Open Access

Complete genome sequence of Arthrobacter phenanthrenivorans type strain (Sphe3)

  • Aristeidis Kallimanis
  • , Kurt M. LaButti
  • , Alla Lapidus
  • , Alicia Clum
  • , Athanasios Lykidis
  • , Kostantinos Mavromatis
  • , Ioanna Pagani
  • , Konstantinos Liolios
  • , Natalia Ivanova
  • , Lynne Goodwin,
  • , Sam Pitluck
  • , Amy Chen
  • , Krishna Palaniappan
  • , Victor Markowitz
  • , Jim Bristow
  • , Athanasios D. Velentzas
  • , Angelos Perisynakis
  • , Christos C Ouzounis,
  • , Nikos C. Kyrpides
  • , Anna I. Koukkou
  • and Constantin Drainas
Corresponding author

DOI: 10.4056/sigs.1393494

Received: 29 April 2011

Published: 29 April 2011

Abstract

Arthrobacter phenanthrenivorans is the type species of the genus, and is able to metabolize phenanthrene as a sole source of carbon and energy. A. phenanthrenivorans is an aerobic, non-motile, and Gram-positive bacterium, exhibiting a rod-coccus growth cycle which was originally isolated from a creosote polluted site in Epirus, Greece. Here we describe the features of this organism, together with the complete genome sequence, and annotation.

Keywords:

ArthrobacterdioxygenasesPAH biodegradationphenanthrene degradation

Introduction

Strain Sphe3T (=DSM 18606T = LMG 23796T) is the type strain of Arthrobacter phenanthrenivorans [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 [2]. Strain Sphe3T is of particular interest because it is able to metabolize phenanthrene at concentrations of up to 400 mg/L as a sole source of carbon and energy, at rates faster than those reported for other Arthrobacter species [3-5]. It appears to internalize phenanthrene with two mechanisms: a passive diffusion when cells are grown on glucose, and an inducible active transport system, when cells are grown on phenanthrene as a sole carbon source [2]. Here we present a summary classification and a set of features for A. phenanthrenivorans strain Sphe3T, together with the description of the complete genome sequencing and annotation.

Classification and features

Figure 1 shows the phylogenetic neighborhood of A. phenanthrenivorans strain Sphe3T in a 16S rRNA based tree.

Figure 1

Phylogenetic tree highlighting the position of A. phenanthrenivorans strain Sphe3T relative to the other type strains within the family. Numbers above branches are support values from 100 bootstrap replicates.

Strain Sphe3T is a Gram-positive, aerobic, non-motile bacterium exhibiting a rod-coccus cycle (Figure 2), with a cell size of approximately 1.0-1.5 x 2.5-4.0 μm. Colonies were slightly yellowish on Luria agar. The temperature range was 40-37oC with optimum growth at 30-37oC. The pH range was 6.5-8.5 with optimal growth at pH 7.0-7.5 (Table 1). Strain Sphe3T was found to be sensitive to various antibiotics, the minimal inhibitory concentrations of which were estimated as follows: ampicillin 20 mgL-1, chloramphenicol 10 mgL-1, erythromycin 10 mgL-1, neomycin 20 mgL-1, rifampicin 10 mgL-1 and tetracycline 10 mgL-1.

Figure 2

Scanning electron micrograph of A. phenanthrenivorans strain Sphe3T

Table 1

Classification and general features of A. phenanthrenivorans strain Sphe3T 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]

   Family Micrococcaceae

   TAS [9-11,13]

   Genus Arthrobacter

   TAS [1,11,14-17]

   Species Arthrobacter phenanthrenivorans

   TAS [1]

   Type strain Sphe3

   TAS [1]

  Gram stain

   positive

   TAS [1]

  Cell shape

   irregular rods, coccoid

   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

   Phenanthrene, glucose, yeast extract

   TAS [1,2]

  Energy source

   Phenanthrene, glucose, yeast extract

   TAS [1,2]

MIGS-6

  Habitat

   Soil

   TAS [1,2]

MIGS-15

  Biotic relationship

   Free-living

   NAS

MIGS-14

  Pathogenicity

   none

   NAS

  Biosafety level

   1

   NAS

  Isolation

   Creosote contaminated soil

   TAS [1,2]

MIGS-4

  Geographic location

   Perivleptos, Epirus, Greece

   TAS [1,2]

MIGS-5

  Sample collection time

   April 2000

   TAS [1,2]

MIGS-4.1

  Latitude

   39.789

   NAS

MIGS-4.2

  Longitude

   20.781

   NAS

MIGS-4.3

  Depth

   10-20 cm

   TAS [1,2]

MIGS-4.4

  Altitude

   500 meters

   TAS [1,2]

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Amylase, 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 and sucrose.

Chemotaxonomy

Menaquinones are the sole respiratory lipoquinones of A. phenanthrenivorans strain Sphe3T. Both MK-8 and MK-9(H2) are present in a ratio of 3.6:1, respectively. Major fatty acids are anteiso-C15:0 (36.2%), iso-C16:0 (15.7%), iso-C15:0 (14.3%), anteiso-C17:0 (12.0%), C16:0 (8.3%), iso-C17:0 (4.0%), C16:1ω7c (2.5%) and C14:0 (1.4%). The major phospholipids were diphospatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), (63.8, 27.5 and 4.0% respectively).

Genome sequencing and annotation

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 [18] 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

    Three genomic libraries:    6kb (pMCL200) and fosmids (pcc1Fos) Sanger libraries     and one 454 pyrosequence standard library

MIGS-29

    Sequencing platforms

    ABI 3730. 454 GS FLX

MIGS-31.2

    Sequencing coverage

    9.33× Sanger, 17.45× pyrosequence

MIGS-30

    Assemblers

    Newbler version 1.1.02.15, Arachne

MIGS-32

    Gene calling method

    Prodigal, GenePRIMP

    INSDC ID

    CP002379

    Genbank Date of Release

    February 16, 2011

    GOLD ID

    Gc01621

    NCBI project ID

    38025

    Database: IMG-GEBA

    2503538005

MIGS-13

    Source material identifier

    DSM 12885

    Project relevance

    Tree of Life, GEBA

Growth conditions and DNA isolation

A. phenanthrenivorans Sphe3T, DSM 18606T was grown aerobically at 30°C on MM M9 containing 0.02% (w/v) phenanthrene. 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 Arthrobacter phenanthrenivorans type strain (Sphe3)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 [19]. Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,967 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 [20]. Possible mis-assemblies were corrected and gaps between contigs were closed by by editing in Consed, by custom primer walks from sub-clones or PCR products. A total of 822 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to 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 26.78 x coverage of the genome. The final assembly contains 44,113 Sanger reads and 599,557 pyrosequencing reads.

Genome annotation

Genes were identified using Prodigal [21] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [22]. 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. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [23].

Genome properties

The genome consists of a 4,250,414 bp long chromosome with a GC content of 66% and two plasmids both with 62% GC content, the larger being 190,450 bp long and the smaller 94,456 bp (Table 3, Figure 3 and Figure 4). Of the 4,288 genes predicted, 4,212 were protein-coding genes, and 76 RNAs; 77 pseudogenes were also identified. The majority of the protein-coding genes (73.8%) were assigned with 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.

Table 3

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

4,535,320

100.00%

DNA Coding region (bp)

4,033,112

88.93%

DNA G+C content (bp)

2,964,596

65.37%

Number of replicons

1

Extrachromosomal elements

2

Total genes

4,288

100.00%

RNA genes

76

1.77%

rRNA operons

4

Protein-coding genes

4,212

98.23%

Pseudo genes

77

1.80%

Genes with function prediction

3,167

73.86%

Genes in paralog clusters

930

21.69%

Genes assigned to COGs

3,075

71.71%

Genes assigned Pfam domains

3,277

76.42%

Genes with signal peptides

978

22.81%

Genes with transmembrane helices

999

23.30%

CRISPR repeats

0

Figure 3

Graphical circular map of the chromosome, not drawn to scale with plasmids. 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 4

The two plasmids, not drawn to scale with chromosome. 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 4

Number of genes associated with the general COG functional categories

Code

   value

   %age

   Description

J

   153

   4.5

   Translation, ribosomal structure and biogenesis

A

   1

   0.0

   RNA processing and modification

K

   308

   9.0

   Transcription

L

   239

   7.0

   Replication, recombination and repair

B

   1

   0.0

   Chromatin structure and dynamics

D

   29

   0.8

   Cell cycle control, cell division, chromosome partitioning

Y

   0

   0.0

   Nuclear structure

V

   45

   1.3

   Defense mechanisms

T

   135

   3.9

   Signal transduction mechanisms

M

   142

   4.1

   Cell wall/membrane/envelope biogenesis

N

   2

   0.0

   Cell motility

Z

   0

   0.0

   Cytoskeleton

W

   0

   0.0

   Extracellular structures

U

   45

   1.3

   Intracellular trafficking and secretion, and vesicular transport

O

   100

   2.9

   Posttranslational modification, protein turnover, chaperones

C

   205

   6.0

   Energy production and conversion

G

   396

   11.6

   Carbohydrate transport and metabolism

E

   329

   9.6

   Amino acid transport and metabolism

F

   87

   2.5

   Nucleotide transport and metabolism

H

   141

   4.2

   Coenzyme transport and metabolism

I

   134

   3.9

   Lipid transport and metabolism

P

   167

   4.9

   Inorganic ion transport and metabolism

Q

   95

   2.8

   Secondary metabolites biosynthesis, transport and catabolism

R

   430

   12.6

   General function prediction only

S

   238

   6. 9

   Function unknown

-

   1,213

   28.3

   Not in COGs

Declarations

Acknowledgements

This work was supported by the program “Pythagoras II” of EPEAEK with 25% National Funds and 75% European Social Funds (ESF). NCK is 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.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Kallimanis A, Kavakiotis K, Perisynakis A, Sproer C, Pukall R, Drainas C and Koukkou AI. Arthrobacter phenanthrenivorans sp. nov., to accommodate the phenanthrene-degrading bacterium Arthrobacter sp. strain Sphe3. Int J Syst Evol Microbiol. 2009; 59:275-279 View ArticlePubMed
  2. Kallimanis A, Frillingos S, Drainas C and Koukkou AI. Taxonomic identification, phenanthrene uptake activity and membrane lipid alterations of the PAH degrading Arthrobacter sp. strain Sphe3. Appl Microbiol Biotechnol. 2007; 76:709-717 View ArticlePubMed
  3. Grifoll M, Casellas M, Bayona JM and Solanas AM. Isolation and Characterization of a Fluorene-Degrading Bacterium: Identification of Ring Oxidation and Ring Fission Products. Appl Environ Microbiol. 1992; 58:2910-2917PubMed
  4. Samanta SK, Chakraborti AK and Jain RK. Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol. Appl Microbiol Biotechnol. 1999; 53:98-107 View ArticlePubMed
  5. Seo JS, Keum YS, Hu Y, Lee SE and Li QX. Phenanthrene degradation in Arthrobacter sp. Pl-1: Initial 1,2-, 3,4- and 9,10-dioxygenation, and meta- and ortho-cleavages of naphthalene-1,2-diol after its formation from naphthalene-1,2-dicarboxylic acid and hydroxyl naphthoic acids. Chemosphere. 2006; 65:2388-2394 View ArticlePubMed
  6. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli 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 domains 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 definition 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. Pribram E. A contribution to the classification of microorganisms. J Bacteriol. 1929; 18:361-394PubMed
  14. Conn HJ and Dimmick I. Soil bacteria similar in morphology to Mycobacterium and Corynebacterium. J Bacteriol. 1947; 54:291-303
  15. Keddie RM. Genus II. Arthrobacter Conn and Dimmick 1947, 300. In: Buchanan RE, Gibbons NE (eds), Bergey's Manual of Determinative Bacteriology, Eighth Edition, The Williams and Wilkins Co., Baltimore, 1974, p. 618-625.
  16. Koch C, Schumann P and Stackebrandt E. Reclassification of Micrococcus agilis (Ali-Cohen 1889) to the genus Arthrobacter as Arthrobacter agilis comb. nov. and emendation of the genus Arthrobacter. Int J Syst Bacteriol. 1995; 45:837-839 View ArticlePubMed
  17. . Opinion 24. Rejection of the Generic Name Arthrobacter Fischer 1895 and Conservation of the Generic Name Arthrobacter Conn and Dimmick 1947. Int Bull Bacteriol Nomencl Taxon. 1958; 8:171-172 View Article
  18. 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. 2009; 38:D346-D354 View ArticlePubMed
  19. JGI website. Web Site
  20. The Arachne assembler. Web Site
  21. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  22. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  23. Markowitz VM, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed