Regulation of alkane degradation pathway by a TetR family repressor via an autoregulation positive feedback mechanism in a Gram-positive Dietzia bacterium

Abstract

n-Alkanes are ubiquitous in nature and serve as important carbon sources for both Gram-positive and Gram-negative bacteria. Hydroxylation of n-alkanes by alkane monooxygenases is the first and most critical step in n-alkane metabolism. However, regulation of alkane degradation genes in Gram-positive bacteria remains poorly characterized. We therefore explored the transcriptional regulation of an alkB-type alkane hydroxylase-rubredoxin fusion gene, alkW1, from Dietzia sp. DQ12-45-1b. The alkW1 promoter was characterized and so was the putative TetR family regulator, AlkX, located downstream of alkW1 gene. We further identified an unusually long 48bp inverted repeat upstream of alkW1 and demonstrated the binding of AlkX to this operator. Analytical ultracentrifugation and microcalorimetric results indicated that AlkX formed stable dimers in solution and two dimers bound to one operator in a positive cooperative fashion characterized by a Hill coefficient of 1.64 (±0.03) [kD=1.06 (±0.16) μM, kD′=0.05 (±0.01) μM]. However, the DNA-binding affinity was disrupted in the presence of long-chain fatty acids (C10-C24), suggesting that AlkX can sense the concentrations of n-alkane degradation metabolites. A model was therefore proposed where AlkX controls alkW1 expression in a metabolite-dependent manner. Bioinformatic analysis revealed that the alkane hydroxylase gene regulation mechanism may be common among Actinobacteria. The alkane hydroxylase gene alkW1 is regulated by a TetR family regulator AlkX via the long-chain fatty acid positive feedback mechanism in Dietzia sp. DQ12-45-1b. In the absence of n-alkane, AlkX is slightly expressed and autoregulates the transcription of alkW1-alkX operon. When n-alkanes are present, they are hydroxylated by AlkW1 at basal concentration, and further oxidized to fatty acids, which can modulate the DNA-binding affinity of AlkX. Thus the transcription of alkW1-alkX operon is initiated.