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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.
Highlights
Neisseria meningitidis, the meningococcus (Mc), is a human-specific opportunistic pathogen and one of the leading causative agents of meningitis and septicaemia worldwide [1].Type IV pili (Tfp) are filamentous appendages emanating from the bacterial surface that are required for adherence of bacteria to human cells and for transformation of DNA [2, 3]
We report that N-terminal PilG exhibits intrinsic DNA uptake sequence (DUS)-independent DNA binding activity and that PilG interacts with the secretin PilQ
To search for DNA binding motifs, we focused on three regions of the PilG aa sequence, residues 1 to 185, 200 to 225 and 250 to 377, and excluded predicted transmembrane helices between these segments
Summary
Type IV pili (Tfp) are filamentous appendages emanating from the bacterial surface that are required for adherence of bacteria to human cells and for transformation of DNA [2, 3]. Biogenesis of Tfp is not well characterized, but several proteins required for pilus assembly, extrusion and retraction have been identified [4]. ComP [6], the secretin PilQ [7,8,9,10], the lipoproteins PilP [11] and PilW [12], the prepilin peptidase PilD [13], the ATPase PilT, which is driving pilus retraction [14, 15], and the adhesin PilC [16]. Tfp pass through the outer membrane secretin pore consisting of the dodecamer PilQ [7, 9, 10, 17]
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