Genome-based analysis for the identification of genes involved in o-xylene degradation in Rhodococcus opacus R7

Alessandra Di Canito,Jessica Zampolli,Patrizia Di Gennaro,Alexander Steinbüchel,Luciano Milanesi,Alessandro Orro,Pasqualina D’Ursi,Guido Sello

doi:10.1186/s12864-018-4965-6

Abstract

BackgroundBacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes. These bacteria, widely distributed in the environment, are known to be a powerhouse of numerous degradation functions, due to their ability to metabolize a wide range of organic molecules including aliphatic, aromatic, polycyclic aromatic compounds (PAHs), phenols, and nitriles. In accordance with their immense catabolic diversity, Rhodococcus spp. possess large and complex genomes, which contain a multiplicity of catabolic genes, a high genetic redundancy of biosynthetic pathways and a sophisticated regulatory network. The present study aimed to identify genes involved in the o-xylene degradation in R. opacus strain R7 through a genome-based approach.ResultsUsing genome-based analysis we identified all the sequences in the R7 genome annotated as dioxygenases or monooxygenases/hydroxylases and clustered them into two different trees. The akb, phe and prm sequences were selected as genes encoding respectively for dioxygenases, phenol hydroxylases and monooxygenases and their putative involvement in o-xylene oxidation was evaluated. The involvement of the akb genes in o-xylene oxidation was demonstrated by RT-PCR/qPCR experiments after growth on o-xylene and by the selection of the R7–50 leaky mutant. Although the akb genes are specifically activated for o-xylene degradation, metabolic intermediates of the pathway suggested potential alternative oxidation steps, possibly through monooxygenation. This led us to further investigate the role of the prm and the phe genes. Results showed that these genes were transcribed in a constitutive manner, and that the activity of the Prm monooxygenase was able to transform o-xylene slowly in intermediates as 3,4-dimethylphenol and 2-methylbenzylalcohol. Moreover, the expression level of phe genes, homologous to the phe genes of Rhodococcus spp. 1CP and UPV-1 with a 90% identity, could explain their role in the further oxidation of o-xylene and R7 growth on dimethylphenols.ConclusionsThese results suggest that R7 strain is able to degrade o-xylene by the Akb dioxygenase system leading to the production of the corresponding dihydrodiol. Likewise, the redundancy of sequences encoding for several monooxygenases/phenol hydroxylases, supports the involvement of other oxygenases converging in the o-xylene degradation pathway in R7 strain.

Highlights

Bacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes
The only o-xylene degradation pathway described in literature for bacteria of Rhodococcus genus is through the dioxygenase system of Rhodococcus sp
DK17 leading to the corresponding dihydrodiol that could be dehydrated to dimethyl phenols (DMPs) [2]

Summary

Introduction

Bacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes These bacteria, widely distributed in the environment, are known to be a powerhouse of numerous degradation functions, due to their ability to metabolize a wide range of organic molecules including aliphatic, aromatic, polycyclic aromatic compounds (PAHs), phenols, and nitriles. While the third pathway is initiated by a ring-hydroxylating monooxygenase at the different position 3, or 4, as performed by the Toluene o-xylene Monooxygenase (ToMo) in P. stutzeri strain OX1 (Fig. 1, routes C1 and C2), leading to the formation of 3,4-dimethylcatechol [1]

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Results

Conclusion