Results: 5

1.
Figure 2

Figure 2. Genetic analysis of pilE G4-associated sRNA.. From: Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae.

The pilE G4-associated sRNA promoter (boxed), start site (+1) and surrounding sequence are shown. Arrows indicate the location of insertions (+) and point mutations (pt mts) while deletions (−) are underlined in red. Whether these mutations resulted in wild-type levels of pilin Av (Av), an Av deficient (Avd), or an Av intermediate (Avi) phenotype is also indicated. These Av phenotypes were determined by a pilus-dependent colony phase variation kinetic assay [16] which is shown in Fig. S1.

Laty A. Cahoon, et al. PLoS Pathog. 2013 January;9(1):e1003074.
2.
Figure 5

Figure 5. A working model of the initiation of pilin Av in N. gonorrhoeae.. From: Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae.

Shown is the pilin expression locus (pilE) and the upstream region containing the pilE G4-associated sRNA promoter. We propose that the initiation of pilin Av begins with transcription of the pilE G4-associated sRNA promoter (i). During transcription, a RNA∶DNA hybrid is made on the C-rich strand of the pilE G4 sequence (ii) facilitating the formation of a G4 structure by the unpaired G-rich strand, which may be mediated by structure specific binding proteins (iii).

Laty A. Cahoon, et al. PLoS Pathog. 2013 January;9(1):e1003074.
3.
Figure 4

Figure 4. The orientation and direction of the pilE G4 sRNA is required for its function.. From: Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae.

A. The pilE G4 sRNA region. The C-rich strand (blue) and G-rich strand (pink) are designated. A triangle indicates a transposon insertion used for selection of transformation. B. The pilE G4 sRNA endogenous locus mutants. A representation of the inverted, reverse, and reverse-inverted sRNA mutants is shown. A triangle indicates a transposon used as a marker for transformation. C. Kinetic pilus-dependent colony phase variation assay. The pilE G4 sRNA inverted, reverse, and reverse-inverted do not allow pilin Av. P<0.05 as determined by two-tailed Student's T-test (asterisks). Error bars represent the standard error of the mean for 10 colonies. A representative assay of n = 3 is shown.

Laty A. Cahoon, et al. PLoS Pathog. 2013 January;9(1):e1003074.
4.
Figure 3

Figure 3. A phage promoter can substitute for the pilE G4-associated sRNA promoter.. From: Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae.

A. In vitro transcription. Shown is a 14% denaturing polyacrylamide gel containing T7 RNA polymerase-transcribed RNA products from a template containing the minimal T7 promoter element. The RNA ladder is labeled in bases. The higher molecular weight band is most likely an alternative start site directed by the T7 polymerase. B. The T7 promoter/T7 RNA polymerase strain. T7 RNA polymerase was cloned downstream of a dual taclac promoter which allows induction with IPTG [26]. The construct was transformed into a strain where the pilE G4 sRNA −10 promoter element was replaced with a minimal T7 promoter and inserted into the neisserial intergenic complementation site (NICS). [A triangle indicates a transposon (TN) encoding kanamycin resistance and was used as a marker for transformation to create the sRNA T7 promoter mutant]. C. Colony morphology of the T7 promoter and/or T7 polymerase strains. Colonies were grown for 46 hours with IPTG. The parental strain shows an Av phenotype, a strain expressing only T7 at the NICS (NICS T7 Pol.) has an Av phenotype, a strain where the G4 sRNA −10 element is replaced by the minimal T7 promoter (T7 Prom.) shows an Av-deficient (Avd) phenotype that is rescued with the expression of T7 polymerase (T7 Prom./NICS T7 Pol.). D. Kinetic pilus-dependent colony phase variation assay. Expression of T7 polymerase at the NICS (NICS T7 Pol.) does not affect phase variation whereas replacement of the G4 sRNA promoter with a minimal T7 promoter (T7 Prom.) causes a decrease in phase variation which is rescued by expression of T7 polymerase at the NICS (T7 Prom./NICS T7 Pol.). P<0.05 as determined by two-tailed Student's T-test (asterisks). Error bars represent the standard error of the mean for 10 colonies. A representative assay of n = 4 is shown.

Laty A. Cahoon, et al. PLoS Pathog. 2013 January;9(1):e1003074.
5.
Figure 1

Figure 1. Analysis of the pilE G4-associated sRNA and effect on pilin Av.. From: Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae.

A. Chromosomal map of the pilE G4-associated sRNA locus. In gonococci, the G4-associated sRNA promoter is located upstream of pilE in the opposite orientation of transcription and is adjacent to the pilE G4. B. Predicted sRNA promoter sequence. Shown are the predicted sRNA −10 and −35 promoter sequences (boxed), the G to T mutation that makes a stronger promoter, and the predicted start sites detected by 5′RACE located within the pilE G4 forming sequence (underlined). Bases numbered and arrowed indicate the number of 5′RACE transformants showing the arrowed base as a start site. C. pilE G4 sRNA promoter mutagenesis. Knock-out mutation of the −10 and −35 promoter elements are shown D. Kinetic pilus-dependent colony phase variation assay. This standard assay measures the average number of visible pilus-dependent colony morphology changes occurring over time and is a surrogate measure for pilin Av [16]. Mutation of the −10 (mt-10) and/or −10 & −35 (mt-10/-35) promoter elements causes a loss in phase variation (these lines overlap), whereas mutation of the −35 promoter element alone results in an intermediate phenotype. P<0.05 as determined by two-tailed Student's T-test indicated by an asterisk. Bacteria having a stronger −10 promoter (str −10) element show the same levels of phase variation as the parental strain. Error bars represent the standard error of the mean for 10 colonies. A representative assay of n = 4 is shown. E. In vitro transcription products initated from sRNA promoter sequences. Shown is a 14% denaturing polyacrylamide gel containing in vitro transcribed RNA products using Escherichia coli RNA polymerase. Transcription was initiated from the wild-type promoter (wt-10), str-10, mt −10/−35, mt-10, mt −35, and a promoter that contains an insertion of 5 base pairs (bps) between the −10 and −35 elements (+5 bp btwn −10/−35). The RNA ladder is labeled in bases. The location of the sRNA transcript is shown by the arrow. The higher molecular weight bands represent irrelevant transcription products.

Laty A. Cahoon, et al. PLoS Pathog. 2013 January;9(1):e1003074.

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