Standards in Genomic Sciences (2011) 4:123-130 DOI:10.4056/sigs.1393494 Complete genome sequence of Arthrobacter phenanthrenivorans type strain (Sphe3) Aristeidis Kallimanis1, Kurt M. LaButti2, Alla Lapidus 2, 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. Koukkou1*, and Constantin Drainas1 1 Sector of Organic Chemistry and Biochemistry, University of Ioannina, Ioannina, Greece 2 DOE Joint Genome Institute, Walnut Creek, California, USA 3 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 4 Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA 5 Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Athens, Greece 6 Centre for Bioinformatics - Department of Informatics - School of Natural & Mathematical Sciences, King's College London (KCL) - London, UK 7 Present address: Computational Genomics Unit, Institute of Agrobiotechnology - Centre for Research & Technology Hellas - Thessaloniki - Greece *Corresponding author: Anna I. Koukkou, email: [email protected] 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 fea- tures 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 Classification and features type strain of Arthrobacter phenanthrenivorans Figure 1 shows the phylogenetic neighborhood of [1]. It was isolated from Perivleptos, a creosote A. phenanthrenivorans strain Sphe3T in a 16S rRNA polluted site in Epirus, Greece (12 Km North of the based tree. city of Ioannina), where a wood preserving indus- Strain Sphe3T is a Gram-positive, aerobic, non- try was operating for over 30 years [2]. Strain motile bacterium exhibiting a rod-coccus cycle Sphe3T is of particular interest because it is able to (Figure 2), with a cell size of approximately 1.0-1.5 metabolize phenanthrene at concentrations of up x 2.5- to 400 mg/L as a sole source of carbon and ener- Luria agar. The temperature range was 40-37o gy, at rates faster than those reported for other with optimum4.0 μm. Colonies growth wereat 30 -slightly37o yellowish on Arthrobacter species [3-5]. It appears to internal- was 6.5-8.5 with optimal growth at pH 7.0-7.5C ize phenanthrene with two mechanisms: a passive (Table 1). Strain Sphe3T was foundC. toThe be pH sensitive range diffusion when cells are grown on glucose, and an to various antibiotics, the minimal inhibitory con- inducible active transport system, when cells are centrations of which were estimated as follows: grown on phenanthrene as a sole carbon source ampicillin 20 mgL-1, chloramphenicol 10 mgL-1, [2]. Here we present a summary classification and erythromycin 10 mgL-1, neomycin 20 mgL-1, ri- a set of features for A. phenanthrenivorans strain fampicin 10 mgL-1 and tetracycline 10 mgL-1. Sphe3T, together with the description of the com- plete genome sequencing and annotation. The Genomic Standards Consortium Arthrobacter phenanthrenivorans type strain (Sphe3) Amylase, catalase and nitrate reductase tests were dase, urease, citrate assimilation and H2S produc- positive, whereas arginine dihydrolase, gelatinase, tion tests were negative. No acid was produced in lipase, lysine and ornithine decarboxylase, oxi- the presence of glucose, lactose and sucrose. 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. Figure 2. Scanning electron micrograph of A. phenanthrenivorans strain Sphe3T 124 Standards in Genomic Sciences Kallimanis et al. Chemotaxonomy Menaquinones are the sole respiratory lipoqui- anteiso- 17:0 16:0 (8.3%), iso- 17:0 (4.0%), nones of A. phenanthrenivorans strain Sphe3T. 16:1 14:0 (1.4%). The major phos- Both MK-8 and MK-9(H2) are present in a ratio of pholipidsC were(12.0%), diphospatidylglycerol C C (DPG), 3.6:1, respectively. Major fatty acids are anteiso- Cphosphatidylω7c (2.5%)glycerol and C (PG) and phosphatidyletha- 15:0 (36.2%), iso- 16:0 (15.7%), iso- 15:0 (14.3%), nolamine (PE), (63.8, 27.5 and 4.0% respectively). C C C Table 1. Classification and general features of A. phenanthrenivorans strain Sphe3T according to the MIGS recommendations [6] MIGS ID Property Term Evidence code Domain Bacteria TAS [7] Phylum Actinobacteria TAS [8] Class Actinobacteria TAS [9] Subclass Actinobacteridae TAS [9,10] Current classification 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. http://standardsingenomics.org 125 Arthrobacter phenanthrenivorans type strain (Sphe3) Genome sequencing and annotation Genome project history This organism was selected for sequencing on the plete genome sequence is deposited in GenBank. basis of its biodegradation capabilities, i.e. meta- Sequencing, finishing and annotation were per- bolizes phenanthrene as a sole source of carbon formed by the DOE Joint Genome Institute (JGI). A and energy. The genome project is deposited in summary of the project information is shown in the Genome OnLine Database [18] and the com- Table 2. Table 2. Genome sequencing project information MIGS ID Property Term MIGS-31 Finishing quality Finished Three genomic libraries: 6kb (pMCL200) and fosmids (pcc1Fos) MIGS-28 Libraries used 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 tom primer walks from sub- d- ucts. A total of 822 Sanger finishing reads were 0.02% (w/v) phenanthrene. DNA was isolated ac- produced to close gaps, to resolveclones orrepetitive PCR pro re- grown aerobically at 30°C on MM M9 containing gions, and to raise the quality of the finished se- Bacterial genom quence. The error rate of the completed genome cording to the standard JGI (CA, USA) protocol for sequence is less than 1 in 100,000. Together, the Genome sequencingic DNA andisolation assembly using CTAB. combination of the Sanger and 454 sequencing The genome of Arthrobacter phenanthrenivorans platforms provided 26.78 × coverage of the ge- type strain (Sphe3)was sequenced using a nome. The final assembly contains 44,113 Sanger combination of Sanger and 454 sequencing plat- reads and 599,557 pyrosequencing reads. forms. All general aspects of library construction and sequencing can be found at the JGI website Genome annotation [19]. Pyrosequencing reads were assembled using Genes were identified using Prodigal [21] as part the Newbler assembler version 1.1.02.15 (Roche). of the Oak Ridge National Laboratory genome an- Large Newbler contigs were broken into 4,967 notation pipeline, followed by a round of manual overlapping fragments of 1,000 bp and entered curation using the JGI GenePRIMP pipeline [22]. into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler n- consensus q-scores with modifications to account The predicted CDSs were translated and used toi- for overlap redundancy and to adjust inflated q- Prot,search TIGR the- National Center for Biotechnology In- scores. A hybrid 454/Sanger assembly was made terProformation databases. (NCBI) Additional nonredundant gene predictiondatabase, anaUnl- using the Arachne assembler [20]. Possible mis- ysis and functionalFam, Pfam, annotation PRIAM, KEGG, were COG, performed and assemblies were corrected and gaps between con- within the Integrated Microbial Genomes - Expert s- Review (IMG-ER) platform [23]. 126tigs were closed by by editing in Consed, by cu Standards in Genomic Sciences Kallimanis et al. Genome properties predicted, 4,212 were protein-coding genes, and The genome consists of a 4,250,414 bp long chro- 76 RNAs; 77 pseudogenes were also identified.