Molecular and biochemical characterization of the tetralin degradation pathway in Rhodococcus sp. strain TFB.
Bottom Line: In addition to tetralin induction, TFB thn structural genes are subject to glucose repression.Primer extension assays and translational thnA1::gfp and thnS::gfp fusions were used to characterize putative promoter regions.A mutational analysis of the thnA1 promoter region allowed us to define nucleotides within the cis regulatory elements that are important for the control of thn gene expression.
Affiliation: Centro Andaluz de Biología del Desarrollo-CSIC, Universidad Pablo de Olavide Carretera de Utrera, Km 1. 41013-Seville, Spain.Show MeSH
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Mentions: Based on the peptide sequences obtained from the proteomic analysis, degenerate primers (HIDFw and HIDRv; Table S1) were designed to amplify a region of the putative thnD gene. A 477 bp PCR product, obtained using total DNA from Rhodococcus sp. strain TFB as template, was cloned and sequenced. Its nucleotide sequence shows 70% identity to the corresponding region of the Rhodococcus sp. RHA1 bphD gene (GenBank CP000433) that encodes a 2‐hydroxy‐6‐oxo‐6‐phenylhexa‐2,4‐dienoate hydrolase. We used the putative thnD PCR product to screen a TFB library, identifying three positive cosmids (Fig. 2A). Cosmid pMPO613 was selected for further subcloning and sequencing. A 23 Kb fragment from this cosmid contained 18 ORFs oriented in the same direction. The deduced amino acid sequences of these ORFs were screened against the peptide sequences obtained from the proteome analysis to determine whether these ORFs encoded any of the proteins induced in tetralin‐grown cells. Remarkably, peptides from spots 1, 2, 3, 4, 5, 6, 7, 8, 11, 12 and 13 were perfect matches to sequences of several of these ORFs (Table 1). As this suggests that the 23 Kb cosmid fragment contains one or more operons specifically induced by growth on tetralin medium, we designated each of these sequences as thn genes. Database comparison revealed high similarity between the TFB thn genes and the bph genes of Rhodococcus sp. RHA1 (Table S2). Based on information from previously characterized Thn proteins (GenBank AF157565), we propose a putative tetralin degradation pathway for strain TFB (Fig. 2C), analogous to the pathway that seems to be operating in S. macrogolitabida strain TFA (Fig. 2B). Nevertheless, differences between the genetic organization of the thn genes in both bacteria are evident. In strain TFB, the structural genes are transcribed in the same direction, although seem to be arranged into two operons: one made up of thnA1A2A3UB and another comprising of a perfect duplication of thnA1A2 (thnA1′A2′) plus the thnCGQILWVDEF genes (Fig. 2A). In addition, the thnU gene, which encodes a putative sterol transfer protein, is missing in strain TFA. We also found that a set of five TFB strain genes (thnQILWV) encodes proteins similar to those involved in β‐oxidation pathways. DNA sequences resembling the remnants of transposons were found between both operons.
Affiliation: Centro Andaluz de Biología del Desarrollo-CSIC, Universidad Pablo de Olavide Carretera de Utrera, Km 1. 41013-Seville, Spain.