3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase.

The enzymes and genes responsible for the catabolism of higher alkylphenols have not been characterized in aerobic bacteria. Pseudomonas sp. strain KL28 can utilize a wide range of alkylphenols, which include the 4-n-alkylphenols (C(1)-C(5)). The genes, designated as lap (for long-chain alkylphenols), encoding enzymes for the catabolic pathway were cloned from chromosomal DNA and sequenced. The lap genes are located in a 13.2 kb region with 14 ORFs in the order lapRBKLMNOPCEHIFG and with the same transcriptional orientation. The lapR gene is transcribed independently and encodes a member of the XylR/DmpR positive transcriptional regulators. lapB, the first gene in the lap operon, encodes catechol 2,3-dioxygenase (C23O). The lapKLMNOP and lapCEHIFG genes encode a multicomponent phenol hydroxylase (mPH) and enzymes that degrade derivatives of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates, respectively. The P(lapB) promoter contains motifs at positions -24(GG) and -12(GC) which are typically found in sigma(54)-dependent promoters. A promoter assay using a P(lapB) : : gfp transcriptional fusion plasmid showed that lapB promoter activity is inducible and that it responds to a wide range of (alkyl)phenols. The structural genes encoding enzymes required for this catabolism are similar (42-69 %) to those encoded on a catabolic pVI150 plasmid from an archetypal phenol degrader, Pseudomonas sp. CF600. However, the lap locus does not include genes encoding HMS hydrolase and ferredoxin. The latter is known to be functionally associated with C23O for use of 4-alkylcatechols as substrates. The arrangement of the lap catabolic genes is not commonly found in other meta-cleavage operons. Substrate specificity studies show that mPH preferentially oxidizes 3- and 4-alkylphenols to 4-alkylcatechols. C23O preferentially oxidizes 4-alkylcatechols via proximal (2,3) cleavage. This indicates that these two key enzymes have unique substrate preferences and lead to the establishment of the initial steps of the lap pathway in strain KL28.