A new method for the detection of alkane-monooxygenase homologous genes (alkB) in soils based on PCR-hybridization.

An improved method was developed that allowed the specific detection of the gene alkB (coding for the rubredoxin dependent alkane monooxygenase) from bacteria without any obvious strain specific discrimination using a combination of PCR and hybridization. This approach enabled a fast culture-independent monitoring of environmental samples for the occurrence of alkB, and an estimation of the gene copy number and the genetic diversity. Both parameters provide useful informations for an assessment of the intrinsic biodegradation potential that is present at a site. The method was applied to soil samples from different uncontaminated sites. alkB was highly abundant and redundant in all soils tested. Potential biodegradation of n-alkanes was also demonstrated for these soils with substrate utilization assays. Cell numbers of hydrocarbon degraders estimated as MPN varied from 10(3) to 10(6)g(-1) soil (dry weight) for the different soils. Gene copy numbers estimated with MPN-PCR ranged within 1-40*10(4)ng(-1) soil DNA. Analysis of the diversity of the alkB sequences obtained from a grassland and an agricultural soil indicated that the alkane degrading microbial populations occurring at these sites were rather diverse. Compared on protein level, three major clusters were distinguishable for both soils that showed highest similarities to AlkB from the Gram-positives Nocardioides and Mycobacterium, and the Gram-negative Alcanivorax. The majority of the cloned AlkB sequences were homologous to proteins from the Gram-positive bacteria. However, significant differences from published sequences were observed; homologies varied from 50% to 90% (identity of amino acids).