ComE, a Competence Protein from Neisseria gonorrhoeae with DNA-Binding Activity

Abstract

Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 ؋ 10 4 -fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.

Specific DNA recognition mediated by a type IV pilin

“…The fusion proteins are directed to the periplasm, which is important for disulfide bond formation, a characteristic feature of type IV pilins (27). After a two-step purification that yielded pure proteins, we tested the ability of these four pilins to interact with DNA using agarose EMSAs (10). As a target, we used the pSY6 plasmid that contains one DUS, which has been widely used to measure transformation efficiencies in Neisseria species (13).…”

“…Bound Tfp were detected by ELISA using the 20D9 monoclonal antibody as previously described (21). Agarose EMSAs were performed essentially as described elsewhere (10). Acrylamide EMSAs were performed as follows.…”

“…The finding that PilT, which is a cytoplasmic ATPase powering the remarkable capacity of Tfp to retract and generate mechanical force (7), is necessary for DNA uptake (8) suggests that Tfp/pseudopili pull DNA through the outer membrane/peptidoglycan on retraction (5). DNA then interacts with ComE/ComEA (9,10) and is delivered to a conserved translocase machinery in the cytoplasmic membrane that further transports it into the cytoplasm. Finally, DNA is integrated in the bacterial chromosome in a RecAdependent manner, but it can also be used as a template for the repair of DNA damage or a source of food (5).…”

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

“…In the cytoplasm the pilus retraction ATPase PilT is necessary for transformation (21). Within the periplasm, the DNAbinding protein ComE is essential for DNA uptake (22). In the absence of transforming DNA, ComE is distributed homogeneously within the periplasm and rapidly colocalizes with imported DNA (23,24).…”

“…Similarly, no ion gradient is maintained over the outer membrane. (ii) The periplasmic DNA-binding protein ComE is essential for DNA import into the periplasm (22,23) and depicts an ideal candidate for biasing the direction of DNA movement through the membrane. (iii) Single-cell studies of DNA uptake show that the secondary structure of DNA has only a minor effect on uptake kinetics (30), arguing against a machine that requires a tight fit on its substrate.…”