GENETIC-RELATIONSHIP BETWEEN THE 53- AND 49-KILODALTON FORMS OF EXOENZYME-S FROM PSEUDOMONAS-AERUGINOSA

Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomona s aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form. The postu lated relationship between the two proteins involves a carboxy-termina l proteolytic cleavage of the 53-kDa precursor to produce an enzymatic ally active 49-kDa protein. To determine the genetic relationship betw een the two forms of exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P. aeruginosa chromosomal digests. Cross-hybridizing bands were detected in chromosomal digests of a strain of P. aeruginosa in which exoS had been deleted by alleli c exchange. A chromosomal bank was prepared from the exoS deletion str ain, 388 Delta exoS::Tc, and screened with a probe internal to exoS. T hirteen clones that cross-hybridized with the exoS probe were identifi ed. One representative clone contained the open reading frame exoT; th is open reading frame encoded a protein of 457 amino acids which showe d 75% amino acid identity to ExoS. The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against exoenzyme S. A histidine-tagged derivative of recombinant Exo53 possessed approximate ly 0.2% of the ADP-ribosyltransferase activity of recombinant ExoS. In activation of exoT in an allelic-replacement strain resulted in an Exo 53-deficient phenotype without modifying the expression of ExoS. These studies prove that the 53- and 49-kDa forms of exoenzyme S are encode d by separate genes. In addition, this is the first report of the fact or-activating-exoenzyme-S-dependent ADP-ribosyltransferase activity of the 53-kDa form of exoenzyme S.