title: Type IV pilus assembly proficiency and dynamics influence pilin subunit phospho-form macro- and microheterogeneity in Neisseria gonorrhoeae.
abstract: The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp) colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE). In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC). Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A) required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae.
authors.affiliation: Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway; Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Åshild
authors.initials: Å
authors.lastname: Vik
authors.affiliation: Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway; Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Jan Haug
authors.initials: JH
authors.lastname: Anonsen
authors.affiliation: Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway; Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Finn Erik
authors.initials: FE
authors.lastname: Aas
authors.affiliation: Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway; Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Finn Terje
authors.initials: FT
authors.lastname: Hegge
authors.affiliation: Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway.
authors.forename: Norbert
authors.initials: N
authors.lastname: Roos
authors.affiliation: Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway; Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Michael
authors.initials: M
authors.lastname: Koomey
authors.affiliation: Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway; Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Marina
authors.initials: M
authors.lastname: Aspholm
mesh_terms: D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004983:Ethanolamines / Q000737:chemistry / Q000378:metabolism; D036781:Fimbriae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006031:Glycosylation; D015151:Immunoblotting; D013058:Mass Spectrometry; D008958:Models, Molecular; D020125:Mutation, Missense; D009344:Neisseria gonorrhoeae / Q000378:metabolism*; D010767:Phosphorylcholine / Q000737:chemistry / Q000378:metabolism; D018933:Pili, Sex / Q000378:metabolism; D011499:Protein Processing, Post-Translational
chemical_list: D004983:Ethanolamines; C074063:pilE protein, Neisseria gonorrhoeae; D010767:Phosphorylcholine; D036781:Fimbriae Proteins; C005448:phosphorylethanolamine