title: The Inner Membrane Protein PilG Interacts with DNA and the Secretin PilQ in Transformation.
abstract: Expression of type IV pili (Tfp), filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.
authors.affiliation: Department of Microbiology, Oslo University Hospital, Oslo, Norway.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Frye
authors.affiliation: Department of Microbiology, Oslo University Hospital, Oslo, Norway.
authors.forename: Emma
authors.initials: E
authors.lastname: Lång
authors.affiliation: Department of Microbiology, Oslo University Hospital, Oslo, Norway.
authors.forename: Getachew Tesfaye
authors.initials: GT
authors.lastname: Beyene
authors.affiliation: Department of Microbiology, Oslo University Hospital, Oslo, Norway.
authors.forename: Seetha V
authors.initials: SV
authors.lastname: Balasingham
authors.affiliation: Department of Microbiology, University of Oslo, Oslo, Norway.
authors.forename: Håvard
authors.initials: H
authors.lastname: Homberset
authors.affiliation: Department of Microbiology, Oslo University Hospital, Oslo, Norway.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Rowe
authors.affiliation: Department of Microbiology, Oslo University Hospital, Oslo, Norway.
authors.forename: Ole Herman
authors.initials: OH
authors.lastname: Ambur
authors.affiliation: Department of Microbiology, Oslo University Hospital, Oslo, Norway; Department of Microbiology, University of Oslo, Oslo, Norway.
authors.forename: Tone
authors.initials: T
authors.lastname: Tønjum
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009345:Neisseria meningitidis / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D008565:Membrane Proteins; D011994:Recombinant Proteins; C084295:pilQ protein, bacteria; D036781:Fimbriae Proteins