Q-RT-PCR Detection of substrate-specific gene expression

The assessment of oil impact in marine environment must take into account two important issues: (1) to establish the type or types of contaminants and (2) verify their bioavailability and transformation by microbial degradation. This chapter discuses the specialization of hydrocarbonoclastic bacteria, in particular Alcanivorax borkumensis SK2, in the selection of metabolic pathway to consume different substrates. The study is focused on the two major systems involved in alkanes degradation, cytochrome P450 and alkane hydrolases alkB and the related genes expression in the presence of two classes of hydrocarbons: phytane (naturally occurring isoprenoids) and n-tetradecane (anthropogenic aliphatics). Results of Q-RT-PCR analysis of alkane hydrolases and cytochromes P450 expression in A. borkumensis revealed drastic changes at early exponential phase compared with those at late exponential phase of growth on tetradecane, for both genes studied, while a different behavior of alkB genes expression in isoprenoid-growing cells was registered with no alkB1 mRNA detection and overexpression of alkB2 and P450 genes at early exponential phase of growth. Moreover, P450(c) showed a significant expression in pristine-growing cells and a "house-keeping" type expression in phytane-growing cells. Remarkably, phytane seems to be a strong inducer of cytochrome P450a gene expression and the highest copy number of its corresponding mRNA (20 ± 3 molecules per cell) was detected.