Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect Type IV pilus assembly, dynamics and associated functions.
Type IV pili (Tfp) are multifunctional surface appendages expressed by many Gram negative species of medical, environmental and industrial importance. The N-terminally localized, so called alpha-helical spine is the most conserved structural feature of pilin subunits in these organelles. Prevailing models of pilus assembly and structure invariably implicate its importance to membrane trafficking, organelle structure and related functions. Nonetheless, relatively few studies have examined the effects of missense substitutions within this domain. Using Neisseria gonorrhoeae as a model system, we constructed mutants with single and multiple amino acid substitutions localized to this region of the pilin subunit PilE and characterized them with regard to pilin stability, organelle expression and associated phenotypes. The consequences of simultaneous expression of the mutant and wild-type PilE forms were also examined. The findings document for the first time in a defined genetic background the phenomenon of pilin intermolecular complementation in which assembly defective pilin can be rescued into purifiable Tfp by coexpression of wild-type PilE. The results further demonstrate that pilin subunit composition can impact on organelle dynamics mediated by the PilT retraction protein via a process that appears to monitor the efficacy of subunit-subunit interactions. In addition to confirming and extending the evidence for PilE multimerization as an essential component for competence for natural genetic transformation, this work paves the way for detailed studies of Tfp subunit-subunit interactions including self-recognition within the membrane and packing within the pilus polymer.
Aas
Finn Erik
FE
Centre for Molecular Biology and Neuroscience, University of Oslo, 0316 Oslo, Norway.
Winther-Larsen
Hanne C
HC
Wolfgang
Matthew
M
Frye
Stephan
S
Løvold
Cecilia
C
Roos
Norbert
N
van Putten
Jos P M
JP
Koomey
Michael
M
D019943:Amino Acid Substitution; D036781:Fimbriae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D010861:Fimbriae, Bacterial / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D009344:Neisseria gonorrhoeae / Q000166:cytology* / Q000378:metabolism
D036781:Fimbriae Proteins