IDENTIFICATION AND CLONING OF GENES INVOLVED IN SPECIFIC DESULFURIZATION OF DIBENZOTHIOPHENE BY RHODOCOCCUS SP STRAIN IGTS8

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to re move sulfur from certain aromatic compounds without breaking carbon-ca rbon bonds. In particular, sulfur is removed from dibenzothiophene (DB T) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurizat ion phenotype was imparted to Escherichia coli. Therefore, IGTS8 was m utagenized, and a new strain (UV1) was selected that had lost the abil ity to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype w ere isolated, though free plasmid could not be isolated. Instead, vect or DNA had integrated into either the chromosome or a large resident p lasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This proce dure identified individual cosmid clones that, when electroporated int o strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which th ese clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicat ed that the desulfurization genes reside on a 4.0-kb DNA fragment. Fin ally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV 1, and R. fascians produced 2-hydroxybiphenyl from DBT when they conta ined appropriate clones, indicating that the genes for the entire path way have been isolated.