CLONING THE STRUCTURAL GENE FOR THE 49-KDA FORM OF EXOENZYME-S (EXOS)FROM PSEUDOMONAS-AERUGINOSA STRAIN 388

We report the purification and proteolytic characterization of the 49- kDa form of exoenzyme S and the cloning of the structural gene for the 49-kDa form of exoenzyme S (exoS). The 49-kDa form of exoenzyme S was purified from SDS-polyacrylamide gels. Conditions were established th at allowed efficient trypsin digestion of the 49-kDa form of exoenzyme S. Amino acid sequence determination of the amino terminus and trypti c peptides of the 49-kDa form of exoenzyme S allowed the generation of degenerate oligonucleotides, which were used to amplify DNA encoding an amino-terminal sequence and an internal sequence of the 49-kDa form of exoenzyme S. These DNA fragments were used to clone the entire str uctural gene for the 49-kDa form of exoenzyme S (exoS) from a cosmid l ibrary of Pseudomonas aeruginosa strain 388. The 49-kDa form of exoenz yme S (ExoS) is predicted to be a 453 amino acid protein. The predicte d amino acid sequence indicates that ExoS is secreted from Pseudomonas without cleavage of an aminoterminal sequence. BESTFIT analysis ident ified three regions of alignment between ExoS and the active site of E scherichia coli heat-labile enterotoxin. One region of homology appear s to be shared among several members of the family of bacterial ADP-ri bosyltransferases.