Open Access

Complete genome sequence of Arthrobacter phenanthrenivorans type strain (Sphe3)

  • Aristeidis Kallimanis1,
  • Kurt M. LaButti2,
  • Alla Lapidus2,
  • Alicia Clum2,
  • Athanasios Lykidis2,
  • Kostantinos Mavromatis2,
  • Ioanna Pagani2,
  • Konstantinos Liolios2,
  • Natalia Ivanova2,
  • Lynne Goodwin2, 3,
  • Sam Pitluck2,
  • Amy Chen4,
  • Krishna Palaniappan4,
  • Victor Markowitz4,
  • Jim Bristow2,
  • Athanasios D. Velentzas5,
  • Angelos Perisynakis1,
  • Christos C. Ouzounis6, 7,
  • Nikos C. Kyrpides2,
  • Anna I. Koukkou1Email author and
  • Constantin Drainas1
Standards in Genomic Sciences20114:4020123

DOI: 10.4056/sigs.1393494

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

Arthrobacter dioxygenases PAH biodegradation phenanthrene 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 [35]. 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 × 2.5–4.0 µm. Colonies were slightly yellowish on Luria agar. The temperature range was 40–37°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 (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

DomainBacteria

TAS [7]

 

PhylumActinobacteria

TAS [8]

 

Class Actinobacteria

TAS [9]

 

Subclass Actinobacteridae

TAS [9,10]

 

Order Actinomycetales

TAS [912]

 

Family Micrococcaceae

TAS [911,13]

 

Genus Arthrobacter

TAS [1,11,1417]

 

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 × 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.
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 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

 
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.

Authors’ Affiliations

(1)
Sector of Organic Chemistry and Biochemistry, University of Ioannina
(2)
DOE Joint Genome Institute
(3)
Bioscience Division, Los Alamos National Laboratory
(4)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory
(5)
Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens
(6)
Centre for Bioinformatics - Department of Informatics - School of Natural & Mathematical Sciences, King’s College London (KCL)
(7)
Computational Genomics Unit, Institute of Agrobiotechnology - Centre for Research & Technology Hellas

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Copyright

© The Author(s) 2011