bLately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteria Arhodomonas sp. strain Seminole and Arhodomonas sp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles. Many hypersaline environments, such as natural saline lakes, salt flats, solar salterns, saline industrial effluents, oil fields, and salt marshes, have been shown to be contaminated with high levels of petroleum hydrocarbons. Among these, oil fields pose a special problem due to their sheer numbers worldwide and due to their high salinity caused by produced water (salty brackish water) generated during oil and natural gas extraction. Produced water is highly saline and contains a complex mixture of hydrocarbons, including alkanes (linear or branched), cycloalkanes, mono-and polyaromatics, asphaltenes, heavy metals, and resins.The ability of microorganisms to degrade aromatic hydrocarbons in terrestrial and marine environments has been studied extensively under oxic conditions (28,43,44,50,56). On the other hand, little is known about hydrocarbon degradation in hypersaline environments. Bioremediation of polluted hypersaline wastewaters and other environments with nonhalophilic microorganisms is difficult because salt inhibits their growth and the degradation of hydrocarbons (41, 57). Therefore, the cleanup of such environments can only be accomplished by stimulating the growth of indigenous microorganisms capable of degrading petroleum hydrocarbons or through the bioaugmentation of halophilic...