Proteogenomic elucidation of the initial steps in the benzene degradation pathway of a novel halophile, Arhodomonas sp. strain Rozel, isolated from a hypersaline environment

Abstract

Lately, 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.

Fig 1

(A) Schematic diagram showing the genetic organization of benzene-degrading ORFs predicted on contig 494 of the Arhodomonas sp. strain Seminole genome. These candidate ORFs are involved in the initial steps of the benzene degradation pathway. The putative functions of the candidate ORFs are listed in Table 1. The ORFs with dark arrows were identified by proteomic analysis. The arrowheads indicate the directions of transcription, and the gene sizes are not proportional to the sizes of the arrows. (B) Proposed benzene degradation pathway by a multicomponent phenol hydroxylase-like enzyme in Arhodomonas sp. strain Seminole. ORFs and the corresponding putative enzymes in bold were identified by genomic and proteomic analyses. Phenol was confirmed as the initial intermediate of benzene degradation by GC-MS. TCA, tricarboxylic acid.

Fig 2

Phylogenetic analysis chart showing the relationships among various monooxygenase components in Arhodomonas sp. strain Seminole and in other aromatic hydrocarbon-degrading nonhalophiles. The unrooted neighbor-joining tree was constructed in MEGA 5 by using predicted amino acid sequences of ORF 1080, ORF 1082, and ORF 1084 from the strain Seminole genome and closely related phenol hydroxylase, toluene monooxygenase, and benzene monooxygenase subunits from nonhalophiles. Sequences for the analysis were obtained from the GenBank and UniProtKB database. Bootstrap values were calculated as a percentage of 1,000 replicates and are shown next to the branches. The enzyme components (and the corresponding GenBank accession numbers) are benzene monooxygenase oxygenase subunit (BAA11761), benzene monooxygenase alpha subunit BtxP (ABG82181), toluene 3-monooxygenase alpha subunit (AAB09618), toluene ortho-monooxygenase subunit (CAA06654), toluene 4-monooxygenase alpha subunit (AAS66660), phenol hydroxylase phL component (AAO47356), phenol hydroxylase phN component (AAO47358), and phenol hydroxylase phP component (AAO47360). The enzyme components (and the UniProtKB accession numbers) are phenol 2-monooxygenase P1 component (P19730), phenol 2-monooxygenase P3 component (P19732), and phenol 2-monooxygenase P5 component (P19734).

Fig 3

A 2D gel image showing candidate protein spots. Progenesis Workstation software was used for protein spot detection, matching, and abundance quantification. (A and B) Sections of the 2D gels with protein spots of the cytosolic proteome of cells grown on benzene (A) and lactate (B). The protein spots detected only in benzene-degrading cells are circled (lane A). The spots were in-gel digested and analyzed by LTQ Orbitrap LC-MS/MS to create peptide mass fingerprints (PMFs). The PMFs were identified by using MASCOT and the translated protein database of the Arhodomonas sp. strain Seminole genome.

Fig 4

(A) Mass spectrum of a BSTFA-derivatized phenol-D₆ standard. (B) A similar mass spectrum of a BSFTA-derivatized metabolite was found in a deuterated benzene (benzene-D₆)-fed strain Rozel culture on day 1, confirming the metabolite as phenol.