of Algeria. This isolate is a thermophilic aerobic bacterium with the capacity to produce extracellular proteolytic enzymes. This strain exhibits up to 99 % similarity with members of the genus Thermoactinomyces, based on 16S rRNA gene sequence similarity. Here we report on the phenotypic features of Thermoactinomyces sp. strain AS95 together with the draft genome sequence and its annotation. The genome of this strain is 2,558,690 bp in length (one chromosome, but no plasmid) with an average G + C content of 47.95 %, and contains 2550 protein-coding and 60 RNA genes together with 64 ORFs annotated as proteases.
Modern metagenomic approaches have provided insights on the evolution and functional capacity of microbial communities resistant to classical culture-based methods . However, these classical techniques remain crucial for understanding the molecular adaptations of microbial guilds, especially those with potential biotechnological applications [2, 3]. Consequently, efforts to isolate novel taxa, particularly from environmentally extreme habitats remain widespread [4, 5].
The genus Thermoactinomyces is a member of the family Thermoactinomycetaceae . The first known representative from this genus ( Thermoactinomyces vulgaris ) was isolated from decaying straw and manure . Since then, a number of isolates, from a wide array of extreme habitats [7–10] have been validly described. Currently, this genus comprises ten validly published species, and a few of these are; Thermoactinomyces vulgaris , Thermoactinomyces intermedius , Thermoactinomyces daqus  and Thermoactinomyces guangxiensis . These species are all Gram-positive, aerobic, non-acid-fast, chemoorganotrophic, filamentous and thermophilic bacteria.
Here, we report the draft genome sequence of Thermoactinomyces sp. strain AS95, which was isolated from a sebkha (endorheic salt pan) in the Thamelaht region of Algeria. We present a summary of the classification and set of phenotypic features for Thermoactinomyces sp. strain AS95 together with the description of the non-contiguous genome sequence and its annotation with particular reference to ORFs encoding proteolytic enzymes.
Classification and features
Thermoactinomyces strain AS95 was isolated from a sebkha water sample collected in June 2013 from the Thamelaht region of Algeria (Table 1). This isolate is a Gram-positive, aerobic, thermophilic, filamentous bacterium (Fig. 1) belonging to the order Bacillales . Based on the 16S rRNA gene sequence similarity searches by BLASTN against the NCBI-NT database, strain AS95 showed 97–99 % sequence similarity to members of the genus Thermoactinomyces . A 16S rRNA gene-based phylogenetic tree of Thermoactinomyces sp. strain AS95 was constructed (Fig. 2), based on neighbor-joining and maximum composite likelihood models with 1000 bootstrap replications using MEGA 7 . The Thermoactinomyces sp. strain AS95 (KU942442) 16S rRNA gene sequence exhibited high identity (99 %) with Thermoactinomyces vulgaris RVH210302 (AY114167), the closest validly published Thermoactinomyces species.
The strain was cultivated on Thermus medium agar containing 2.0 g NaCl, 4.0 g yeast extract, 8.0 g peptone and 30.0 g agar per liter of distilled water. The bacterium grew optimally at 55 °C, with a broad temperature growth range of between 40 and 65 °C (Table 1). The strain grew in liquid media at pH values from 5.6 to 8.6, but optimal growth occurred at a pH of 7.2. Morphologically, the isolate forms white colonies and abundant aerial mycelia with the appearance of well-developed, branched and septate substrate mycelia. The micromorphology of the cells was examined using scanning electron microscopy (Fig. 1). The predominant menaquinone was MK-7. Major fatty acids included iso-C15:0, and significant amounts of iso-C17:0 were also present.
Genome sequencing information
Genome project history
A high-quality draft genome sequence is deposited at DDBJ/EMBL/GenBank under the accession LSVF00000000 and consists of 11 scaffolds of 11 contigs. A summary of the project information and its association with MIGS version 2.0 compliance are shown in Table 2 .
Growth conditions and genomic DNA preparation
Thermoactinomyces sp. strain AS95 was grown aerobically on Thermus medium agar (pH 7.2) at 55 °C for 24 h. Genomic DNA was extracted using a modification of a previously described protocol . The quantity and quality of the genomic DNA was measured using a NanoDrop Spectrophotometer and a Qubit™ Fluorometer (Thermo Fisher Scientific Inc.).
Genome sequencing and assembly
Genomic DNA samples of Thermoactinomyces sp. strain AS95 were sequenced at MR DNA (Shallowater, TX, USA). Genome sequencing was performed on a MiSeq (Illumina, Inc.) generating 2 x 300 bp paired-end libraries. The sequencing run produced a total of 5,085,250 reads, with a mean length of 265.58 bp. The raw paired-end sequences were subjected to the fastxtools software  for quality trimming using a phred quality score ≥ 20. After trimming, a total of 3,013,639 reads with a mean length of 171.11 bp were assembled using SPAdes, version 3.5.0 . The final assembly resulted in a total of 11 scaffolds, which generated a genome size of 2.56 Mb.
Genome annotation was carried out on the RAST server  and using the NCBI Prokaryotic Genome Annotation Pipeline tools . This Whole Genome Shotgun sequence project has been deposited at DDBJ/EMBL/GenBank under accession LSVF00000000. The version described in this paper is version LSVF00000000.
The genome is composed of 2,558,690 nucleotides with 47.95 % G + C content (Table 3) and comprised 11 scaffolds of 11 contigs. The genome contains a total of 2649 genes, 2550 of which were protein coding, 39 pseudogenes and 60 RNA coding genes. The majority of protein-coding genes (75.45 %) were assigned a putative function while the remaining genes were annotated as hypothetical. The distribution of genes in COGs functional categories is presented in Table 4.
A blastp comparison was conducted against the MEROPS database. A total of 64 protein-coding genes (2.4 %) were predicted to share homology with various categories of proteases (Table 5). Of these predictions indicated that 36 were putatively secreted in a classical pathway (SignalP), whereas the other 28 were secreted in a non-classical pathway (SecretomeP). Only 2 of the 64 protein-coding genes share sequence similarities with proteases of the Thermoactinomyces vulgaris and sp. E79 families of peptidases in the MEROPS database.
This study describes the draft genome sequence of Thermoactinomyces sp. strain AS95, which is associated with a high level of extracellular proteolytic activities. To date, only a few metabolic pathways involved in protein degradation have been characterized for the genus Thermoactinomyces . The genome sequence and characteristics of strain AS95 will provide new insights into the mechanisms of protein degradation in the genus Thermoactinomycetes, and towards establishing a comprehensive genomic catalog of the metabolic diversity of the genus Thermoactinomyces .
Cowan DA, Ramond J-B, Makhalanyane TP, De Maayer P. Metagenomics of extreme environments. Curr Opin Microbiol. 2015;25:97–102.
Taylor MP, Eley KL, Martin S, Tuffin MI, Burton SG, Cowan DA. Thermophilic ethanologenesis: future prospects for second-generation bioethanol production. Trends Biotechnol. 2009;27(7):398–405.
Hahn MW, Lünsdorf H, Wu Q, Schauer M, Höfle MG, Boenigk J, Stadler P. Isolation of novel ultramicrobacteria classified as Actinobacteria from five freshwater habitats in Europe and Asia. Appl Environ Microbiol. 2003;69(3):1442–51.
Harrison JP, Gheeraert N, Tsigelnitskiy D, Cockell CS. The limits for life under multiple extremes. Trends Microbiol. 2013;21(4):204–12.
Dash HR, Mangwani N, Chakraborty J, Kumari S, Das S. Marine bacteria: potential candidates for enhanced bioremediation. Appl Microbiol Biotechnol. 2013;97(2):561–71.
Tsilinsky P. On the thermophilic moulds. Ann Inst Pasteur. 1899;13:500–5.
Yao S, Liu Y, Zhang M, Zhang X, Li H, Zhao T, Xin C, Xu L, Zhang B, Cheng C. Thermoactinomyces daqus sp. nov., a thermophilic bacterium isolated from high-temperature Daqu. Int J Syst Evol Microbiol. 2014;64(1):206–10.
Wu H, Liu B, Pan S. Thermoactinomyces guangxiensis sp. nov., a thermophilic actinomycete isolated from mushroom compost. Int J Syst Evol Microbiol. 2015;65(9):2859–64.
Mokrane S, Bouras N, Meklat A, Lahoum A, Zitouni A, Verheecke C, Klenk HP. Thermoactinomyces khenchelensis sp. nov., a filamentous bacterium isolated from soil sediment of a terrestrial hot spring. Antonie van Leeuwenhoek. 2016;109(2):311–317.
Yao S, Xu Y, Xin C, Xu L, Liu Y, Li H, Li J, Zhao J, Cheng C. Genome sequence of Thermoactinomyces daqus H-18, a novel thermophilic species isolated from high-temperature Daqu. Genome announcements. 2015;3(1):e01394–01314.
Kurup V, Hollick G, Pagan E. Thermoactinomyces intermedius, a new species of amylase negative thermophilic actinomycetes. Science-Ciencia Bol Cien Sur. 1980;7:104–8.
Kumar S, Stecher G, Tamura K. Kumar S, Stecher G, Tamura K. MEGA7. Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–1874.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26(5):541–7.
Miller D, Bryant J, Madsen E, Ghiorse W. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol. 1999;65(11):4715–24.
FASTX-Toolkit T: http://hannonlab.cshl.edu/fastx_toolkit/. Accessed Mar 2016.
Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455–77.
Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2014;42(Database issue):D206–214.
Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Ciufo S, Li W. Prokaryotic genome annotation pipeline. 2013.
Białkowska A, Gromek E, Florczak T, Krysiak J, Szulczewska K, Turkiewicz M. Extremophilic Proteases: Developments of Their Special Functions, Potential Resources and Biotechnological Applications. In: Biotechnology of Extremophiles. Switzerland: Springer; 2016: 399–444.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87(12):4576–9.
Gibbons NE, Murray RGE. Proposals Concerning the Higher Taxa of Bacteria. Int J Syst Bacteriol. 1978;28(1):1–6.
Garrity GM, Holt JG. The Road Map to the Manual. In: Boone DR, Castenholz RW, Garrity GM, editors. Bergey’s Manual® of Systematic Bacteriology: Volume One: The Archaea and the Deeply Branching and Phototrophic Bacteria. New York, NY: Springer New York; 2001. p. 119–66.
Murray R. The higher taxa, or, a place for everything. In: Bergey’s Manual of Systematic Bacteriology. 1984.
Ludwig WW, Whitman WB. Bacilli class nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB, editors. Bergey’s manual of systematic bacteriology, vol. 3. 2nd ed. New York: Springer; 2009. p. 19–20.
Euzeby J. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006;56(5):925–7.
Skerman V, McGowan V, Sneath PHA. Approval lists of bacterial names. Int J Syst Bacteriol. 1980;30:255–420.
Hauduroy P, Ehringer G. Dictionnaire des bactéries pathogènes. Paris: Masson; 1953.
Goodfellow M, Jones AL. “Thermoactinomycetaceae”. Bergey's Manual of Systematics of Archaea and Bacteria. New York: John Wiley & Sons, Ltd; 2015. p. 1–18.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25–9.
We wish to acknowledge the following organizations for providing financial support for this project: The Genomics Research Institute and the University of Pretoria (OKIB, DAC, TPM), the National Research Foundation (MWVG, DAC, TPM). The Algerian Ministry of Higher Education and Scientific Research is also acknowledged for funding (MAG and KK).
OKIB performed the analysis, and led the drafting of the manuscript. MAG isolated the strain and conducted confirmatory analysis using 16S rRNA gene sequencing. RP performed the assembly and annotation. MWVG performed the SEM and helped draft the manuscript. KK supervised the isolation of the strain. DAC provided support in drafting the manuscript. TPM conceived the study and provided support in drafting the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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Bezuidt, O.K.I., Gomri, M.A., Pierneef, R. et al. Draft genome sequence of Thermoactinomyces sp. strain AS95 isolated from a Sebkha in Thamelaht, Algeria. Stand in Genomic Sci 11, 68 (2016). https://doi.org/10.1186/s40793-016-0186-2
- Thermoactinomyces sp. strain AS95
- Proteolytic activity