Abstract
Subunits of type IV pili and a subset of proteins of the type II extracellular protein secretion apparatus undergo two consecutive post-translational modifications: leader peptide cleavage, followed by methylation of the newly created N-terminal amino acid. These two reactions are carried out by a single bifunctional enzyme encoded in Pseudomonas aeruginosa by the pilD gene. Properties of PilD mutants at positions Gly95 and/or Lys96 which were differentially affected in leader peptidase and N-methyltransferase function were characterized. None of the single amino acid substitutions showed a significant alteration in their 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 was detected in PilD having substitutions at Gly95. Mutants with substitutions at position Lys96 showed a variable effect on N-methyltransferase activity with an apparent requirement for any charged amino acid at this position. Absence of N-methyltransferase activity did not appear to interfere with the ability of P. aeruginosa to assemble functional pili. Moreover, pilin monomers isolated from P. aeruginosa expressing PilD with Gly95 substitutions were not methylated. Although complete methylation does not appear to be absolutely required for pilus assembly in P. aeruginosa, this modification may be important for pilus function in its natural habitat.
Highlights
The respiratory distress and morbidity in individuals suffering from cystic fibrosis is most often caused by the opportunistic pathogen Pseudomonas aeruginosa [1]
Site-directed Mutagenesis of Conserved Amino Acids within a Potential N-Methyltransferase Box of PilD—The post-translational modifications carried out by the bifunctional enzyme PilD of P. aeruginosa are necessary for type IV pilus biogenesis and for the assembly of a functional apparatus of the general secretory pathway
The glycine at position (Gly95) is invariant, and the lysine at position (Lys96) was found in half of the homologs, whereas the other half had a conserved change to another basic amino acid, arginine
Summary
The respiratory distress and morbidity in individuals suffering from cystic fibrosis is most often caused by the opportunistic pathogen Pseudomonas aeruginosa [1] This organism has at its disposal a variety of adhesins [2], including type IV pili, to establish an infection in a susceptible host [3]. P. aeruginosa expressing any one of the different PilD mutants showed no defect in pilus biogenesis or extracellular protein secretion. This indicates that PilD substrates may not need to be fully N-methylated for these proteins to be functional
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
