Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation.
Bottom Line: However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes in pilE transcript or protein level.By using Northern blotting and RT-PCR analysis, we found that the RNA is expressed in stationary phase or following salt stress.Our work also indicates that this RNA does not significantly affect pilE or pilin expression levels but instead appears to modulate pilin variation.
Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.Show MeSH
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Mentions: To determine whether the AS promoter is functional, we first examined the ability of this sequence to mediate transcription in E. coli. A 595-bp region from N. meningitidis, including the putative AS promoter and 386 bp of the 3′ end of the pilE coding sequence (but excluding the pilE promoter sequence) was introduced into pEGFP-N2 to make pEGFP-N2(insert1); this plasmid only harbors a cytomegalovirus early-immediate promoter for transcription in eukaryotic but not prokaryotic cells. To examine promoter activity, E. coli strain DH5α was transformed with pEGFP-N2(insert1), grown for 3 h to an OD600 of 0.5 to 0.6 in liquid medium, and then subjected to acid, NaCl, temperature, envelope, or oxidative stress for 10 min. The presence of the AS transcript was detected by Northern blotting by using a 50-nt probe that hybridized 39 nt downstream of the predicted −10 sequence. The AS transcript was significantly more abundant when the E. coli cells were exposed to high NaCl concentrations (0.5 M for 10 min) but not after other stresses (Fig. 2A). In addition, we generated plasmids in which the predicted −35 sequence (TTGATT → TCCCTT; Mut1), the −10 sequence (TATAAT → TCACAT; Mut2), or both (Mut3) had been modified in an attempt to abolish promoter activity (Fig. 2B). No transcript was detected in E. coli harboring pEGFP-N2 alone (empty vector) with or without salt stress. A transcript of the expected size was detected in E. coli containing the plasmid with the unmodified promoter sequence (wild type [WT]) or with an alteration in the putative −35 sequence (Mut1), indicating that the promoter is functional and that the −35 sequence is dispensable for expression of the RNA. In contrast, mutation of the −10 sequence (in Mut 2 and Mut 3) was sufficient to abolish transcription.
Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.