Novel organization of genes in a phthalate degradation operon of Mycobacterium vanbaalenii PYR-1

1 Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA 2 Institute of Molecular Biology, Slovak Academy of Sciences, 845 51 Bratislava, Slovakia Correspondence Ashraf A. Khan Ashraf{at}nctr.fda.gov Mycobacterium vanbaalenii PYR-1 is capable of degrading polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites. This study identified and characterized a putative phthalate degradation operon in the M. vanbaalenii PYR-1 genome. A putative regulatory protein ( phtR ) was encoded divergently with five tandem genes: phthalate dioxygenase large subunit ( phtAa ), small subunit ( phtAb ), phthalate dihydrodiol dehydrogenase ( phtB ), phthalate dioxygenase ferredoxin subunit ( phtAc ) and phthalate dioxygenase ferredoxin reductase ( phtAd ). A 6·7 kb Eco RI fragment containing these genes was cloned into Escherichia coli and converted phthalate to 3,4-dihydroxyphthalate. Homologues to the operon region were detected in a number of PAH-degrading Mycobacterium spp. isolated from various geographical locations. The operon differs from those of other Gram-positive bacteria in both the placement and orientation of the regulatory gene. In addition, the M. vanbaalenii PYR-1 pht operon contains no decarboxylase gene and none was identified within a 37 kb region containing the operon. This study is the first report of a phthalate degradation operon in Mycobacterium spp. Abbreviations: PAH, polycyclic aromatic hydrocarbon; PFGE, pulsed field gel electrophoresis; TMS, trimethylchlorosilane The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY365117 ( M. vanbaalenii PYR-1 pht operon region), AY372761 and AY372763 ( Mycobacterium sp. PAH2.135 phtAa and phtB PCR products, respectively), and AY372762 and AY372764 ( M. flavescens PYR-GCK phtAa and phtB PCR products, respectively).