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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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Effects on antigenic variation of AS mutation and AS induction under salt stress. (A) Av assays were performed with wild-type N. meningitidis strain 8013 and an isogenic mutant with a kanamycin resistance cassette inserted into the guanine quartet (G4) sequence. pilE sequences obtained were compared to the potential pilS donor sequences, and the number of variation events was calculated. No pilE variation events were detected in the strain with the G4 mutation. (B) Av assays were performed on WT_ery(RecA6) and Mut_ery(RecA6) with and without salt stress. Results were analyzed by fitting generalized linear models. NaCl stress resulted in a statistically significant reduction in antigenic variation in WT_ery(RecA6) (P = 0.0149) but not in Mut_ery(RecA6).
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Figure 6: Effects on antigenic variation of AS mutation and AS induction under salt stress. (A) Av assays were performed with wild-type N. meningitidis strain 8013 and an isogenic mutant with a kanamycin resistance cassette inserted into the guanine quartet (G4) sequence. pilE sequences obtained were compared to the potential pilS donor sequences, and the number of variation events was calculated. No pilE variation events were detected in the strain with the G4 mutation. (B) Av assays were performed on WT_ery(RecA6) and Mut_ery(RecA6) with and without salt stress. Results were analyzed by fitting generalized linear models. NaCl stress resulted in a statistically significant reduction in antigenic variation in WT_ery(RecA6) (P = 0.0149) but not in Mut_ery(RecA6).

Mentions: The G4 sequence and position relative to pilE is conserved in N. meningitidis strain 8013, and so we first performed Av assays using 8013 and 8013ΔG4, which has a kanamycin resistance marker with transcriptional terminators inserted into the G4 sequence (see Fig. S1 in the supplemental material). pilE variation was detected in 8013, while no Av events occurred in 8013ΔG4 (Fig. 6A), indicating that the G4 is required for Av in meningococcus and that Av is detectable by our methods. We therefore examined Av in WT_ery(RecA6) and Mut_ery(RecA6) in the presence of IPTG to induce RecA expression, with or without NaCl to induce the expression of the AS RNA. The sequence of pilE in the resulting colonies was compared to the potential pilS donor sequences in the N. meningitidis 8013 genome, and the number of variation events was determined. Table 3 and Fig. 6B show results from three independent assays (a total of 1,150 pilE sequences). We fitted a binomial GLH which proved to be satisfactory (goodness of fit statistic = 5.8392 on 6 degrees of freedom; P = 0.4414). On their own, neither the presence of NaCl nor the mutation resulted in a statistically significant change in levels of antigenic variation. However, we found that there was a significantly reduced level of antigenic variation when WT_ery was induced with NaCl (z = 2.434, P = 0.0149) but this was not observed for Mut_ery, indicating that the AS RNA transcript or transcription may influence pilin Av.


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)

Effects on antigenic variation of AS mutation and AS induction under salt stress. (A) Av assays were performed with wild-type N. meningitidis strain 8013 and an isogenic mutant with a kanamycin resistance cassette inserted into the guanine quartet (G4) sequence. pilE sequences obtained were compared to the potential pilS donor sequences, and the number of variation events was calculated. No pilE variation events were detected in the strain with the G4 mutation. (B) Av assays were performed on WT_ery(RecA6) and Mut_ery(RecA6) with and without salt stress. Results were analyzed by fitting generalized linear models. NaCl stress resulted in a statistically significant reduction in antigenic variation in WT_ery(RecA6) (P = 0.0149) but not in Mut_ery(RecA6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402397&req=5

Figure 6: Effects on antigenic variation of AS mutation and AS induction under salt stress. (A) Av assays were performed with wild-type N. meningitidis strain 8013 and an isogenic mutant with a kanamycin resistance cassette inserted into the guanine quartet (G4) sequence. pilE sequences obtained were compared to the potential pilS donor sequences, and the number of variation events was calculated. No pilE variation events were detected in the strain with the G4 mutation. (B) Av assays were performed on WT_ery(RecA6) and Mut_ery(RecA6) with and without salt stress. Results were analyzed by fitting generalized linear models. NaCl stress resulted in a statistically significant reduction in antigenic variation in WT_ery(RecA6) (P = 0.0149) but not in Mut_ery(RecA6).
Mentions: The G4 sequence and position relative to pilE is conserved in N. meningitidis strain 8013, and so we first performed Av assays using 8013 and 8013ΔG4, which has a kanamycin resistance marker with transcriptional terminators inserted into the G4 sequence (see Fig. S1 in the supplemental material). pilE variation was detected in 8013, while no Av events occurred in 8013ΔG4 (Fig. 6A), indicating that the G4 is required for Av in meningococcus and that Av is detectable by our methods. We therefore examined Av in WT_ery(RecA6) and Mut_ery(RecA6) in the presence of IPTG to induce RecA expression, with or without NaCl to induce the expression of the AS RNA. The sequence of pilE in the resulting colonies was compared to the potential pilS donor sequences in the N. meningitidis 8013 genome, and the number of variation events was determined. Table 3 and Fig. 6B show results from three independent assays (a total of 1,150 pilE sequences). We fitted a binomial GLH which proved to be satisfactory (goodness of fit statistic = 5.8392 on 6 degrees of freedom; P = 0.4414). On their own, neither the presence of NaCl nor the mutation resulted in a statistically significant change in levels of antigenic variation. However, we found that there was a significantly reduced level of antigenic variation when WT_ery was induced with NaCl (z = 2.434, P = 0.0149) but this was not observed for Mut_ery, indicating that the AS RNA transcript or transcription may influence pilin Av.

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