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Evidence for sigma factor competition in the regulation of alginate production by Pseudomonas aeruginosa.

Yin Y, Withers TR, Wang X, Yu HD - PLoS ONE (2013)

Bottom Line: However, compared to the wild type AlgU, AlgU(A61V) had a reduced activity in promoting alginate production in PAO1ΔalgU.SspA and three other anti-σ(70) orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149.Altogether, our results indicate that the anti-σ(70) factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia, USA.

ABSTRACT
Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU (AlgU(A61V)). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgU(A61V), 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ(70)). Induction of AlgU(A61V) in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgU(A61V) is functional in activating alginate production. Furthermore, the level of AlgU(A61V) was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgU(A61V) had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ(70) orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (PssrA ) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ(70) factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.

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The effect of mucA on the mucoid suppression in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).AlgUA61V–induced mucoidy was suppressed by the wild type mucA in trans. The mucA gene was over-expressed in trans in CF149(+algU), CF149(−rpoD) and CF149(+sspA) by adding 0.1% L-Ara in PIA supplemented with 300 µg/ml carbenicllin. The plate was incubated for 24 hrs at 37°C.
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pone-0072329-g004: The effect of mucA on the mucoid suppression in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).AlgUA61V–induced mucoidy was suppressed by the wild type mucA in trans. The mucA gene was over-expressed in trans in CF149(+algU), CF149(−rpoD) and CF149(+sspA) by adding 0.1% L-Ara in PIA supplemented with 300 µg/ml carbenicllin. The plate was incubated for 24 hrs at 37°C.

Mentions: In wild type strain PAO1, AlgU can be sequestered by wild type MucA, thereby preventing it from activating the alginate biosynthetic operon [12], [38], [39]. Since all three mutants of CF149 have an increased level of AlgUA61V, we next investigated whether the wild type MucA can still exert an inhibitory effect on AlgUA61V. The wild type mucA gene was transferred into these mucoid strains: CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD). Over-expression of mucA suppressed mucoidy (Figure 4) suggesting that the mucoidy of all three mutants is due to the activation of the AlgU pathway. But the mutant MucA in CF149 has 128 amino acid residues, and carries the intact trans-membrane domain of MucA84–104. We also noticed that the promoter activity of PalgW in CF149 (−rpoD) was increased compared to CF149 (Figure S3). To test if intramembrane proteolysis has a role in cleaving the periplasmic portion of MucA in CF149, we overexpressed proteases algW, mucP, clpX, clpP and clpP2 and found this had no effect on mucoid induction in CF149. Together, these results suggest that these proteases have a minimal role in regulating the mucoid conversion in CF149, or they may require a mechanism of activation which is absent in CF149.


Evidence for sigma factor competition in the regulation of alginate production by Pseudomonas aeruginosa.

Yin Y, Withers TR, Wang X, Yu HD - PLoS ONE (2013)

The effect of mucA on the mucoid suppression in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).AlgUA61V–induced mucoidy was suppressed by the wild type mucA in trans. The mucA gene was over-expressed in trans in CF149(+algU), CF149(−rpoD) and CF149(+sspA) by adding 0.1% L-Ara in PIA supplemented with 300 µg/ml carbenicllin. The plate was incubated for 24 hrs at 37°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750012&req=5

pone-0072329-g004: The effect of mucA on the mucoid suppression in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).AlgUA61V–induced mucoidy was suppressed by the wild type mucA in trans. The mucA gene was over-expressed in trans in CF149(+algU), CF149(−rpoD) and CF149(+sspA) by adding 0.1% L-Ara in PIA supplemented with 300 µg/ml carbenicllin. The plate was incubated for 24 hrs at 37°C.
Mentions: In wild type strain PAO1, AlgU can be sequestered by wild type MucA, thereby preventing it from activating the alginate biosynthetic operon [12], [38], [39]. Since all three mutants of CF149 have an increased level of AlgUA61V, we next investigated whether the wild type MucA can still exert an inhibitory effect on AlgUA61V. The wild type mucA gene was transferred into these mucoid strains: CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD). Over-expression of mucA suppressed mucoidy (Figure 4) suggesting that the mucoidy of all three mutants is due to the activation of the AlgU pathway. But the mutant MucA in CF149 has 128 amino acid residues, and carries the intact trans-membrane domain of MucA84–104. We also noticed that the promoter activity of PalgW in CF149 (−rpoD) was increased compared to CF149 (Figure S3). To test if intramembrane proteolysis has a role in cleaving the periplasmic portion of MucA in CF149, we overexpressed proteases algW, mucP, clpX, clpP and clpP2 and found this had no effect on mucoid induction in CF149. Together, these results suggest that these proteases have a minimal role in regulating the mucoid conversion in CF149, or they may require a mechanism of activation which is absent in CF149.

Bottom Line: However, compared to the wild type AlgU, AlgU(A61V) had a reduced activity in promoting alginate production in PAO1ΔalgU.SspA and three other anti-σ(70) orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149.Altogether, our results indicate that the anti-σ(70) factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia, USA.

ABSTRACT
Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU (AlgU(A61V)). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgU(A61V), 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ(70)). Induction of AlgU(A61V) in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgU(A61V) is functional in activating alginate production. Furthermore, the level of AlgU(A61V) was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgU(A61V) had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ(70) orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (PssrA ) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ(70) factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.

Show MeSH
Related in: MedlinePlus