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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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psl transcripts are regulated by RpoS.A. pslA transcript levels were measured using quantitative real-time PCR. Transcripts increased in stationary phase compared with mid-log phase in PAO1, but did not increase in ΔrpoS.B. Mid-log phase grown cells constitutively overexpressing RpoS by the introduction of pMW105 increased psl transcription compared with the pEX1.8 vector control (VC) strains. The y-axis unit is described as β-galactosidase activity divided by total mg of protein from the cell lysates.C. Strains constitutively overexpressing RpoS from pMW105 confer RSCV phenotypes compared with smooth colonies of pEX1.8 VC strains when grown on VBMM Congo Red plates. Scale bar = 0.5 mm.D. α-Psl immunoblot indicated an increase in Psl production by PAO1 in stationary phase, but not by ΔrpoS.E. α-Psl immunoblot demonstrated an increase of Psl production by RpoS overexpression strains compared with their respective vector control strains in PAO1 and ΔrpoS.
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fig03: psl transcripts are regulated by RpoS.A. pslA transcript levels were measured using quantitative real-time PCR. Transcripts increased in stationary phase compared with mid-log phase in PAO1, but did not increase in ΔrpoS.B. Mid-log phase grown cells constitutively overexpressing RpoS by the introduction of pMW105 increased psl transcription compared with the pEX1.8 vector control (VC) strains. The y-axis unit is described as β-galactosidase activity divided by total mg of protein from the cell lysates.C. Strains constitutively overexpressing RpoS from pMW105 confer RSCV phenotypes compared with smooth colonies of pEX1.8 VC strains when grown on VBMM Congo Red plates. Scale bar = 0.5 mm.D. α-Psl immunoblot indicated an increase in Psl production by PAO1 in stationary phase, but not by ΔrpoS.E. α-Psl immunoblot demonstrated an increase of Psl production by RpoS overexpression strains compared with their respective vector control strains in PAO1 and ΔrpoS.

Mentions: In P. aeruginosa, RpoS is an alternative σ-factor that regulates expression of a number of genes in the stationary phase (Schuster et al., 2004). Schuster et al. observed that all the genes in the psl operon (pslA-L) were upregulated during stationary-phase growth in wild-type (WT) PAO1, but not in an isogenic ΔrpoS mutant strain. We therefore tested whether RpoS controls psl transcription. As shown by quantitative real-time polymerase chain reaction (PCR) in Fig. 3A, psl transcripts increased about threefold in stationary phase compared with mid-log phase in WT cultures, but ΔrpoS failed to show an increase in psl transcription during stationary phase. To confirm a functional consequence for the RpoS-dependent increase in psl transcription in stationary phase, we analysed relative Psl levels using Psl-specific antisera (Fig. 3D). As predicted from the quantitative real-time PCR data, the amount of Psl produced by WT and ΔrpoS were low during mid-log phase, and only WT increased during stationary phase.


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)

psl transcripts are regulated by RpoS.A. pslA transcript levels were measured using quantitative real-time PCR. Transcripts increased in stationary phase compared with mid-log phase in PAO1, but did not increase in ΔrpoS.B. Mid-log phase grown cells constitutively overexpressing RpoS by the introduction of pMW105 increased psl transcription compared with the pEX1.8 vector control (VC) strains. The y-axis unit is described as β-galactosidase activity divided by total mg of protein from the cell lysates.C. Strains constitutively overexpressing RpoS from pMW105 confer RSCV phenotypes compared with smooth colonies of pEX1.8 VC strains when grown on VBMM Congo Red plates. Scale bar = 0.5 mm.D. α-Psl immunoblot indicated an increase in Psl production by PAO1 in stationary phase, but not by ΔrpoS.E. α-Psl immunoblot demonstrated an increase of Psl production by RpoS overexpression strains compared with their respective vector control strains in PAO1 and ΔrpoS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: psl transcripts are regulated by RpoS.A. pslA transcript levels were measured using quantitative real-time PCR. Transcripts increased in stationary phase compared with mid-log phase in PAO1, but did not increase in ΔrpoS.B. Mid-log phase grown cells constitutively overexpressing RpoS by the introduction of pMW105 increased psl transcription compared with the pEX1.8 vector control (VC) strains. The y-axis unit is described as β-galactosidase activity divided by total mg of protein from the cell lysates.C. Strains constitutively overexpressing RpoS from pMW105 confer RSCV phenotypes compared with smooth colonies of pEX1.8 VC strains when grown on VBMM Congo Red plates. Scale bar = 0.5 mm.D. α-Psl immunoblot indicated an increase in Psl production by PAO1 in stationary phase, but not by ΔrpoS.E. α-Psl immunoblot demonstrated an increase of Psl production by RpoS overexpression strains compared with their respective vector control strains in PAO1 and ΔrpoS.
Mentions: In P. aeruginosa, RpoS is an alternative σ-factor that regulates expression of a number of genes in the stationary phase (Schuster et al., 2004). Schuster et al. observed that all the genes in the psl operon (pslA-L) were upregulated during stationary-phase growth in wild-type (WT) PAO1, but not in an isogenic ΔrpoS mutant strain. We therefore tested whether RpoS controls psl transcription. As shown by quantitative real-time polymerase chain reaction (PCR) in Fig. 3A, psl transcripts increased about threefold in stationary phase compared with mid-log phase in WT cultures, but ΔrpoS failed to show an increase in psl transcription during stationary phase. To confirm a functional consequence for the RpoS-dependent increase in psl transcription in stationary phase, we analysed relative Psl levels using Psl-specific antisera (Fig. 3D). As predicted from the quantitative real-time PCR data, the amount of Psl produced by WT and ΔrpoS were low during mid-log phase, and only WT increased during stationary phase.

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