Isolation and characterization of Gordonia SoCg n-alkane degradation cluster

Abstract

Gordonia strain SoCg is a Gram-positive GC rich n-alkane degrader, isolated from a long-term contaminated beach in Sicily for her ability to degrade long (dodecane C12) and very long chain n-alkanes up to hexatriacontane (C36) (P.Quatrini et al., J. Appl. Microbiol., 2007). PCR analysis, using degenerated primers, reveled that it carries one alkane-hydroxylase gene (alkB); PFGE and Southern analysis showed that this gene is localized on the chromosome. In order to isolate the alk cluster of Gordonia strain SoCg, an enriched gene bank was constructed in E.coli DH10B by isolating restriction fragments of the desired size from a preparative gel. One clone, containing a DNA insert of about 7 kb was detected by colony PCR and Southern hybridization and completely sequenced. The Gordonia SoCg alk locus shows 84% similarity with a 4.5 kb fragment from Gordonia TF6, that was necessary to confer n-alkanes biotrasformation ability in E.coli. (T.Fujii et al., Biosci. Biotechnol. Biochem., 2004) The expression of alk genes in SoCg was monitored by qRT-PCR analysis, using total RNA extracted throughout the growth in the presence of n-alkanes or fructose, respectively, as sole C source. The potential role of the alkane hydroxylase AlkB in long chain n-alkanes degradation was explored by comparing the alkB gene expression with hexadecane (C16) and triacontane (C30) consumption, determined by GC-MS analysis. Alkanes longer than C16 support growth of many microorganisms, but the enzyme systems required for their degradation are still unknown. This work will allow to gain insight in the molecular mechanisms of long chain n-alkanes catabolic pathways in GC rich Gram positive bacteria, recognized as ideal candidates for biodegradation of hydrocarbons in soils