Identification of neisserial DNA binding components

Abstract

Neisseria meningitidis, a causative agent of meningitis and septicaemia, expresses type IV pili, a feature correlating with the uptake of exogenous DNA from the environment by natural transformation. The outer membrane complex PilQ, through which pili are extruded and retracted, has previously been shown to bind DNA in its pore region. In order to further elucidate how DNA is transported across the membranes, we searched for DNA binding proteins within the meningococcal inner membrane. Inner membrane fractions from a panel of neisserial strains were subjected to a solid-phase overlay assay with DNA substrates, and MS was subsequently employed to identify proteins that bind DNA. A number of DNA binding components were detected, including the pilus biogenesis component PilG, the competence protein ComL, and the cell division ATP-binding protein FtsE, as well as two hypothetical proteins. The DNA binding activity of these components was not dependent on the presence of the neisserial DNA uptake sequence. Null mutants, corresponding to each of the proteins identified, were constructed to assess their phenotypes. Only mutants defective in pilus biogenesis were non-competent and non-piliated. The DNA binding activity of the pilus biogenesis components PilQ and PilG and the phenotypes of their respective null mutants suggest that these proteins are directly involved as players in natural transformation, and not only indirectly, through pilus biogenesis.

Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus

“…These findings provide clear evidence that in T. thermophilus pilus structures are not required for DNA uptake. This is different from the findings in other Gram-negative bacteria, such as N. gonorrhoeae and V. cholerae (36,37). Here the pilus structures are suggested to be implicated in DNA uptake.…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…Well-studied examples include those of Haemophilus, Neisseria, Legionella, Bacillus, and Thermus (8,33,82,150,338,367). DNA uptake occurs by a series of steps that require both inner and outer membrane components of the T4P assembly system, some of which have been shown to bind DNA directly (24,245). The exact mechanism by which T4P participate in DNA uptake is not yet clear, but mutants lacking pili or that are incapable of pilus retraction lose competency (70).…”

Specific DNA recognition mediated by a type IV pilin

“…(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). Nevertheless, the fact that Neisseria species and Pasteurellaceae favor uptake of homotypic DNA by selectively recognizing genus-specific DNA uptake sequence (DUS) motifs [or uptake signal sequence (USS) as they are known in Pasteurellaceae] is clear evidence of the existence of specific surfaceexposed DNA receptors (5,17,18).…”