Molecular and biochemical characterization of the tetralin degradation pathway in Rhodococcus sp. strain TFB

• Published in: Microbial biotechnology Vol. 2; no. 2; pp. 262 - 273
• Main Authors: Tomás-Gallardo, Laura, Santero, Eduardo, Camafeita, Emilio, Calvo, Enrique, Schlömann, Michael, Floriano, Belén
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.03.2009; John Wiley & Sons, Inc
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Biodegradation; Biodegradation, Environmental; Carbon; Catabolite repression; Cloning; Dehydrogenases; Electrophoresis, Gel, Two-Dimensional; Gene expression; Gene Expression Regulation, Bacterial; Genes; Genetic engineering; Genetics; Genomes; Glucose; Mass spectrometry; Metabolic Networks and Pathways; Metabolism; Molecular Sequence Data; Mutation; Nucleotides; Operon; Operons; Plasmids; Promoter Regions, Genetic; Proteins; Proteomics; Regulatory sequences; Rhodococcus; Rhodococcus - chemistry; Rhodococcus - genetics; Rhodococcus - metabolism; Scientific imaging; Tetrahydronaphthalenes - metabolism; Tetralin; Transcription; Japan
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/j.1751-7915.2009.00086.x

Summary The tetralin biodegradation pathway in Rhodococcus sp. strain TFB, a Gram‐positive bacterium resistant to genetic manipulation, was characterized using a proteomic approach. Relative protein expression in cell free extracts from tetralin‐ and glucose‐grown cells was compared using the 2D‐DIGE technique. Identification of proteins specifically expressed in tetralin‐grown cells was used to characterize a complete set of genes involved in tetralin degradation by reverse genetics. We propose a tetralin degradation pathway analogous to that described for Sphingomonas macrogolitabida strain TFA. TFB thn genes are organized into three operons; two contain all of the structural genes and are transcribed in the same direction, while the third operon, thnST, is transcribed in the opposite direction and encodes a two‐component regulatory system, whose transcription is higher in tetralin‐grown cells. 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.