Cloning of an alkane hydroxylase system in a long chain n-alkane- degrader Gordonia sp.

Abstract

Five Gram-positive GC rich n-alkane degraders were isolated from a long-term accidentally contaminated beach in Sicily and identified as one Nocardia, two Rhodococcus and two Gordonia strains (Quatrini et al., 2008 J. Appl. Microbiol. 104:251-9). All the isolates were able to grow on long and very long chain n-alkanes up to C36. Diverging alkane-hydroxylase encoding genes (alkB) were detected by PCR using degenerated primers in all the strains. Multiple sequences were obtained from the Nocardia strain while only one alkB gene was detected in Rhodococcus and Gordonia. The aim of this work is to genetically characterize the alk cluster in one of the two Gordonia strains called SoCg. Pulsed Field Gel Electrophoresis analysis revealed an extrachromosomal element (apparent size: 150Kb) but Southern hybridization excluded that alkB is located on this large plasmid. In order to isolate the alk cluster, an enriched mini-library was constructed in E.coli DH10B and screened by colony-PCR using an alkB probe; sequencing of the positive clones is in progress. To investigate alkB gene expression in SoCg, qRT-PCR analysis was carried out using total RNA extracted throughout the growth phase in the presence of hexadecane, triacontane and fructose, respectively, as the sole C sources. The expression profiles were related to HC degradation analyzed by GC-MS. These analysis will allow to gain insight in the molecular mechanisms of long chain alkanes catabolic pathways in GC rich gram positive bacteria, recognized as ideal candidates for biodegradation of hydrocarbons.