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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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Mucoid mutants of P. aeruginosa display a reduced growth rate compared to the isogenic nonmucoid strain PAO1.The growth curves were created by growth of PAO1 (nonmucoid), VE2 (mucoid) and PAO581 (mucoid) in PIB. The horizontal axis represents time in hrs, while the vertical axis is the optical density at 600 nm. *, represents a significant difference compared to PAO1 (P<0.05).
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pone-0072329-g006: Mucoid mutants of P. aeruginosa display a reduced growth rate compared to the isogenic nonmucoid strain PAO1.The growth curves were created by growth of PAO1 (nonmucoid), VE2 (mucoid) and PAO581 (mucoid) in PIB. The horizontal axis represents time in hrs, while the vertical axis is the optical density at 600 nm. *, represents a significant difference compared to PAO1 (P<0.05).

Mentions: Since the transcription from two promoters, PalgD and PssrA is AlgU-dependent and RpoD-dependent, respectively, we cloned these two promoters into a lacZ fusion vector (pLP170) and the reporter β-galactosidase activity was measured [26]. As seen in Figure 5A, alginate production in the mucoid strains VE2 and PAO581 was higher than that in the non-mucoid strain PAO1. Similarly, the promoter activity of PalgD also was higher in these mucoid strains (Figure 5A). However, the promoter activity of PssrA was inversely related to PalgD activity and alginate production (Figure 5B), suggesting that the RpoD-dependent promoter was less active in mucoid cells than in the isogenic non-mucoid cells. We also measured the growth curve for mucoid and non mucoid strains. As seen in Figure 6, the growth rate for mucoid strains was reduced after 16 hrs growth for VE2 and PAO581 in comparison with nonmoucid strain PAO1.


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)

Mucoid mutants of P. aeruginosa display a reduced growth rate compared to the isogenic nonmucoid strain PAO1.The growth curves were created by growth of PAO1 (nonmucoid), VE2 (mucoid) and PAO581 (mucoid) in PIB. The horizontal axis represents time in hrs, while the vertical axis is the optical density at 600 nm. *, represents a significant difference compared to PAO1 (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0072329-g006: Mucoid mutants of P. aeruginosa display a reduced growth rate compared to the isogenic nonmucoid strain PAO1.The growth curves were created by growth of PAO1 (nonmucoid), VE2 (mucoid) and PAO581 (mucoid) in PIB. The horizontal axis represents time in hrs, while the vertical axis is the optical density at 600 nm. *, represents a significant difference compared to PAO1 (P<0.05).
Mentions: Since the transcription from two promoters, PalgD and PssrA is AlgU-dependent and RpoD-dependent, respectively, we cloned these two promoters into a lacZ fusion vector (pLP170) and the reporter β-galactosidase activity was measured [26]. As seen in Figure 5A, alginate production in the mucoid strains VE2 and PAO581 was higher than that in the non-mucoid strain PAO1. Similarly, the promoter activity of PalgD also was higher in these mucoid strains (Figure 5A). However, the promoter activity of PssrA was inversely related to PalgD activity and alginate production (Figure 5B), suggesting that the RpoD-dependent promoter was less active in mucoid cells than in the isogenic non-mucoid cells. We also measured the growth curve for mucoid and non mucoid strains. As seen in Figure 6, the growth rate for mucoid strains was reduced after 16 hrs growth for VE2 and PAO581 in comparison with nonmoucid strain PAO1.

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