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DNA processing chain A (DprA) is a DNA-binding protein that is ubiquitous in bacteria and expressed in some archaea. DprA is active in many bacterial species that are competent for transformation of DNA, but its role in Neisseriameningitidis (Nm) is not well characterized. An Nm mutant lacking DprA was constructed, and the phenotypes of the wild-type and ΔdprA mutant were compared. The salient feature of the phenotype of dprA null cells is the total lack of competence for genetic transformation shown by all of the donor DNA substrates tested in this study. Here, Nm wild-type and dprA null cells appeared to be equally resistant to genotoxic stress. The gene encoding DprANm was cloned and overexpressed, and the biological activities of DprANm were further investigated. DprANm binds ssDNA more strongly than dsDNA, but lacks DNA uptake sequence-specific DNA binding. DprANm dimerization and interaction with the C-terminal part of the single-stranded binding protein SSBNmwere demonstrated. dprA is co-expressed with smg, a downstream gene of unknown function, and the gene encoding topoisomerase 1, topA.

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.

Neisseria meningitidis can be the causative agent of meningitis or septicemia. This bacterium expresses type IV pili, which mediate a variety of functions, including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but it also requires auxiliary proteins for its assembly and localization in the outer membrane. Here we have studied the physical properties of the lipoprotein PilP and examined its interaction with PilQ. We found that PilP was an inner membrane protein required for pilus expression and transformation, since pilP mutants were nonpiliated and noncompetent. These mutant phenotypes were restored by the expression of PilP in trans. The pilP gene is located upstream of pilQ, and analysis of their transcripts indicated that pilP and pilQ were cotranscribed. Furthermore, analysis of the level of PilQ expression in pilP mutants revealed greatly reduced amounts of PilQ only in the deletion mutant, exhibiting a polar effect on pilQ transcription. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide. A three-dimensional reconstruction of the PilQ-PilP interacting complex was obtained at low resolution by transmission electron microscopy, and PilP was shown to localize around the cap region of the PilQ oligomer. These findings suggest a role for PilP in pilus biogenesis. Although PilQ does not need PilP for its stabilization or membrane localization, the specific interaction between these two proteins suggests that they might have another coordinated activity in pilus extrusion/retraction or related functions.