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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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Over-expression of SspA induces mucoidy in CF149.The sspA and sspB genes from P. aeruginosa were cloned behind the PBAD promoter in the pHERD20T vector, and conjugated into CF149. (A) Western blot analysis of SspA and SspB proteins using an anti-HA monoclonal antibody. Lanes 1, 2 and 3 represent total cellular proteins extracted from CF149 carrying pHERD20T-sspA, pHERD20T-sspB, and pHERD20T, respectively. (B) Morphology of CF149 containing pHERD20T-sspA, pHERD20T-sspB and pHERD20T. These strains were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin, 0.1% L-Ara and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods.
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pone-0072329-g002: Over-expression of SspA induces mucoidy in CF149.The sspA and sspB genes from P. aeruginosa were cloned behind the PBAD promoter in the pHERD20T vector, and conjugated into CF149. (A) Western blot analysis of SspA and SspB proteins using an anti-HA monoclonal antibody. Lanes 1, 2 and 3 represent total cellular proteins extracted from CF149 carrying pHERD20T-sspA, pHERD20T-sspB, and pHERD20T, respectively. (B) Morphology of CF149 containing pHERD20T-sspA, pHERD20T-sspB and pHERD20T. These strains were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin, 0.1% L-Ara and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods.

Mentions: The operon of sspAB encodes the stringent starvation proteins A and B that function in response to amino acid starvation [32]. The sspA and sspB genes share the same promoter, and are co-expressed in E. coli[32]. SspB is also a specificity-enhancing factor for the protease ClpXP in E. coli[33]. ClpXP has been reported to regulate alginate production in P. aeruginosa[23]. In order to determine which gene is responsible for the mucoid conversion in CF149 (+sspA), P. aeruginosa sspA and sspB were cloned behind the PBAD promoter in the shuttle vector pHERD20T [34]. As seen in Figure 2A, we detected the expression of SspA and SspB in CF149 after induction with 0.1% L-arabinose (L-Ara). However, we observed a mucoid phenotype only with the overexpression of sspA (Figure 2B), indicating that SspA is an inducer of alginate production when overexpressed 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)

Over-expression of SspA induces mucoidy in CF149.The sspA and sspB genes from P. aeruginosa were cloned behind the PBAD promoter in the pHERD20T vector, and conjugated into CF149. (A) Western blot analysis of SspA and SspB proteins using an anti-HA monoclonal antibody. Lanes 1, 2 and 3 represent total cellular proteins extracted from CF149 carrying pHERD20T-sspA, pHERD20T-sspB, and pHERD20T, respectively. (B) Morphology of CF149 containing pHERD20T-sspA, pHERD20T-sspB and pHERD20T. These strains were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin, 0.1% L-Ara and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750012&req=5

pone-0072329-g002: Over-expression of SspA induces mucoidy in CF149.The sspA and sspB genes from P. aeruginosa were cloned behind the PBAD promoter in the pHERD20T vector, and conjugated into CF149. (A) Western blot analysis of SspA and SspB proteins using an anti-HA monoclonal antibody. Lanes 1, 2 and 3 represent total cellular proteins extracted from CF149 carrying pHERD20T-sspA, pHERD20T-sspB, and pHERD20T, respectively. (B) Morphology of CF149 containing pHERD20T-sspA, pHERD20T-sspB and pHERD20T. These strains were streaked on PIA plates supplemented with 300 µg/ml of carbenicillin, 0.1% L-Ara and incubated overnight at 37°C. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods.
Mentions: The operon of sspAB encodes the stringent starvation proteins A and B that function in response to amino acid starvation [32]. The sspA and sspB genes share the same promoter, and are co-expressed in E. coli[32]. SspB is also a specificity-enhancing factor for the protease ClpXP in E. coli[33]. ClpXP has been reported to regulate alginate production in P. aeruginosa[23]. In order to determine which gene is responsible for the mucoid conversion in CF149 (+sspA), P. aeruginosa sspA and sspB were cloned behind the PBAD promoter in the shuttle vector pHERD20T [34]. As seen in Figure 2A, we detected the expression of SspA and SspB in CF149 after induction with 0.1% L-arabinose (L-Ara). However, we observed a mucoid phenotype only with the overexpression of sspA (Figure 2B), indicating that SspA is an inducer of alginate production when overexpressed 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