Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB

Abstract

Rhodococcus sp. strain TFB is a metabolic versatile bacterium able to grow on naphthalene as the only carbon and energy source. Applying proteomic, genetic and biochemical approaches, we propose in this paper that, at least, three coordinated but independently regulated set of genes are combined to degrade naphthalene in TFB. First, proteins involved in tetralin degradation are also induced by naphthalene and may carry out its conversion to salicylaldehyde. This is the only part of the naphthalene degradation pathway showing glucose catabolite repression. Second, a salicylaldehyde dehydrogenase activity that converts salicylaldehyde to salicylate is detected in naphthalene-grown cells but not in tetralin- or salicylate-grown cells. Finally, we describe the chromosomally located nag genes, encoding the gentisate pathway for salicylate conversion into fumarate and pyruvate, which are only induced by salicylate and not by naphthalene. This work shows how biodegradation pathways in Rhodococcus sp. strain TFB could be assembled using elements from different pathways mainly because of the laxity of the regulatory systems and the broad specificity of the catabolic enzymes.

Figure 1

Proteomic analysis of TFB cells.A. Naphthalene-versus glucose-grown cells were compared using DIGE technology. Green spots are those specifically induced by naphthalene.B. Silver stained gel of naphthalene-versus glucose-grown cells used for DIGE analysis with the identified spots marked with a circle.C. Tetralin-versus naphthalene-grown cells using DIGE technology where yellow spots are those induced by both substrates, the green spots are induced only by tetralin, and the red spots are induced only by naphthalene.

Figure 2

Proteomic analysis of TFB cells grown on salicylate (A) or naphthalene (B). Silver stained 2D gels where analysed with Image Master Platinum version 7.0 (GE Healthcare) and selected spots induced by each substrate were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Figure 3

Genetic organization, chromosomal location and expression profile of nag genes in TFB.A. Genetic organization of nag genes: nagI (gentisate dioxygenase), nagK (fumarylpyruvate hydrolase) and nagL (mycothiol-dependent maleylpyruvate isomerase). Other ORFs: orf1, putative benzoate transporter; orf2, 3-hydroxybenzoate 6-monooxygenase; orf3, CorA-like putative magnesium transport protein; orf4, IclR-like regulatory protein.B. Genomic location of nagI. Southern blot hybridization showing the chromosomal location of nag genes. Lane 1, size markers. Lane 2, separated total TFB DNA after ethidium bromide staining. Lane 3, hybridization of Lane 2 with a nagI probe.C. Expression profile. RNA was isolated from mid-log glucose (G), salicylate (S) or naphthalene (N) grown cells. Intergenic amplified regions (1, 2 and 3) are shown with brackets. Amplification of 16S ribosomal RNA (rrn) was used as control of the amount of cDNA. Genomic DNA was used as template in control PCR (+).

Figure 4

Inducer profile of TFB thn genes. Fluorescence of TFB cells carrying plasmid pMPO633 (with a gfp::thnA1 translational fusion) in the presence of each compound was measured in relative units (RUs).

Figure 5

Naphthalene degradation pathway proposed in Rhodococcus sp. strain TFB. Reactions catalysed by unknown enzymes are shown with dotted lines. HCCA, 2-hydroxycromene-2-carboxylate.