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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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Alginate induction by CF149 AlgUA61V, the detection of AlgUA61V and the promoter activity of PalgD-lacZ in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).(A) AlgUA61V retained the function of inducing alginate production. The wild type algU gene of PAO1 and its variant of CF149 were cloned into pHERD20T, and conjugated into PAO1ΔalgU. Strains containing pHERD20T-algUWT (PAO1), pHERD20T-algUA61V (CF149), and pHERD20T were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. (B) The level of AlgU in CF149, CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) was detected using Western blot with anti-AlgU monoclonal antibody [16]. (C) Measurement of the activity of the algD promoter in the respective strains. The PalgD promoter was inserted into pLP170 vector containing the promoterless lacZ gene. The pLP170 PalgD-lacZ was transferred into the respective strains via triparental conjugation. The β-galactosidase activity was measured as described in Materials and Methods. *, represents a significant difference compared to CF149 (P<0.05).
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pone-0072329-g003: Alginate induction by CF149 AlgUA61V, the detection of AlgUA61V and the promoter activity of PalgD-lacZ in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).(A) AlgUA61V retained the function of inducing alginate production. The wild type algU gene of PAO1 and its variant of CF149 were cloned into pHERD20T, and conjugated into PAO1ΔalgU. Strains containing pHERD20T-algUWT (PAO1), pHERD20T-algUA61V (CF149), and pHERD20T were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. (B) The level of AlgU in CF149, CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) was detected using Western blot with anti-AlgU monoclonal antibody [16]. (C) Measurement of the activity of the algD promoter in the respective strains. The PalgD promoter was inserted into pLP170 vector containing the promoterless lacZ gene. The pLP170 PalgD-lacZ was transferred into the respective strains via triparental conjugation. The β-galactosidase activity was measured as described in Materials and Methods. *, represents a significant difference compared to CF149 (P<0.05).

Mentions: One explanation for mucoidy in CF149 (+algU) is that AlgUA61V retains some function despite the amino acid substitution. To test this, we compared the function of AlgUA61V vs. wild type AlgU by measuring the amount of alginate induced by two forms of AlgU in the PAO1ΔalgU strain. CF149 AlgUA61V kept the ability to induce mucoidy, albeit with a reduced amount of alginate (Figure 3A). To explain how the three mutants become mucoid, we next measured the level of AlgUA61V in the total cell lysates of CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) through Western blot. The AlgU protein level was increased in CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) compared to the parent CF149 (Figure 3B). The promoter activity of PalgD-lacZ also increased in these mucoid strains (Figure 3C).We also tested the hypothesis that the mucoidy in all three mutants was due to the increased expression of AlgUA61V. To do so, we introduced pHERD20T-AlgUA61V into CF149. As predicted, CF149 carrying pHERD20T-AlgUA61V displayed a mucoid phenotype (data not shown). Furthermore, the absence of AlgUA61V in Figure 3B is consistent with non-mucoidy of CF149 on PIA, which is probably due to the reduced auto-regulatory activity of AlgUA61V[38].


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)

Alginate induction by CF149 AlgUA61V, the detection of AlgUA61V and the promoter activity of PalgD-lacZ in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).(A) AlgUA61V retained the function of inducing alginate production. The wild type algU gene of PAO1 and its variant of CF149 were cloned into pHERD20T, and conjugated into PAO1ΔalgU. Strains containing pHERD20T-algUWT (PAO1), pHERD20T-algUA61V (CF149), and pHERD20T were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. (B) The level of AlgU in CF149, CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) was detected using Western blot with anti-AlgU monoclonal antibody [16]. (C) Measurement of the activity of the algD promoter in the respective strains. The PalgD promoter was inserted into pLP170 vector containing the promoterless lacZ gene. The pLP170 PalgD-lacZ was transferred into the respective strains via triparental conjugation. The β-galactosidase activity was measured as described in Materials and Methods. *, represents a significant difference compared to CF149 (P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750012&req=5

pone-0072329-g003: Alginate induction by CF149 AlgUA61V, the detection of AlgUA61V and the promoter activity of PalgD-lacZ in CF149 (+algU), CF149 (+sspA) and CF149 (−rpoD).(A) AlgUA61V retained the function of inducing alginate production. The wild type algU gene of PAO1 and its variant of CF149 were cloned into pHERD20T, and conjugated into PAO1ΔalgU. Strains containing pHERD20T-algUWT (PAO1), pHERD20T-algUA61V (CF149), and pHERD20T were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. (B) The level of AlgU in CF149, CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) was detected using Western blot with anti-AlgU monoclonal antibody [16]. (C) Measurement of the activity of the algD promoter in the respective strains. The PalgD promoter was inserted into pLP170 vector containing the promoterless lacZ gene. The pLP170 PalgD-lacZ was transferred into the respective strains via triparental conjugation. The β-galactosidase activity was measured as described in Materials and Methods. *, represents a significant difference compared to CF149 (P<0.05).
Mentions: One explanation for mucoidy in CF149 (+algU) is that AlgUA61V retains some function despite the amino acid substitution. To test this, we compared the function of AlgUA61V vs. wild type AlgU by measuring the amount of alginate induced by two forms of AlgU in the PAO1ΔalgU strain. CF149 AlgUA61V kept the ability to induce mucoidy, albeit with a reduced amount of alginate (Figure 3A). To explain how the three mutants become mucoid, we next measured the level of AlgUA61V in the total cell lysates of CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) through Western blot. The AlgU protein level was increased in CF149 (+algU), CF149 (−rpoD) and CF149 (+sspA) compared to the parent CF149 (Figure 3B). The promoter activity of PalgD-lacZ also increased in these mucoid strains (Figure 3C).We also tested the hypothesis that the mucoidy in all three mutants was due to the increased expression of AlgUA61V. To do so, we introduced pHERD20T-AlgUA61V into CF149. As predicted, CF149 carrying pHERD20T-AlgUA61V displayed a mucoid phenotype (data not shown). Furthermore, the absence of AlgUA61V in Figure 3B is consistent with non-mucoidy of CF149 on PIA, which is probably due to the reduced auto-regulatory activity of AlgUA61V[38].

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