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.

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

“…PilG is a polytopic inner membrane protein which is required for DNA uptake [68] and it has been suggested that PilG is involved in counter-retraction of the pilus fibre, thereby antagonizing the function of the pilus retraction motor PilT [69]. PilG exhibits intrinsic DUS-independent DNA-binding activity and interacts with the PilQ secretin in the outer membrane [70]. Recently, a link between pilus biogenesis and processing of incoming DNA was proposed based on the observation that knockout of pilG and dprA , which loads RecA onto single-stranded DNA, resulted in reciprocal downregulation of the corresponding proteins [71].…”

“…PilG is a polytopic inner membrane protein which is required for DNA uptake [68] and it has been suggested that PilG is involved in counterretraction of the pilus fibre, thereby antagonizing the function of the pilus retraction motor PilT [69]. PilG exhibits intrinsic DUS-independent DNA-binding activity and interacts with the PilQ secretin in the outer membrane [70].…”

The RpoN2‐PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae

“…Homologous proteins that play roles in twitching motility and virulence in other bacteria have also been studied. For example, the PilG/H/L/J proteins are required for twitching signal transduction in Acinetobacter baylyi (Leong et al , ), and PilG is involved in T4P‐mediated transformation by interacting with PilQ and target DNAs in Neisseria meningitidis (Collins et al , ; Frye et al , ). PilM/N/O/P proteins form an inner membrane complex that affects the stability of the P. aeruginosa T4P secretin (Ayers et al , ).…”

Comparative proteomic analysis of Neisseria meningitidis wildtype and dprA null mutant strains links DNA processing to pilus biogenesis

“…5 ). We have previously shown that PilG is absolutely required for Nm pilus biogenesis [ 26 ] and directly binds DNA [ 40 , 41 ] and the secretin PilQ in transformation [ 40 , 42 ]. The neisserial pilG and pilT mutants and the Thermus thermophilus pilM mutant are defective for transformation [ 43 – 46 ].…”