A novel phenanthrene-degrading bacterium, designated strain Sphe3 T , was isolated from a creosote-contaminated soil in Greece. Cells were non-motile, Gram-positive, aerobic, and rod-to coccus-shaped. The strain was isolated on the basis of formation of a clear zone on agar plates sprayed with phenanthrene. Optimal growth occurred at 30 6C. The G+C content of the DNA was 65.7 mol%. The polar lipid pattern of strain Sphe3 T consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant fatty acids were iso-C 15 : 0 , anteiso-C 15 : 0 , iso-C 16 : 0 , C 16 : 0 and anteiso-C 17 : 0 , representing .86 % of the total fatty acids. The predominant isoprenoid quinone of strain Sphe3 T was menaquinone-8 (MK-8). Based on 16S rRNA gene sequence analysis, strain Sphe3 T showed 99 and 98.9 % similarity to the type strains of Arthrobacter oxydans and Arthrobacter polychromogenes, respectively. Strain Sphe3 T showed 91 % similarity to homologues of A. oxydans and A. polychromogenes based on recA gene sequence analysis. Based on 16S rRNA and recA gene sequence analysis and DNA-DNA hybridization analysis, as well as physiological and chemotaxonomic characteristics, it is concluded that strain Sphe3 T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter phenanthrenivorans sp. nov. is proposed. The type strain is Sphe3 T (5DSM 18606 T 5LMG 23796 T ).Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are found in many polluted soils as a result of natural or industrial activities, including those of creosote wood-treatment facilities (Mueller et al., 1989). PAHs have attracted considerable attention because of their potential toxicity for higher organisms and resistance to microbial degradation (Kanaly & Harayama, 2000). A wide range of micro-organisms have been discovered that are able to degrade highly stable, toxic organic compounds such as polycyclic and aliphatic hydrocarbons (Habe & Omori, 2003;Kanaly & Harayama, 2000;Van Hamme et al., 2003). Among these micro-organisms, several Arthrobacter species are able to degrade PAHs (Grifoll et al., 1992;Seo et al., 2006). We have previously reported the phenanthrene uptake activity and membrane lipid alterations of a PAH-degrading Arthrobacter strain, Sphe3, isolated from a creosote-contaminated soil in Greece (Kallimanis et al., 2007). In the present study, on the basis of phylogenetic analysis of 16S rRNA and recA gene sequences, together with physiological and chemotaxonomic characteristics and DNA-DNA hybridization analysis, we demonstrate that strain Sphe3 represents a novel species of the genus Arthrobacter.Strain Sphe3 T was isolated from Perivleptos, a creosotepolluted site in Epirus, Greece (12 km north of the city of Ioannina), in which a wood-preserving industry had operated for over 30 years (Kallimanis et al., 2007). Soil samples (10 g) were mixed with 100 ml of minimal medium M9 (Sambrook et al., 1989) supplemented with 0.01 % (w/v) phenanthrene (in crystal form) as the sole ca...

A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent K m of 35 M for Diox1 and 29 M for Diox2, whereas they showed similar kinetic turnover characteristics with K cat /K m values of 11 ؋ 10 6 M ؊1 s ؊1 and 12 ؋ 10 6 M ؊1 s ؊1 , respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans.