PCR Detection of Polycyclic Aromatic Hydrocarbon-Degrading Mycobacteria

Abstract

Polymerase chain reaction (PCR) methods based on the 16S rRNA genes of Mycobacterium sp. PYR-1 and Mycobacterium sp. PAH135, known PAH-degrading bacteria, were developed. An efficient mycobacterial cell lysis procedure was used for the PCR assay. The PCR methods were positive with the target species, but negative for the other 45 bacterial species tested including other Mycobacterium spp. The PCR sensitivity for pure cultures was 20 cells for Mycobacterium sp. PAH135 and 200 cells for Mycobacterium sp. PYR-1. The PCR with a simple sample preparation procedure was used to monitor Mycobacterium sp. PYR-1 cell concentrations in soil slurries amended with [14C]pyrene. The pyrene mineralization correlated with the Mycobacterium PYR-1 cell concentrations in the soil slurries. When the PCR titer (the maximum dilution for positive PCR results) reached 10-4−10-5 at 5−10 days incubation, approximately 50% of the [14C]pyrene had been mineralized to 14CO2. However, without inoculation with Mycobacterium PYR-1 cells, both the sterile and nonsterile soils had negative PCR results and no pyrene mineralization.