Characterization of a Novel Antisense RNA in the Major Pilin Locus of Neisseria meningitidis Influencing Antigenic Variation

The AS transcript is induced by salt stress in E. coli and N. meningitidis. (A) A 595-bp region, including the AS promoter from N. meningitidis 8013, was cloned into pEGFP-N2 and transformed into E. coli; transformants were subjected to different stresses to identify conditions under which the AS promoter is functional. Northern blot analysis of total RNA was performed using a probe complementary to the cloned insert (AS) or transfer mRNA (tmRNA) as a loading control. The AS transcript was upregulated following NaCl stress. (B) Northern blot analysis of total RNA from E. coli strains containing plasmids with mutations to the −35 site (Mut1), the −10 site (Mut2), or both (Mut3) of the N. meningitidis pilE AS promoter with (+) and without (−) NaCl stress. No transcript was detected upon mutation of the −10 sequence. (C) Northern blot analysis of total RNA from wild-type N. meningitidis strain 8013 subjected to different stresses. The AS transcript was detected using a 50-nt probe [(AS)pilE-1] that hybridizes specifically to the AS RNA 533 nt downstream of the AS promoter. tmRNA was used as a loading control. A transcript corresponding to the AS RNA was detected following exposure to NaCl and KCl. (D) Analysis of AS RNA expression in N. meningitidis following a 10-min incubation with different concentrations of NaCl. AS RNA levels were increased at higher salt concentrations (0.4 and 0.5 M NaCl).

Expression of the AS transcript in N. meningitidis. (A) Growth curves of WT_ery and Mut_ery cultured at 37°C in BHI broth. Arrows indicate times at which RNA was extracted for Northen blot analysis of AS RNA expression. (B) Northern blot analysis results for total RNA isolated at different time points during growth (h 1.75 [a], h 3 [b], h 5 [c], h 6 [d], and after growth overnight [O/N] for 21.5 h). The AS transcript was detected using a 50-nt probe [(AS)pilE-1] that hybridizes specifically to the AS RNA 533 nt downstream of the AS promoter. tmRNA was used as a loading control. (C) Representative Northern blot analysis of total RNA from N. meningitidis strains WT_ery and Mut_ery, cultured with and without NaCl stress. (D) Strand-specific qRT-PCR analysis of AS transcript levels in WT_ery and Mut_ery strains grown with and without NaCl stress. Amount of transcript is expressed as the R value standardized to that of the tmRNA control. P = 0.0003, Student's t test. The AS transcript was upregulated under NaCl stress in WT_ery; no transcript was detected in Mut_ery.

Characterization of the 5′ and 3′ ends of the AS transcript. (A) Primer extension analysis identified the transcriptional start site of the AS RNA in WT_ery following salt stress. The −10 and −35 sequences are indicated, and the transcriptional start site is shown (red arrow). No product was obtained using RNA from Mut_ery. (B, top panel) Mapping of the AS 3′ end by RT-PCR. Black arrows correspond to the hybridization positions of primers F and R1 to R4.5 used for PCR. The scale bar indicates the distance from the AS transcriptional start site. (Bottom panels) Agarose gel analysis of PCR products amplified using cDNAs from N. meningitidis WT_ery total RNA (upper panel) or in vitro T7-synthesized AS RNA (used as a template) (lower panel). The presence (+) or absence (−) of reverse transcriptase is indicated. A full-size 888-nt product obtained from amplification with primers F and R4 was detected using in vitro-synthesized RNA (red asterisks) but not total RNA from N. meningitidis, indicating that the in vivo transcript terminates before or within the sequence corresponding to primer R4. Green asterisks indicate nonspecific products amplified from rRNA, and orange and blue asterisks indicate truncated PCR products ascertained by sequencing. (C) Sequence of the region at the 3′ end of the AS RNA and location of primers R2 to R4.5. The G4 sequence is indicated in purple. The promoters of pilE and the G4-associated sRNA are depicted in red and purple boxes, respectively. The AS RNA is predicted to terminate within the shaded region.

Effect of the AS RNA on pilE mRNA and PilE protein levels in N. meningitidis. (A) Northern blot analysis of RNA from WT_ery and Mut_ery taken at different time points during growth (shown in Fig. 3A). pilE transcript was detected using the pilE_probe. tmRNA was used as a loading control. (B) Detection of pilE transcript in N. meningitidis with and without NaCl stress. (C) Strand-specific qRT-PCR analysis of pilE mRNA levels in WT_ery and Mut_ery with and without NaCl stress. Average R values were calculated and analyzed as described in the text. The pilE transcript was downregulated under NaCl stress in both WT_ery and Mut_ery. (D) Western blot analysis of whole-cell lysates of WT_ery and Mut_ery taken at different time points during growth. GroEL was used as a loading control. (E) Western blot analysis results for WT_ery and Mut_ery with and without NaCl stress. GroEL was used as a loading control. Mutation of the AS promoter or NaCl stress did not result in a detectable difference in PilE protein levels.