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Pseudomonas aeruginosa biofilm matrix polysaccharide Psl is regulated transcriptionally by RpoS and post-transcriptionally by RsmA.

Irie Y, Starkey M, Edwards AN, Wozniak DJ, Romeo T, Parsek MR - Mol. Microbiol. (2010)

Bottom Line: In this study, we demonstrate that the alternative σ-factor RpoS is a positive transcriptional regulator of psl gene expression.Furthermore, we show that psl mRNA has an extensive 5' untranslated region, to which the post-transcriptional regulator RsmA binds and represses psl translation.This constitutes a novel mechanism for translational repression by this family of regulators.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

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RsmA binding to the 5′ UTR of psl mRNA may promote double-stranded RNA binding between SD and anti-SD sequences.A. Possible two-dimensional structure of psl mRNA translational control region predicted by mFOLD. The translational start codon is shown in bold font and underlined. The RsmA binding site is located in the top portion of the stem-loop structure, with the essential GG pair in the loop region. The anti-SD sequence (UGCUCU) is boxed. The anti-SD sequence was mutated to CUGCAG (anti-SD mutant) in order to prevent its base-pairing with the SD sequence.B. Representative images of RsmA–psl repression model. In WT cells, RsmA homodimers bind to the RsmA binding site located in the 5′ UTR of psl mRNA. This results in the formation of a stem-loop structure, which base-pairs the SD sequence with the anti-SD sequence. The double-stranded RNA form blocks ribosome access to the SD site, preventing ribosome assembly and therefore inhibiting translation of psl. In the ΔrsmA mutant, the stem-loop structure is not stable enough to prevent ribosome access to the SD site, therefore initiating psl translation. Constructs designed to prevent SD base-pairing (anti-SD mutant) may still allow RsmA binding to the 5′ UTR of psl resulting in a smaller stem-loop structure, but the SD site remains accessible for ribosome to initiate translation of psl.C. Transcriptional lacZ fusion constructs of the anti-SD mutant does not display altered levels of psl transcription in PAO1 and isogenic ΔrsmA backgrounds.D. Translational lacZ fusion constructs of the anti-SD mutant demonstrate an increase of psl translation compared with WT in PAO1 background, similar to the psl translation level of ΔrsmA. The stem disruption mutation did not significantly affect the levels of psl translation in the ΔrsmA background.
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fig11: RsmA binding to the 5′ UTR of psl mRNA may promote double-stranded RNA binding between SD and anti-SD sequences.A. Possible two-dimensional structure of psl mRNA translational control region predicted by mFOLD. The translational start codon is shown in bold font and underlined. The RsmA binding site is located in the top portion of the stem-loop structure, with the essential GG pair in the loop region. The anti-SD sequence (UGCUCU) is boxed. The anti-SD sequence was mutated to CUGCAG (anti-SD mutant) in order to prevent its base-pairing with the SD sequence.B. Representative images of RsmA–psl repression model. In WT cells, RsmA homodimers bind to the RsmA binding site located in the 5′ UTR of psl mRNA. This results in the formation of a stem-loop structure, which base-pairs the SD sequence with the anti-SD sequence. The double-stranded RNA form blocks ribosome access to the SD site, preventing ribosome assembly and therefore inhibiting translation of psl. In the ΔrsmA mutant, the stem-loop structure is not stable enough to prevent ribosome access to the SD site, therefore initiating psl translation. Constructs designed to prevent SD base-pairing (anti-SD mutant) may still allow RsmA binding to the 5′ UTR of psl resulting in a smaller stem-loop structure, but the SD site remains accessible for ribosome to initiate translation of psl.C. Transcriptional lacZ fusion constructs of the anti-SD mutant does not display altered levels of psl transcription in PAO1 and isogenic ΔrsmA backgrounds.D. Translational lacZ fusion constructs of the anti-SD mutant demonstrate an increase of psl translation compared with WT in PAO1 background, similar to the psl translation level of ΔrsmA. The stem disruption mutation did not significantly affect the levels of psl translation in the ΔrsmA background.

Mentions: Predicted RsmA binding site in the psl 5′ UTR.A. The predicted position of SD is indicated by the underlined 7–12 bases upstream of the start codon of pslA ORF. Predicted RsmA binding site is boxed, 24–37 bases upstream of the start codon. The asterisks indicate the GG nucleotide pair required for CsrA/RsmA binding to its RNA target, which was mutated to the CC pair for the experiments demonstrated in Fig. 11. +1 indicates the transcriptional start site (in bold font) and the corresponding −10 and −35 regions of the promoter (underlined).B. Sequence alignment of psl RsmA binding site with the CsrA consensus sequence (R = A or G) as previously reported (Dubey et al., 2005).


Pseudomonas aeruginosa biofilm matrix polysaccharide Psl is regulated transcriptionally by RpoS and post-transcriptionally by RsmA.

Irie Y, Starkey M, Edwards AN, Wozniak DJ, Romeo T, Parsek MR - Mol. Microbiol. (2010)

RsmA binding to the 5′ UTR of psl mRNA may promote double-stranded RNA binding between SD and anti-SD sequences.A. Possible two-dimensional structure of psl mRNA translational control region predicted by mFOLD. The translational start codon is shown in bold font and underlined. The RsmA binding site is located in the top portion of the stem-loop structure, with the essential GG pair in the loop region. The anti-SD sequence (UGCUCU) is boxed. The anti-SD sequence was mutated to CUGCAG (anti-SD mutant) in order to prevent its base-pairing with the SD sequence.B. Representative images of RsmA–psl repression model. In WT cells, RsmA homodimers bind to the RsmA binding site located in the 5′ UTR of psl mRNA. This results in the formation of a stem-loop structure, which base-pairs the SD sequence with the anti-SD sequence. The double-stranded RNA form blocks ribosome access to the SD site, preventing ribosome assembly and therefore inhibiting translation of psl. In the ΔrsmA mutant, the stem-loop structure is not stable enough to prevent ribosome access to the SD site, therefore initiating psl translation. Constructs designed to prevent SD base-pairing (anti-SD mutant) may still allow RsmA binding to the 5′ UTR of psl resulting in a smaller stem-loop structure, but the SD site remains accessible for ribosome to initiate translation of psl.C. Transcriptional lacZ fusion constructs of the anti-SD mutant does not display altered levels of psl transcription in PAO1 and isogenic ΔrsmA backgrounds.D. Translational lacZ fusion constructs of the anti-SD mutant demonstrate an increase of psl translation compared with WT in PAO1 background, similar to the psl translation level of ΔrsmA. The stem disruption mutation did not significantly affect the levels of psl translation in the ΔrsmA background.
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fig11: RsmA binding to the 5′ UTR of psl mRNA may promote double-stranded RNA binding between SD and anti-SD sequences.A. Possible two-dimensional structure of psl mRNA translational control region predicted by mFOLD. The translational start codon is shown in bold font and underlined. The RsmA binding site is located in the top portion of the stem-loop structure, with the essential GG pair in the loop region. The anti-SD sequence (UGCUCU) is boxed. The anti-SD sequence was mutated to CUGCAG (anti-SD mutant) in order to prevent its base-pairing with the SD sequence.B. Representative images of RsmA–psl repression model. In WT cells, RsmA homodimers bind to the RsmA binding site located in the 5′ UTR of psl mRNA. This results in the formation of a stem-loop structure, which base-pairs the SD sequence with the anti-SD sequence. The double-stranded RNA form blocks ribosome access to the SD site, preventing ribosome assembly and therefore inhibiting translation of psl. In the ΔrsmA mutant, the stem-loop structure is not stable enough to prevent ribosome access to the SD site, therefore initiating psl translation. Constructs designed to prevent SD base-pairing (anti-SD mutant) may still allow RsmA binding to the 5′ UTR of psl resulting in a smaller stem-loop structure, but the SD site remains accessible for ribosome to initiate translation of psl.C. Transcriptional lacZ fusion constructs of the anti-SD mutant does not display altered levels of psl transcription in PAO1 and isogenic ΔrsmA backgrounds.D. Translational lacZ fusion constructs of the anti-SD mutant demonstrate an increase of psl translation compared with WT in PAO1 background, similar to the psl translation level of ΔrsmA. The stem disruption mutation did not significantly affect the levels of psl translation in the ΔrsmA background.
Mentions: Predicted RsmA binding site in the psl 5′ UTR.A. The predicted position of SD is indicated by the underlined 7–12 bases upstream of the start codon of pslA ORF. Predicted RsmA binding site is boxed, 24–37 bases upstream of the start codon. The asterisks indicate the GG nucleotide pair required for CsrA/RsmA binding to its RNA target, which was mutated to the CC pair for the experiments demonstrated in Fig. 11. +1 indicates the transcriptional start site (in bold font) and the corresponding −10 and −35 regions of the promoter (underlined).B. Sequence alignment of psl RsmA binding site with the CsrA consensus sequence (R = A or G) as previously reported (Dubey et al., 2005).

Bottom Line: In this study, we demonstrate that the alternative σ-factor RpoS is a positive transcriptional regulator of psl gene expression.Furthermore, we show that psl mRNA has an extensive 5' untranslated region, to which the post-transcriptional regulator RsmA binds and represses psl translation.This constitutes a novel mechanism for translational repression by this family of regulators.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

Show MeSH