Screening A Cosmid Library From Rhodococcus I24 for Genes Involved in Indene Bioconversion

Rhodococcus I24 and B264-1 are able to stereospecifically bioconvert indene to indandiol,a potential precursor to CRIXIVAN. Indene bioconversion is catalyzed by the multicomponent enzymes, naphthalene d ioxygenase and toluene dioxygenase. The genes for these enzy mes have not been identified in Rhodococcus, but sequences are known in Pseudomonas. The objective of these experiments is to screen a cosmid library from Rhodococcus I24 for homologs to genes from Pseudomonas that encode naphthalene dioxygenase (nahAa) and toluene dioxygenase (todA). DNA from cosmids making up the I24 l ibrary were isolated and prepared for hybridization studies. Probes to nahAa and todA were prepared by polymerase chain reaction (PCR) and labeled. Dot blots identified which cosmids hybridized to the probes, and Southern blots determined whether these hybridizations were due to sequence-specific homology or to a nonspecific, bulk DNA effect (binding due to the presence of a large amount of DNA from multiples sources). Many dots from the dot blots showed hybridization to each probe. Analysis of these dots by Southern hybridization suggested that hybridization was due to nonspecific binding to pRhodoCOS, the cosmid vector. However, comparing the two membranes used in the dot blots suggested that some sequence-specific interactions existed. Screening A Cosmid Library From Rhodococcus I24 for Genes Involved in Indene Bioconversion