Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains

We have developed highly degenerate oligonucleotides for polymerase chain r eaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of h ighly conserved sequence motifs. In all Gram-negative and in two out of thr ee Gram-positive strains able to grow on medium- (C-6-C-11) Or long-chain n -alkanes (C-12-C-16), PCR products Of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 4 3.2-93.8% sequence identity to the corresponding fragment of the P. oleovor ans GPo1 alkane hydroxyiase. Strains that were unable to grow on n-alkanes did not yield non products with homology to alkane hydroxylase genes. The a lkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomona s putida P1 were cloned using the PCR products as probes. The two genes all ow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an E scherichia coil recombinant containing all P. oleovorans alk genes except a lkB, respectively, to grow on n-alkanes, showing that the cloned genes do i ndeed encode alkane hydroxylases.