Transcriptomic Analysis of Rhodococcus opacus R7 Grown on o-Xylene by RNA-Seq

Abstract

Xylenes are considered one of the most common hazardous sources of environmental contamination. The biodegradation of these compounds has been often reported, rarer the ability to oxidize the ortho-isomer. Among few o-xylene-degrading bacteria, Rhodococcus opacus R7 is well known for its capability to degrade diverse aromatic hydrocarbons and toxic compounds, including o-xylene as only carbon and energy source. This work shows for the first time the RNA-seq approach to elucidate the genetic determinants involved in the o-xylene degradation pathway in R. opacus R7. Transcriptomic data showed 542 differentially expressed genes that are associated with the oxidation of aromatic hydrocarbons and stress response, osmotic regulation and central metabolism. Gene ontology (GO) enrichment and KEGG pathway analysis confirmed significant changes in aromatic compound catabolic processes, fatty acid metabolism, beta-oxidation, TCA cycle enzymes, and biosynthesis of metabolites when cells are cultured in the presence of o-xylene. Interestingly, the most up-regulated genes belong to the akb gene cluster encoding for the ethylbenzene (Akb) dioxygenase system. Moreover, the transcriptomic approach allowed identifying candidate enzymes involved in R7 o-xylene degradation for their likely participation in the formation of the metabolites that have been previously identified. Overall, this approach supports the identification of several oxidative systems likely involved in o-xylene metabolism confirming that R. opacus R7 possesses a redundancy of sequences that converge in o-xylene degradation through R7 peculiar degradation pathway. This work advances our understanding of o-xylene metabolism in bacteria belonging to Rhodococcus genus and provides a framework of useful enzymes (molecular tools) that can be fruitfully targeted for optimized o-xylene consumption.

Keywords: RNA-seq; Rhodococcus opacus; environmental contamination; o-xylene; oxygenases; stress response.

FIGURE 1

Expression profile of R. opacus R7 grown on o-xylene respect to malate condition in a V-plot with annotation based on GO analysis. V-plot shows R. opacus R7 statistically significant genes (p-value < 0.05) in the higher part of the plot (low p-value in the y axis); the right and the left parts of the plot (x axis) represent the DEGs under o-xylene or malate condition, respectively. The following GO categories are in the V-plot: Aromatic compound catabolic process, Toluene catabolic process, Xylene catabolic process, Toluene dioxygenase activity, Benzene 1,2-dioxygenase activity, Biphenyl 2,3-dioxygenase activity, Protocatechuate 3,4-dioxygenase activity, Catechol 1,2-dioxygenase, and Oxygenase.

FIGURE 2

Distribution of main GO categories with respect to R. opacus R7 response to o-xylene and malate. Barplot shows the percentage of DEGs grouped in relevant GO categories, including the following categories: “Molecular functions,” “Biological processes,” “Cellular components,” “Catalytic activity,” “Metabolic processes,” and “Cellular processes.”

FIGURE 3

Distribution of R. opacus R7 responses to o-xylene and malate according to physiological categories. Barplot shows the number of up- and down-regulated genes grouped in relevant KEGG categories, such as microbial metabolism in diverse environments, metabolic pathway, degradation of aromatic compounds, carbon metabolism, biosynthesis of secondary metabolites, toluene degradation, xylene degradation, methane metabolism, styrene degradation, benzoate degradation, chlorocyclohexane and chlorobenzene degradation, chloroalkane and chloroalkene degradation, fluorobenzoate degradation, dioxin degradation, steroid degradation, propanoate metabolism, butanoate metabolism, phenylalanine metabolism, citrate cycle (TCA cycle), pyruvate metabolism, pentose phosphate pathway, fatty acid metabolism, fatty acid degradation, fatty acid biosynthesis, lipid metabolism, biosynthesis of antibiotics, biosynthesis amino acids, terpenoid backbone biosynthesis, synthesis and degradation of ketone bodies, DNA replication, biotin metabolism, transporter, ABC transporter.

FIGURE 4

Expression levels of R7 selected DEGs by RT-qPCR analysis. The selected genes are akbA1a, padAa, akbC, catA, pcaH, and czcO6 genes encoding for the ethylbenzene dioxygenase, the phthalate 3,4-dioxygenase, the meta-cleavage dioxygenase, the catechol 1,2-dioxygenase, the protocatechuate 3,4-dioxygenase, and the cyclohexanone monooxygenase.

FIGURE 5

Proposed metabolic pathways of R. opacus R7 for o-xylene degradation. The black genes represent the confirmed genes involved in the o-xylene pathway: akbA1A2A3A4, multicomponent o-xylene dioxygenase; akbB, dihydrodiol dehydrogenase; akbC, meta-cleavage dioxygenase; akbD, a meta-cleavage hydrolase; akbE, a hydratase component; akbF, aldolase; prmACBD, monooxygenase; phe, phenol hydroxylase. The blue genes were identified on the basis of transcriptomic data and are potentially involved in the o-xylene pathway: padAaAbAcAd, multicomponent phthalate dioxygenase; padB, phthalate dihydrodiol dehydrogenase; czcO, cyclohexanone monooxygenases; luxA, alkanal monooxygenase alpha-chain; rutA, predicted monooxygenase; fmoB, flavin reductase (NADH) monooxygenases; catA, cathecol 1,2-dioxygenase; catB, muconate cycloisomerase; pcaB, 3-carboxy-cis,cis-muconate cycloisomerase; pcaG and pcaH, alpha and beta subunits of the protocatechuate 3,4-dioxygenase, respectively. Dashed arrow indicates a spontaneous dehydration. (1) o-xylene; (2) cis-3,4-dihydrodiol; (3) 2,3-dimethylphenol; (4) 3,4-dimethylphenol; (5) 2-methylbenzylalcohol; (6) 3,4-dimethylcatechol; (7) 5-methyl-2,6-dioxohept-3-enoic acid; (8) 2,3-dimethylhexa-2,4-dienedioic acid.