Genetic investigation of the catabolic pathway for degradation of abietane diterpenoids by Pseudomonas abietaniphila BKME-9.
We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. The dit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the alpha and beta subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8, 13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylE transcriptional fusion strains showed induction of ditA1 and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12, 14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the dit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, a ditR::Km(r) mutation in a ditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation.
Martin
V J
VJ
Department of Microbiology and Immunology and Pulp and Paper Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Mohn
W W
WW
D045784:Abietanes; D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial; D049308:Dioxygenases; D004224:Diterpenes / Q000378:metabolism*; D005288:Ferredoxins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D016296:Mutagenesis; D016366:Open Reading Frames; D010105:Oxygenases / Q000235:genetics*; D010616:Phenanthrenes / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
C118787:7-oxo-11,12-dihydroxy-8,13-abietadien acid; D045784:Abietanes; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004224:Diterpenes; D005288:Ferredoxins; D010616:Phenanthrenes; D010105:Oxygenases; D049308:Dioxygenases; C118788:DitA protein, Pseudomonas abietaniphila; C095375:7-oxodehydroabietic acid