Transcriptional initiation of a small RNA, not R‐loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae

Abstract

Summary 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.

PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae

“…Mutation of the −35 sequence of the G4 sRNA promoter ( garP − 35 ) in the same FA1090 background showed reduced levels of pilin Av of 6.13 and 5.73%, which is consistent with the reduced levels of gar RNA produced when the −35 sequences was mutated. (Table 2) (33). This reduction in pilin Av frequency is significantly reduced compared to FA1090 by chi-square analysis (Table 2).…”

“…gonorrhoeae strains were maintained on gonococcal medium base (Difco) modified with Kellogg supplements as described previously (64). FA1090 G4 mutant, gar −10 and gar −13 were constructed as described in reference 33.…”

“…Mutation of any of the individual GC base pairs necessary to form the pilE G4 structure prevents pilin Av, while mutation of any of the TA base pairs within the sequence, which are not required to form the structure, allows normal levels of pilin Av (31). Pilin Av also depends on a promoter located adjacent to the G4-forming sequence that initiates transcription within the G4 sequence resulting in a small, noncoding RNA that can only function in cis (32, 33).…”

Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

“…Cells that successfully recombined the multisite G4 mutations were identified by screening the piliated clones in pools by PCR. Briefly, clones were individually stored in glycerol at −80°C, and pools of 10 clones were tested by using a primer, multimutG4_2, that anneals only to G4 sites that carry the desired mutations, paired with RTG4-3R ( 42 ), which binds in the beginning of the pilE locus. Positive pools were recreated by PCR using individual clones as the templates; then the promoter was amplified and sequenced with USS2 ( 43 ) and pilAREV, and the pilE locus was amplified and sequenced with PilRBS and SP3A ( 39 ).…”

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

“…A cis-acting small RNA called gar was detected in the upstream region of the major pilin locus pilE which harbours 12 GC base pairs with the potential to form a guanine quadruplex (G4) structure. It could be shown that transcription of gar opens the DNA duplex, thereby allowing the now single-stranded G-rich strand to form the G4 structure which is required for the homologous recombination reaction by which silent pilS copies are inserted into the pilE expression locus [20,21]. The sRNA NrrF was first observed in a screen for transcripts controlled by the ferric uptake regulator protein Fur in the closely related pathogen N. meningitidis [22] and later its expression was also reported in Ng [23].…”