Isolation and characterization of alkane monooxygenase (alkB) genotypes from Arctic contaminated soils by culture-independent methods

Alkane monooxygenases (encoded by the alkB gene) are a group of microbial enzymes that catalyze the first reaction of alkane degradation. Studies to determine the diversity and prevalence of alkB genotypes in the environment have focused on culturable organisms. The goal of this research was to use culture-independent methods (DGGE, clone library) to identify and characterize alkB genes, and to determine their prevalence in Arctic contaminated soils. General alkB PCR degenerate primers (alkB-Mc) were designed using the conserved nucleotide sequences of the Histidine I Box and Histidine III Box. General alkB-Mc and alkM (Acinetobacter spp. alkane monooxygenase genes) primers were used to screen the soils for the presence of alkane monooxygenase genotypes. A predominance of the Rhodococcus spp. alkB genotypes and the absence of alkM genotypes in these soils was found. alkB PCR fragments amplified from the soils were analyzed by DGGE (Denaturing Gradient Gel Electrophoresis). BlastN and blastX results of the DGGE bands sequences showed that they were similar to Rhodococcus spp. alkB genotypes (~80-90% DNA identity and ~80-90% amino acid homology). An alkB clone library was built using the general alkB-Mc primer set, screened by RFLP (Restriction Fragment Length Polymorphism) and characterized by sequencing of alkB clones. BlastN and blastX results of the alkB clone sequences showed the presence of divergent alkB genotypes (≤ 70% DNA identity and ≤ 67% of amino acid homology to data base sequences). The alignment of the clone-derived amino acid sequences to confirm functional alkane monooxygenase sequences revealed the presence of Histidine Box II and the HYG motif in all of the deduced amino acid clone sequences. These results indicate that the alkB sequences from the clone library represent novel alkB sequences. Both alkB DGGE and clone library techniques were independently able to identify alkB genotypes from High G+C microorganisms as predominant in the 1A03 soil sample. Nevertheless, only the clone library approach identified putative novel alkB sequences. Mineralization of hexadecane and naphthalene was clearly observed at subzero temperatures (-5ºC) in Arctic contaminated soils, proving that the indigenous microbial communities could mineralize these representative hydrocarbons at subzero temperatures in an environment that is predominantly frozen for most of the year