AMINO-ACID SUBSTITUTIONS IN PILD, A BIFUNCTIONAL ENZYME OF PSEUDOMONAS-AERUGINOSA - EFFECT ON LEADER PEPTIDASE AND N-METHYLTRANSFERASE ACTIVITIES IN-VITRO AND IN-VIVO

Subunits of type IV pill and a subset of proteins of the type II extra cellular protein secretion apparatus undergo two consecutive post-tran slational modifications: leader peptide cleavage, followed by methylat ion of the newly created N-terminal amino acid. These two reactions ar e carried out by a single bifunctional enzyme encoded in Pseudomonas a eruginosa by the pilD gene. Properties of PilD mutants at positions Gl y(95) and/or Lys(96) which were differentially affected in leader pept idase and N-methyltransferase function were characterized. None of the single amino acid substitutions showed a significant alteration in th eir ability to cleave the prepilin leader peptide; however, two double mutants did exhibit a modest reduction in the efficiency of cleavage. In contrast, a significant decrease of N-methyltransferase activity w as detected in PilD having substitutions at Gly(95). Mutants with subs titutions at position Lys(96) showed a variable effect on N-methyltran sferase activity with an apparent requirement for any charged amino ac id at this position. Absence of N-methyltransferase activity did not a ppear to interfere with the ability of P. aeruginosa to assemble funct ional pill. Moreover, pilin monomers isolated from P. aeruginosa expre ssing PilD with Gly(95) substitutions were not methylated. Although co mplete methylation does not appear to be absolutely required for pilus assembly in P. aeruginosa, this modification may be important for pil us function in its natural habitat.