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


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.


ArthrobacterdioxygenasesPAH biodegradationphenanthrene degradation


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]




   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


   TAS [1]

  Cell shape

   irregular rods, coccoid

   TAS [1]


   Non motile

   TAS [1]




  Temperature range


   TAS [1]

  Optimum temperature


   TAS [1]



   TAS [1]


  Oxygen requirement


   TAS [1]

  Carbon source

   Phenanthrene, glucose, yeast extract

   TAS [1,2]

  Energy source

   Phenanthrene, glucose, yeast extract

   TAS [1,2]




   TAS [1,2]


  Biotic relationship







  Biosafety level




   Creosote contaminated soil

   TAS [1,2]


  Geographic location

   Perivleptos, Epirus, Greece

   TAS [1,2]


  Sample collection time

   April 2000

   TAS [1,2]











   10-20 cm

   TAS [1,2]



   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.


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





    Finishing quality



    Libraries used

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


    Sequencing platforms

    ABI 3730. 454 GS FLX


    Sequencing coverage

    9.33× Sanger, 17.45× pyrosequence



    Newbler version, Arachne


    Gene calling method

    Prodigal, GenePRIMP



    Genbank Date of Release

    February 16, 2011



    NCBI project ID


    Database: IMG-GEBA



    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 (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



% of Total

Genome size (bp)



DNA Coding region (bp)



DNA G+C content (bp)



Number of replicons


Extrachromosomal elements


Total genes



RNA genes



rRNA operons


Protein-coding genes



Pseudo genes



Genes with function prediction



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


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








   Translation, ribosomal structure and biogenesis




   RNA processing and modification








   Replication, recombination and repair




   Chromatin structure and dynamics




   Cell cycle control, cell division, chromosome partitioning




   Nuclear structure




   Defense mechanisms




   Signal transduction mechanisms




   Cell wall/membrane/envelope biogenesis




   Cell motility








   Extracellular structures




   Intracellular trafficking and secretion, and vesicular transport




   Posttranslational 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



   6. 9

   Function unknown




   Not in COGs



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.


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