RESUMEN
Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av.
Asunto(s)
Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Neisseria gonorrhoeae/genética , Variación Antigénica/genética , Fimbrias Bacterianas/metabolismo , Conversión Génica/genética , Gonorrea/genética , Neisseria gonorrhoeae/metabolismo , Estructuras R-Loop/genética , ARN/metabolismo , Estabilidad del ARN/genética , Recombinación Genética/genéticaRESUMEN
Force production by type IV pilus retraction is critical for infectivity of Neisseria gonorrhoeae and DNA transfer. We investigated the roles of pilus number and the retraction motor, PilT, in force generation in vivo at the single-molecule level and found that individual retraction events are generated by a single pilus fiber, and only one PilT complex powers retraction. Retraction velocity is constant at low forces but decreases at forces greater than 40 pN, giving a remarkably high average stall force of 110 +/- 30 pN. Further insights into the molecular mechanism of force generation are gained from the effect of ATP-depletion, which reduces the rate of retraction but not the stall force. Energetic considerations suggest that more than one ATP is involved in the removal of a single pilin subunit from a pilus. The results are most consistent with a model in which the ATPase PilT forms an oligomer that disassembles the pilus by a cooperative conformational change.
