Regulation of tetralin biodegradation and identification of genes essential for expression of thn operons

Abstract

The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys4[2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NAD-binding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell.

FIG. 1.

Schematic representation of the two divergent strain TFA operons, which bear tetralin biodegradation genes. Genes identified in this work are shown in enlargement at the bottom. Chromosomal insertions of plasmids bearing transcriptional or translational lacZ gene fusions to thnB or thnC by a single recombination event are also schematically represented at the top.

FIG. 2.

Tetralin induction of lacZ fusions to thn genes. The β-galactosidase activity of strains bearing a transcriptional thnB-lacZ fusion (▪), a translational thnB-lacZ fusion (□), a transcriptional thnC-lacZ fusion (•), or a translational thnC-lacZ fusion (○) after the strains were transferred to mineral medium with tetralin as the only carbon and energy source is shown.

FIG. 3.

Carbon catabolite repression of tetralin biodegradation genes. The results of tetralin gene induction in the strain bearing the translational thnC-lacZ fusion while growing in mineral medium supplemented with 8 mM (•), 20 mM (♦), or 40 mM (▪) β-hydroxybutyrate, in rich MML medium (□), or in LB medium (○) are shown.

FIG. 4.

Effect of nitrogen limitation on catabolic repression of tetralin biodegradation genes. The results of tetralin gene induction in the strain bearing the translational thnC-lacZ fusion during growth in mineral medium with 8 mM β-hydroxybutyrate and ammonium (•), urea (♦), or nitrate (▪) as the nitrogen source are shown.

FIG. 5.

Dendrogram showing the best tree obtained by the neighbor-joining method from the alignment of 17 sequences showing significant similarity to that of ThnR. The ThnR sequence is boxed. GenBank accession numbers for other sequences are as follows: for NagR (Ralstonia sp. strain U2), AF036940.2; for NbzR (Comamonas sp. strain JS765), AY223675.1; for NahR (Pseudomonas putida AN10), AF039534.1; for NahR (P. putida pNAH7), A32837; for HybR (Pseudomonas aeruginosa), AF087482.1; for MidR (Ralstonia sp. strain TAL1145), AF312768.2; for PcpR (Sphingomonas chlorophenolica ATCC39723), U12290.2; for PnbR (P. putida TW3), AF292094.1; for SalR (Acinetobacter sp. strain ADP1), AF150928.2; for CatR (P. putida), A35118; for AphT (Comamonas testosteroni TA441), BAA88500; for BenM (Acinetobacter sp. strain ADP1), AAC46441; for ClcR (P. putida pAC27), A40641; for PhnS (Burkholderia sp. strain RP007), AAD09867; for TcbR (Pseudomonas sp. strain P51), A38861; for HcaR (E. coli K-12), Q47141.

FIG. 6.

Tetralin induction of the translational thnC-lacZ fusion in different thn mutants. β-Galactosidase activity of strains bearing a translational thnC-lacZ fusion was measured 20 h after transferring them to mineral medium with 8 mM β-hydroxybutyrate and tetralin.

FIG. 7.

Reverse transcription-PCR of thnB, thnC, thnR, and ribosomal 16S genes. Two different amounts (0.8 and 4.8 μg) of cDNA obtained by retrotranscription of RNA isolated from strain TFA growing in MM-8 mM βHB-tetralin (lanes 2 and 4) or MM-40 mM βHB (lanes 3 and 5) were used. Amplification of the 16S ribosomal gene was used as a control to ensure equivalent amounts of cDNA between different growth conditions. Lane 1, 1-kb Plus DNA ladder (GibcoBRL).