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Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation.

Tan FY, Wörmann ME, Loh E, Tang CM, Exley RM - J. Bacteriol. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

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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. meningitidispilE 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).
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Figure 2: 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. meningitidispilE 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).

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.


Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation.

Tan FY, Wörmann ME, Loh E, Tang CM, Exley RM - J. Bacteriol. (2015)

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. meningitidispilE 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).
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Figure 2: 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. meningitidispilE 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).
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

Show MeSH
Related in: MedlinePlus