Open Access

Draft genome sequence of Thermoactinomyces sp. strain AS95 isolated from a Sebkha in Thamelaht, Algeria

  • Oliver K. I. Bezuidt1, 2,
  • Mohamed A. Gomri3,
  • Rian Pierneef4,
  • Marc W. Van Goethem1,
  • Karima Kharroub3,
  • Don A. Cowan1 and
  • Thulani P. Makhalanyane1Email author
Standards in Genomic Sciences201611:68

https://doi.org/10.1186/s40793-016-0186-2

Received: 10 May 2016

Accepted: 27 August 2016

Published: 9 September 2016

Abstract

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.

Keywords

Thermoactinomyces sp. strain AS95 Genome Thermophilic Proteolytic activity Taxonomo-genomics

Introduction

Modern metagenomic approaches have provided insights on the evolution and functional capacity of microbial communities resistant to classical culture-based methods [1]. 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 [6]. Since then, a number of isolates, from a wide array of extreme habitats [710] have been validly described. Currently, this genus comprises ten validly published species, and a few of these are; Thermoactinomyces vulgaris [6], Thermoactinomyces intermedius [11], Thermoactinomyces daqus [7] and Thermoactinomyces guangxiensis [8]. 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.

Organism information

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 [12]. 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.
Table 1

Classification and general features of Thermoactinomyces sp. strain AS95

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain: Bacteria

TAS [20]

  

Phylum: Firmicutes

TAS [2123]

  

Class: Bacilli

TAS [24, 25]

  

Order: Bacillales

TAS [26, 27]

  

Family: Thermoactinomycetaceae

TAS [25, 28]

  

Genus: Thermoactinomyces

TAS [6]

  

Species: Thermoactinomyces sp.

IDA

  

Strain: AS95

IDA

 

Gram stain

Positive

IDA

 

Cell shape

Filamentous

IDA

 

Motility

Non-motile

IDA

 

Sporulation

Endospores on unbranched sporophores

IDA

 

Temperature range

40–65 °C (Thermophilic)

IDA

 

Optimum temperature

55 °C

IDA

 

pH range; Optimum

5.6–8.6; 7.2

IDA

 

Carbon source

Peptides

IDA

GS-6

Habitat

Saline water

IDA

MIGS-6.3

Salinity

5.0 % total salt (w/v)

IDA

MIGS-22

Oxygen requirement

Aerobic

IDA

MIGS-15

Biotic relationship

Free-living

IDA

MIGS-14

Pathogenicity

Non-pathogen

IDA

MIGS-4

Geographic location

Thamelaht,, Algeria

IDA

MIGS-5

Sample collection time

20 June 2013

IDA

MIGS-4.1

Latitude

36°32'18.29"N

IDA

MIGS-4.2

Longitude

5°11'48.89"E

IDA

MIGS-4.4

Altitude

890 m above sea level

IDA

aEvidence codes – IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e. a direct report exists in the literature). These evidence codes are from the Gene Ontology Project [29]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements

Fig. 1

Scanning electron microscopy of Thermoactinomyces sp. strain AS95 using a Cryo-SEM (JEOL)

Fig. 2

Phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain AS95 (1435 bp) and strains of related genera of the family Thermoactinomycetaceae. The strains and their corresponding Genbank accession numbers are shown following the organism name and indicated in parentheses. The phylogenetic tree was made using the neighbor-joining method with maximum composite likelihood model implemented in MEGA 7. The tree includes the 16S rRNA gene sequence of Sulfobacillus acidophilus DSM 10332T as outgroup. Bootstrap consensus trees were inferred from 1000 replicates, only bootstrap values >50 % are indicated. The scale bar represents 0.02 nucleotide changes per position. (♦) indicates the isolate assessed in the current study, Thermoactinomyces sp. strain AS95

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 [13].
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

One paired-end 300 bp library

MIGS-29

Sequencing platforms

MiSeq-Illumina

MIGS-31.2

Fold coverage

40.0×

MIGS-30

Assemblers

SPAdes 3.5.0

MIGS-32

Gene calling method

NCBI Prokaryotic Genome, Annotation Pipeline

Genbank ID

LSVF00000000

Genbank Date of Release

April 04, 2016

BIOPROJECT

PRJNA312744

GOLD ID

Gs0118400

MIGS-13

Project relevance

Biotechnological, Environmental

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 [14]. 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 [15] 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 [16]. The final assembly resulted in a total of 11 scaffolds, which generated a genome size of 2.56 Mb.

Genome annotation

Genome annotation was carried out on the RAST server [17] and using the NCBI Prokaryotic Genome Annotation Pipeline tools [18]. 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.

Genome properties

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.
Table 3

Genome statistics of the Thermoactinomyces sp. strain AS95

Attribute

Value

% of totala

Genome size (bp)

2,558,690

100.00

DNA coding region (bp)

2,214,681

86.56

DNA G + C (bp)

1,226,817

47.95

DNA scaffolds

11

 

Total genes

2,649

100.00

Protein coding genes

2,550

96.26

RNA genes

60

2.26

Pseudo genes

39

1.47

Genes in internal clusters

ND

ND

Genes with function prediction

1,296

50.82

Genes with Pfam domains

2,001

78.47

Genes assigned to COGs

1,924

75.45

Genes with signal peptides

164

6.43

Genes with transmembrane helices

655

25.69

CRISPR repeats

2

ND

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome. ND: Not determined

Table 4

Number of genes associated with general COG functional categories

Code

Value

% of totala

Description

J

154

9.96

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

145

5.68

Transcription

L

100

3.92

Replication, recombination and repair

B

0

0.00

Chromatin structure and dynamics

D

27

1.05

Cell cycle control, mitosis and meiosis

V

32

1.25

Defense mechanisms

T

71

2.78

Signal transduction mechanisms

M

99

3.88

Cell wall/membrane biogenesis

N

8

0.31

Cell motility

Z

0

0.03

Cytoskeleton

U

33

1.29

Intracellular trafficking and secretion

O

85

3.33

Posttranslational modification, protein turnover, chaperones

C

135

5.29

Energy production and conversion

G

122

4.78

Carbohydrate transport and metabolism

E

213

8.35

Amino acid transport and metabolism

F

70

2.74

Nucleotide transport and metabolism

H

108

4.23

Coenzyme transport and metabolism

I

109

4.27

Lipid transport and metabolism

P

101

3.96

Inorganic ion transport and metabolism

Q

53

2.07

Secondary metabolites biosynthesis, transport and catabolism

R

249

9.76

General function prediction only

S

196

7.68

Function unknown

-

626

24.54

Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome

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.
Table 5

The four major types of proteases predicted in Thermoactinomyces sp. strain AS95

Type

Classical (SignalP)

Non-classical (SecretomeP)

Cysteine

6

3

Metallo

18

12

Serine

11

10

Threonine

0

2

Conclusions

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 [19]. 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 .

Declarations

Acknowledgements

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).

Authors’ contributions

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.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Centre for Microbial Ecology and Genomics, Department of Genetics, University of Pretoria
(2)
Biotechnology Platform, Agricultural Research Council
(3)
Equipe Métabolites des Extrêmophiles, Laboratoire de Recherche Biotechnologie et Qualité des Aliments, INATAA, Université Frères Mentouri Constantine
(4)
Centre for Bioinformatics and Computational Biology, Department of Biochemistry, University of Pretoria

References

  1. Cowan DA, Ramond J-B, Makhalanyane TP, De Maayer P. Metagenomics of extreme environments. Curr Opin Microbiol. 2015;25:97–102.View ArticlePubMedGoogle Scholar
  2. 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.View ArticlePubMedGoogle Scholar
  3. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Harrison JP, Gheeraert N, Tsigelnitskiy D, Cockell CS. The limits for life under multiple extremes. Trends Microbiol. 2013;21(4):204–12.View ArticlePubMedGoogle Scholar
  5. 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.View ArticlePubMedGoogle Scholar
  6. Tsilinsky P. On the thermophilic moulds. Ann Inst Pasteur. 1899;13:500–5.Google Scholar
  7. 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.View ArticlePubMedGoogle Scholar
  8. 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.View ArticlePubMedGoogle Scholar
  9. 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.Google Scholar
  10. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  11. 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.Google Scholar
  12. 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.Google Scholar
  13. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  14. 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.PubMedPubMed CentralGoogle Scholar
  15. FASTX-Toolkit T: http://hannonlab.cshl.edu/fastx_toolkit/. Accessed Mar 2016.
  16. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  17. 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.View ArticlePubMedGoogle Scholar
  18. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Ciufo S, Li W. Prokaryotic genome annotation pipeline. 2013.Google Scholar
  19. 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.Google Scholar
  20. 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.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Gibbons NE, Murray RGE. Proposals Concerning the Higher Taxa of Bacteria. Int J Syst Bacteriol. 1978;28(1):1–6.View ArticleGoogle Scholar
  22. 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.Google Scholar
  23. Murray R. The higher taxa, or, a place for everything. In: Bergey’s Manual of Systematic Bacteriology. 1984.Google Scholar
  24. 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.Google Scholar
  25. 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.View ArticleGoogle Scholar
  26. Skerman V, McGowan V, Sneath PHA. Approval lists of bacterial names. Int J Syst Bacteriol. 1980;30:255–420.Google Scholar
  27. Hauduroy P, Ehringer G. Dictionnaire des bactéries pathogènes. Paris: Masson; 1953.Google Scholar
  28. Goodfellow M, Jones AL. “Thermoactinomycetaceae”. Bergey's Manual of Systematics of Archaea and Bacteria. New York: John Wiley & Sons, Ltd; 2015. p. 1–18.Google Scholar
  29. 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.View ArticlePubMedPubMed CentralGoogle Scholar

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© The Author(s). 2016