Open Access

Complete genome sequence of Mycobacterium sp. strain (Spyr1) and reclassification to Mycobacterium gilvum Spyr1

  • Aristeidis Kallimanis
  • , Eugenia Karabika
  • , Kostantinos Mavromatis
  • , Alla Lapidus
  • , Kurt M. LaButti
  • , Konstantinos Liolios
  • , Natalia Ivanova
  • , Lynne Goodwin,
  • , Tanja Woyke
  • , Athanasios D. Velentzas
  • , Angelos Perisynakis
  • , Christos C. Ouzounis,
  • , Nikos C. Kyrpides
  • , Anna I. Koukkou
  • and Constantin Drainas
Corresponding author

DOI: 10.4056/sigs.2265047

Received: 01 October 2011

Published: 15 October 2011

Abstract

Mycobacterium sp.Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated.

Keywords:

Mycobacterium gilvumPAH biodegradationpyrene degradation

Introduction

Strain Spyr1 (=LMG 24558, =DSM 45189) is a new strain which based on its morphological and genomic features, belongs to the genus Mycobacterium [1]. It was isolated from Perivleptos, a creosote polluted site in Epirus, Greece (12 Km North of the city of Ioannina), where a wood preserving industry was operating for over 30 years. Strain Spyr1 is of particular interest because it is able to utilize a wide range of PAH substrates as sole sources of carbon and energy, including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Microbial degradation is one of the major routes by which Polycyclic Aromatic Hydrocarbons (PAHs) can be removed from the environment. Strain Spyr1 metabolizes pyrene to 1-Hydroxy-2-naphthoic acid which subsequently is degraded via o-phthalic acid, a pathway also proposed for other Mycobacterium strains [1] exhibiting desirable PAH degradation properties as follows. Complete degradation of pyrene at concentrations 80 mg/L occurred within eight days of incubation in the dark [1]. The extrapolated degradation rate for the growth-phase can be averaged to 10 gml-1day-1, a value similar to that reported for other Mycobacterium species [2,3]. Addition of vitamins or trace amounts of yeast extract were not required for the growth of Spyr1 on any PAH, unlike other Mycobacterium spp. [4]. Use of free or entrapped cells of strain Spyr1 resulted in total removal of PAH from spiked soil samples [1]. Here a summary classification and a set of features for strain Spyr1, along with the description of the complete genome sequence and annotation are presented.

Classification and Features

The phylogenetic tree of strain Spyr1 according to 16S rDNA sequences is depicted in Figure 1.

Figure 1

Phylogenetic location of strain Spyr1 among other Mycobacterium species. Corynebacterium glutamicum was used as the outgroup. The scale bar indicates the number of substitutions per nucleotide position (Number of bootstrap analysis: 1000).

The sequence identity of the 16S rRNA genes of strain Spyr1 to those from the two M. gilvum strains is 99%, while the average nucleotide identity (ANI) [5] between strain Spyr1 and M. gilvum PYR-GCK is 98.5. This information indicates that Spyr1 is a strain of M gilvum. Accordingly, we propose the renaming of the Spy1 strain to M. gilvum Spyr1. The ANI values between strain Spyr1 and other sequenced Mycobacteria are depicted in Figure 2.

Figure 2

ANI values between Mycobacterium sp. Spyr1 and other Mycobacteria. The red line is drawn at ANI 95 a suggested threshold for species.

Strain Spyr1 is an aerobic, non-motile rod, with a cell size of approximately 1.5-2.0 × 3.5-5.0 μm and produces only a weakly positive result under Gram staining. (Figure 3). Colonies were slightly yellowish on Luria agar. The temperature range for growth was 4-37°C with optimum growth at 30-37°C. The pH range was 6.5-8.5 with optimal growth at pH 7.0-7.5. Strain Spyr1 was found to be sensitive to various antibiotics, the minimal inhibitory concentrations were reported as follows: chlorampenicol 10 mgL-1, erythromycin 10 mgL-1, rifampicin 10 mgL-1 and tetracycline 10 mgL-1.

Figure 3

Scanning electron micrograph of Mycobacterium gilvum strain Spyr1.

Catalase and nitrate reductase tests were positive, whereas arginine dihydrolase, gelatinase, lipase, lysine and ornithine decarboxylase, oxidase, urease, citrate assimilation and H2S production tests were negative. No acid was produced in the presence of glucose, lactose, sucrose, arabinose, galactose, glycerol, myo-inositol, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose and xylose (see also Table 1).

Table 1

Classification and general features of strain Spyr1 according to the MIGS recommendations [6]

MIGS ID

   Property

     Term

    Evidence code

   Current classification

     Domain Bacteria

    TAS [7]

     Phylum Actinobacteria

    TAS [8]

     Class Actinobacteria

    TAS [9]

     Subclass Actinobacteridae

    TAS [9,10]

     Order Actinomycetales

    TAS [9-12]

     Suborder Corynebacterineae

    TAS [9,10]

     Family Mycobacteriaceae

    TAS [9-11,13]

     Genus Mycobacterium

    TAS [11,14,15]

     Species Mycobacterium gilvum

    TAS [11,13]

     strain Spyr1

    TAS [1]

   Gram stain

      Weakly positive

    TAS [1]

   Cell shape

     irregular rods

    TAS [1]

   Motility

     Non motile

    TAS [1]

   Sporulation

     nonsporulating

    NAS

   Temperature range

     mesophile

    TAS [1]

   Optimum temperature

     30°C

    TAS [1]

   Salinity

     normal

    TAS [1]

MIGS-22

   Oxygen requirement

     aerobic

    TAS [1]

   Carbon source

     Pyrene, fluoranthene, phenanthrene, anthracene, glucose, yeast extract

    TAS [1]

   Energy source

     Pyrene, fluoranthene, phenanthrene, anthracene, glucose, yeast extract

    TAS [1]

MIGS-6

   Habitat

     Soil

    TAS [1]

MIGS-15

   Biotic relationship

     Free-living

    NAS

MIGS-14

   Pathogenicity

     none

    NAS

   Biosafety level

     1

    NAS

   Isolation

     Creosote contaminated soil

    TAS [1]

MIGS-4

   Geographic location

     Perivleptos, Epirus, Greece

    TAS [1]

MIGS-5

   Sample collection time

     April 2000

    TAS [1]

MIGS-4.1

   Latitude

     39.789

    NAS

MIGS-4.2

   Longitude

     20.781

    NAS

MIGS-4.3

   Depth

     10-20 cm

    TAS [1]

MIGS-4.4

   Altitude

     500 m

    TAS [1]

Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [16].

Chemotaxonomy

Strain Spyr1 major fatty acids are C16:1 (16.7%), C16:0 (32,9%), C18:1(47.5%), C18:0 (1.0%) and C19:0 cyclo(1.1%). The major phospholipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and diphospatidylglycerol (DPG) (80.4, 4.7 and 15.0% respectively).

Genome sequencing information

Genome project history

This organism was selected for sequencing on the basis of its biodegradation capabilities, i.e. metabolizes phenanthrene as a sole source of carbon and energy. The genome project is deposited in the Genome OnLine Database [17] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information

MIGS ID

    Property

    Term

MIGS-31

    Finishing quality

    Finished

MIGS-28

    Libraries used

    Tree genomic libraries: Sanger 9 kb pMCL200, fosmids and 454 standard library

MIGS-29

    Sequencing platforms

    ABI3730, 454 GS FLX

MIGS-31.2

    Sequencing coverage

    10.26 × Sanger; 43.3 × pyrosequence

MIGS-30

    Assemblers

    Newbler version 1.1.02.15, Arachne

MIGS-32

    Gene calling method

    Prodigal 1.4, GenePRIMP

    Genbank ID

    CP002385, CP002386, CP002387

    Genbank Date of Release

    December 21, 2010

    GOLD ID

    Gc01567

    NCBI project ID

    28521

    Database: IMG

    649633070

MIGS-13

    Source material identifier

    DSM 45189

    Project relevance

    Bioremediation, PAH degradation

Growth conditions and DNA isolation

Mycobacterium gilvum Spyr1, DSM 45189 was grown aerobically at 30°C on MM M9 containing 0.01% (w/v) pyrene. DNA was isolated according to the standard JGI (CA, USA) protocol for bacterial genomic DNA isolation using CTAB.

Genome sequencing and assembly

The genome of Mycobacterium gilvum Spyr1 strain was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [18]. Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 6,290 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the Arachne assembler [19]. Possible mis-assemblies were corrected and gaps between contigs were closed by editing in Consed, with custom primer walks from sub-clones or PCR products. A total of 346 Sanger finishing reads were produced to close gaps, resolve repetitive regions, and raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Sanger and 454 sequencing platforms provided 53.56 x coverage of the genome. The final assembly contains 61,443 Sanger reads and 1,300,893 pyrosequencing reads.

Genome annotation

Genes were identified using Prodigal [20] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [21]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Comparative analysis was performed within the Integrated Microbial Genomes (IMG) platform [22].

Genome properties

The genome consists of a 5,547,747 bp long circular chromosome with a G+C content of 68% and two plasmids (Figure 4, Figure 5, Figure 6, and Table 3). The larger is 211,864 bp long with 66% G+C content and the smaller 23,681 bp with 64% G+C content. Of the 5,434 genes predicted, 5,379 were protein-coding genes, and 55 RNAs; 30 pseudogenes were also identified. The majority of the protein-coding genes (67.3%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Figure 4

Graphical circular map of the chromosome of strain Spyr1. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Figure 5

Graphical circular map of first plasmid of strain Spyr1. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Figure 6

Graphical circular map of second plasmid of strain Spyr1. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 3

Genome Statistics

Attribute

    Value

     % of Total

Genome size (bp)

    5,783,292

     100.00%

DNA coding region (bp)

    5,256,086

     90.88%

DNA G+C content (bp)

    3,918,840

     67.76%

Number of replicons

    1

Extrachromosomal elements

    2

Total genes

    5,434

     100.00%

RNA genes

    55

     1.01%

rRNA operons

    2

Protein-coding genes

    5,379

     98.99%

Pseudo genes

    30

     0.55%

Genes with function prediction

    3,657

     67.30%

Genes in paralog clusters

    403

     7.42%

Genes assigned to COGs

    4,038

     74.31%

Genes assigned Pfam domains

    4,188

     77.07%

Genes with signal peptides

    1,617

     29.76%

Genes with transmembrane helices

    1,185

     33.80%

CRISPR repeats

    0

Table 4

Number of genes associated with the general COG functional categories

Code

    value

     %age

      Description

J

    154

     3.4

      Translation, ribosomal structure and biogenesis

A

    20

     0.4

      RNA processing and modification

K

    398

     8.7

      Transcription

L

    305

     6.7

      Replication, recombination and repair

B

    1

     0.0

      Chromatin structure and dynamics

D

    34

     0.7

      Cell cycle control, cell division, chromosome partitioning

Y

    0

     0.0

      Nuclear structure

V

    46

     1.0

      Defense mechanisms

T

    193

     4.2

      Signal transduction mechanisms

M

    176

     3.9

      Cell wall/membrane/envelope biogenesis

N

    10

     0.2

      Cell motility

Z

    1

     0.0

      Cytoskeleton

W

    0

     0.0

      Extracellular structures

U

    38

     0.8

      Intracellular trafficking, secretion and vesicular transport

O

    132

     2.9

      Posttranslational modification, protein turnover, chaperones

C

    303

     6.6

      Energy production and conversion

G

    198

     4.3

      Carbohydrate transport and metabolism

E

    320

     7.0

      Amino acid transport and metabolism

F

    81

     1.8

      Nucleotide transport and metabolism

H

    170

     3.7

      Coenzyme transport and metabolism

I

    412

     9.0

      Lipid transport and metabolism

P

    216

     4.7

      Inorganic ion transport and metabolism

Q

    362

     7.9

      Secondary metabolites biosynthesis, transport and catabolism

R

    636

     14.0

      General function prediction only

S

    351

     7.7

      Function unknown

-

    1,396

     25.7

      Not in COGs

Declarations

Acknowledgements

This work was funded by the program “Pythagoras II” of EPEAEK with 25% National Funds and 75% European Social Funds (ESF) and partly supported by the European Commission FP7 Collaborative Project MICROME (grant agreement number 222886-2). Sequencing and annotation was supported by the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396.


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References

  1. Karabika E, Kallimanis A, Dados A, Pilidis G, Drainas C and Koukkou AI. Taxonomic identification and use of free and entrapped cells of a new Mycobacterium sp., strain Spyr1 for degradation of polycyclic aromatic hydrocarbons (PAH). Appl Biochem Biotechnol. 2009; 159:155-167 View ArticlePubMed
  2. Dean-Ross D and Cerniglia CE. Degradation of pyrene by Mycobacterium flavescens. Appl Microbiol Biotechnol. 1996; 46:307-312 View ArticlePubMed
  3. Vila J, Lopez Z, Sabate J, Minuillon C, Solanas A and Grifoll M. Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons. Appl Environ Microbiol. 2001; 67:5497-5505 View ArticlePubMed
  4. Heitkamp MA, Franklin W and Cerniglia CE. Microbial metabolism of polycyclic aromatic hydrocarbons: isolation and characterization of a pyrene-degrading bacterium. Appl Environ Microbiol. 1988; 54:2549-2555PubMed
  5. Konstantinidis KT and Tiedje JM. Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci USA. 2005; 102:2567-2572 View ArticlePubMed
  6. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and An-giuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  7. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms: proposal for the do-mains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  8. Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 119-169.
  9. Stackebrandt E, Rainey FA and Ward-Rainey NL. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol. 1997; 47:479-491 View Article
  10. Zhi XY, Li WJ and Stackebrandt E. An update of the structure and 16S rRNA gene sequence-based de-finition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol. 2009; 59:589-608 View ArticlePubMed
  11. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  12. Buchanan RE. Studies in the nomenclature and classification of bacteria. II. The primary subdivisions of the Schizomycetes. J Bacteriol. 1917; 2:155-164PubMed
  13. Chester FD. Report of mycologist: bacteriological work. Delaware Agricultural Experiment Station Bulletin. 1897; 9:38-145
  14. Lehmann KB, Neumann R. Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik, First Edition, J.F. Lehmann, München, 1896, p. 1-448.
  15. Runyon EH, Wayne LG, Kubica GP. Genus I. Mycobacterium Lehmann and Neumann 1896, 363. In: Buchanan RE, Gibbons NE (eds), Bergey's Manual of Determinative Bacteriology, Eighth Edition, The Williams and Wilkins Co., Baltimore, 1974, p. 682-701.
  16. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  17. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM and Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2010; 38:D346-D354 View ArticlePubMed
  18. . Web Site
  19. Batzoglou S, Jaffe DB, Stanley K, Butler J, Gnerre S, Mauceli E, Berger B, Mesirov JP and Lander ES. ARACHNE: a whole-genome shotgun assembler. Genome Res. 2002; 12:177-189 View ArticlePubMed
  20. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal Prokaryotic Dynamic Programming Genefinding Algorithm. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  21. Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  22. Markowitz VM, Chen IMA, Palaniappan K, Chu K, Szeto E, Grechkin Y, Rather A, Anderson I, Lykidis A and Mavromatis K. The Integrated Microbial Genomes (IMG) system: an expanding comparative analysis resource. Nucleic Acids Res. 2010; 38:D382-D390 View ArticlePubMed
  23. Pribram E. A contribution to the classification of microorganisms. J Bacteriol. 1929; 18:361-394PubMed
  24. Conn HJ and Dimmick I. Soil bacteria similar in mor-phology to Mycobacterium and Corynebacterium. J Bacteriol. 1947; 54:291-303
  25. Stanford JL and Gunthorpe WJ. A study of some fast-growing scotochromogenic mycobacteria including species descriptions of Mycobacterium gilvum (new species) and Mycobacterium duvalii (new species). Br J Exp Pathol. 1971; 52:627-637PubMed