The outer membrane secretin PilQ from Neisseria meningitidis binds DNA

Abstract

Neisseria meningitidis is naturally competent for transformation throughout its growth cycle. Transformation in neisserial species is coupled to the expression of type IV pili, which are present on the cell surface as bundled filamentous appendages, and are assembled, extruded and retracted by the pilus biogenesis components. During the initial phase of the transformation process, binding and uptake of DNA takes place with entry through a presumed outer-membrane channel into the periplasm. This study showed that DNA associates only weakly with purified pili, but binds significantly to the PilQ complex isolated directly from meningococcal membranes. By assessing the DNA-binding activity of the native complex PilQ, as well as recombinant truncated PilQ monomers, it was shown that the N-terminal region of PilQ is involved in the interaction with DNA. It was evident that the binding of ssDNA to PilQ had a higher affinity than the binding of dsDNA. The binding of DNA to PilQ did not, however, depend on the presence of the neisserial DNA-uptake sequence. It is suggested that transforming DNA is introduced into the cell through the outer-membrane channel formed by the PilQ complex, and that DNA uptake occurs by non-specific introduction of DNA coupled to pilus retraction, followed by presentation to DNA-binding component(s), including PilQ.

Specific DNA recognition mediated by a type IV pilin

“…Because pilus/pseudopilus retraction powers DNA uptake, these organelles must interact with DNA at an early stage. However, the biological significance of the weak interactions previously reported between Tfp and DNA remains unclear (13,14), and no interaction with DNA has ever been reported for a purified pilus component. Our finding that the minor pilin ComP displays intrinsic DNA binding activity is, thus, highly significant, because it sheds light on the earliest step in DNA transformation.…”

“…(i) High nonspecific binding of DNA to whole bacteria (11,12) has been a hinder to genetic studies. (ii) ELISA using purified filaments has revealed only weak DNA binding with biological significance that is unclear (13,14). (iii) Various biochemical approaches have only identified DNA binding proteins that are involved at late stages during transformation (15,16).…”

The Extra-Membranous Domains of the Competence Protein HofQ Show DNA Binding, Flexibility and a Shared Fold with Type I KH Domains

“…35 It is highly conserved and predicted to contain several -strands 36 embedded in the outer membrane to form the actual pore through which transport occurs. 21 The N-terminal periplasmic region displays conservation only in secretins from related secretion pathways 29 and may be involved in substrate recognition, 28,37 gating of the proposed channel 35,38 and DNA binding 39 as well as contributing to subunit oligomerization. 35,40 The atomic structures of periplasmic fragments from two secretins, EscC from enteropathogenic E. coli and GspD from enterotoxigenic E. coli, have previously been determined.…”

“…DNA binding to the secretin PilQ has previously been demonstrated in both N. meningitidis and Thermus thermophilus HB27. 39,53 In the case of Neisseria meningitidis PilQ, this binding has been mapped to the extra-membranous region…”

“…The N-terminus of PilQ in N. meningitidis contains many small basic repeat elements that are thought to be critical for DNA binding. 39 Such elements are missing from emHofQ, indicating that DNA binding must be mediated via a different recognition motif.…”