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

Abstract

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.

Figure 1.

gar transcript levels influence pilin Av frequencies A. Diagram of the G4 gar, pilE gene and G4 motif (blue box and highlighted in yellow). gar, the pilE G4-associated sRNA, is long and poorly expressed, as represented by the red dashed line indicating decreasing RNA transcription. In the mutants, the garP₋₁₀ sequence has been mutated to CCGTCC and the garP₋₃₅ sequence has been mutated to CCACTC. The G4 sRNA initiates within the G4 motif, using the C-rich side of the DNA as the template. B. Alignment of the −35 promoter element to the G4 motif of common N. gonorrhoeae (Ng) and N. meningitidis (Nm) strains using CLC sequence viewer. The −35 and −10 promoter elements are denoted with a line along the top. C. Transcript levels in the parent strain (recA6) and mutant strains garP₋₁₀ and garP₋₃₅ were measured using qRT-PCR. Strains were grown in liquid medium to obtain similar growth phase to maintain RNA quality. Primers were designed to only amplify within the sRNA region. The reported value represents the mean of three biological replicates +/− one standard deviation as indicated by the error bars. There is no significant difference between the transcript levels of garP₋₁₀ and garP₋₃₅ but the average garP₋₃₅ transcript levels (0.38) are 6x higher than the garP₋₁₀ (0.06)_.Unpaired student t-test **p<0.01 D. Cartoon showing the gar DNA sequence with a terminator sequence added (Green box) and the resulting RNA products. We constructed strains to create a84nt, 72 nucleotides and 32 nucleotides terminated gar . The 84 nucleotides sequence was used to determine the E. coli terminator efficiency. Pilin Av frequencies of the 72 and 32 nucleotides strains were determined by PacBio sequencing.

Figure 2.

Transcription and modulation of rnhA modulates R-loop levels A. R-loop levels of garP₋₁₀ and garP₋₃₅ strains relative to the parental strain (FA1090). Relative R-loop levels are calculated by determining the percent input after ChIP pulldown for each sample and the dividing by the percent input of FA1090. The reported value represents the mean of five biological replicates (individual dots) +/− one standard deviation as indicated by the error bars. There is no significant difference between garP₋₁₀and garP₋₃₅ . Unpaired student t-test **p<0.01 *p<0.05 B. The rnhA+nicsP_lac::rnhA strain allows modulation of RNase HI. rnhA expression measured using qRT-PCR with Gc grown with different levels of IPTG in solid media. The average fold change compared to FA1090 (±standard deviation) for each IPTG level are: 0 IPTG-0.30 (0.09), 0.001 mM IPTG 0.61 (0.32), 0.005 mM IPTG 1.4 (0.5), 0.01 mM IPTG 4.3 (2.2), 1 mM IPTG 61.9 (33). The reported value represents the mean of five biological replicates (individual dots) +/− one standard deviation as indicated by the error bars. Unpaired student t-test . **p<0.01 *p<0.05 C. R-loop levels determined by R-loop ChIP when rnhA expression is modulated. The reported value represents the mean of six biological replicates (individual dots) +/− one standard deviation as indicated by the error bars. Unpaired student t-test **p<0.01

Figure 3.

G4 ChIP detects intracellular G4 levels The sequence of the G4 motif is 5’-GGGTGGGTTGGGTGGG and G4 mutant is 5’-GTGGGGGTGTGGTGGG with 3 of 4 G-tracts interrupted. G4 levels were determined from plate grown bacteria using G4 ChIP with the 1H6 Ab. The parental strain (recA6) and G4 mutant, and the double helicase mutant (recQ/rep) were tested for G4 levels. An alternate region of the genome (ectopic site) was amplified to test for non-specific DNA affinity by the Ab. IgG was used to determine general Ab binding. The reported value represents the mean of four biological replicates (individual dots) +/− one standard deviation as indicated by the error bars. Unpaired student t-test **p<0.01 *p<0.05

Figure 4.

Transcription initiation, but not R-loop stability, influences G4 levels A. G4 levels were determined using G4 ChIP on garP₋₁₀ and garP₋₃₅ with the G4 specific MAb, 1H6). Relative G4 levels are calculated by determining the % input after ChIP pulldown for each sample and the dividing by the % input of FA1090. Bar The reported value represents the mean of six biological replicates (individual dots) +/− one standard deviation as indicated by the error bars. Unpaired student t-test **p<0.01 B. G4 ChIP was used to determine G4 levels under low rnhA and high rnhA expression (rnhA+nicsP_lac::rnhA 0 IPTG, and rnhA+nicsP_lac::rnhA 1 mM IPTG). The reported value represents the mean of six biological replicates (individual dots) +/− one standard deviation as indicated by the error bars. Unpaired student t-test**p<0.01

Figure 5.

Summary of results Transcription of the G4 sRNA is the first step in Av. In A, RNA polymerase (grey oval) opens the DNA duplex. The sRNA transcript (red line) remains bound to the C rich strand of DNA, while the other DNA strand can fold into a G4 structure (grey structure with three square planes and zig zag loops). In B, reduced transcription causes reduced G4 formation and subsequent Av. In C, RNase H levels are low so R-loops are high, however, R-loop stability is dispensable for G4 levels and pilin Av.