Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression

Abstract

SummaryThe mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transformation have been defined in Gram-negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram-negative transformation pathway. In the human pathogen Neisseria gonorrhoeae , sequence-specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit-like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNAbinding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence-specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non-specific DNA-binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.

Specific DNA recognition mediated by a type IV pilin

“…It can be predicted that, in the absence of such a receptor (when the corresponding gene is mutated), DNA will not interact with Tfp and be pulled through the outer membrane on Tfp retraction and that the transformation will be dramatically affected. Accordingly, as shown here, the fibers produced by a comP mutant display a dramatically reduced ability to bind DNA, and as shown previously, comP mutants are defective for DNA uptake (23) and noncompetent (20,21). It is important to note that comP mutants are otherwise indistinguishable from WT for piliation levels or any other Tfp-linked property (20,21), underlining the exclusive role of ComP in competence.…”

“…It is important to note that comP mutants are otherwise indistinguishable from WT for piliation levels or any other Tfp-linked property (20,21), underlining the exclusive role of ComP in competence. Consistent with this scenario, ComP overexpression enhances transformation frequencies by increasing both DNA binding and uptake (21,23).…”

“…(i) ComP is one of three minor (low abundance) pilins in pathogenic Neisseria species (with PilV and PilX) that modulate Tfp-mediated properties without affecting Tfp biogenesis (19)(20)(21)(22). ComP has a prominent role in competence (20,21) at the level of DNA uptake (23). (ii) ComP displays unparalleled sequence conservation for a surface-exposed protein in N. meningitidis, because it was found to be virtually identical in a large collection of disease isolates (24).…”

Structure of the Neisseria meningitidis Outer Membrane PilQ Secretin Complex at 12 Å Resolution

“…The expression of Tfp is vital to the pathogenicity of these organisms, and they play a pivotal role in cellular adhesion to host epithelial cells. Neisseria also utilizes Tfp in several diverse processes, including cellular agglutination (2), twitching motility (3), and DNA uptake during transformation (4). Tfp are composed predominantly of a helical polymer of the pilin subunit PilE; a model for the helix with five pilin subunits/turn has been proposed based on the crystal structure of PilE (5).…”

Functional Analysis of PilT from the Toxic Cyanobacterium Microcystis aeruginosa PCC 7806

“…Tfp proteins are also responsible for mediating a number of other bacterial physiological processes, such as twitching motility. Tfp proteins occur in a wide range of gram-negative bacteria, including Pseudomonas aeruginosa and Neisseria gonorrhoeae, the genetics of which have been well characterized (1,12,22,23). Evidence for the association of the Tfp system and natural transformability is substantial, and often the presence of a Tfp system is an indication of natural competence (11,19).…”