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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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Schematic diagram for mucoid conversion in CF149 caused by competition between two sigma factors, RpoD and AlgU.Stress or starvation can affect the expression of anti-σ70 factors resulting in a decrease in the activity of RpoD, which in turn increases the chances for the alternative sigma factor AlgU to bind to the core RNAP to initiate transcription for alginate biosynthesis. MucA is an anti-sigma factor that has the ability to inhibit the activity of AlgU in alginate production. Competition for RNAP between RpoD and AlgU can determine which promoter is activated. RpoD binds to core RNAP to promote the transcription of housekeeping genes, while AlgU binds to core RNAP at the PalgD promoter to activate alginate production.
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pone-0072329-g007: Schematic diagram for mucoid conversion in CF149 caused by competition between two sigma factors, RpoD and AlgU.Stress or starvation can affect the expression of anti-σ70 factors resulting in a decrease in the activity of RpoD, which in turn increases the chances for the alternative sigma factor AlgU to bind to the core RNAP to initiate transcription for alginate biosynthesis. MucA is an anti-sigma factor that has the ability to inhibit the activity of AlgU in alginate production. Competition for RNAP between RpoD and AlgU can determine which promoter is activated. RpoD binds to core RNAP to promote the transcription of housekeeping genes, while AlgU binds to core RNAP at the PalgD promoter to activate alginate production.

Mentions: Individuals with CF are thought to acquire initial colonization of P. aeruginosa from environmental sources [9]. These early colonizing strains display a non-mucoid phenotype with a wild-type MucA [17]. Due to strong selective pressure in CF lungs, mucoid mucA mutants eventually become a dominant population [14], [40]. However, secondary mutations that suppress alginate overproduction have been reported [8], [19]. One presumed advantage with non-mucoid suppressors is the loss of mucoid status is coupled with the presence of the flagella, which may promote the colonization of new niches in the lungs [41], [42]. Through screening a transposon library, we found that overexpression of sspA and CF149 algU, and reduced expression of rpoD, are functionally equivalent in causing mucoid conversion in the non-mucoid clinical isolate CF149. This mucoid phenotype can be suppressed by overexpression of the anti-sigma factor MucA [12]. We propose that the mechanism for mucoid conversion mediated by AlgU, SspA and RpoD in CF149 may be related to the competition between sigma factors RpoD and AlgU for the core RNAP binding site (Figure 7). Because of the differential binding ability among sigma factors for core RNAP [43], σ factor competition exists within a cell at any given time [44]. We investigated whether this competition is also present in CF149 and CF149(−rpoD), by measuring the promoter activity of Palgw whose activation depends on RpoN [45], and PalgD which is driven by AlgU [11]. As seen in Figures 3C and S3, activities of both PalgD and PalgW were increased in CF149 (−rpoD). Furthermore, the mucoid suppression resulting from the overexpression of RpoD can be attributed to the competition between two sigma factors (Table 2). Table S2 illustrates that sigma factors besides RpoD can also exert the same effect on mucoid suppression in mucA plus and minus mucoid strains. Thus, any major shift in the intracellular level of sigma factors can potentially affect mucoid conversion, because the pool of core RNAP, which is made up of five different subunits, must be in a limiting amount inside bacterial cells. However, data in Figure S1 demonstrate that induction of FlgM, which is the anti-sigma factor for RpoF responsible for the transcription initiation of flagella biosynthesis [46], failed to induce mucoidy in PAO1 and CF149. This may be due to the fact that the impact on the pool of RNAP is somewhat different between a minor sigma factor RpoF and a major sigma factor RpoD. Therefore not all anti-sigma factors are functionally equal in terms of alginate induction.


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)

Schematic diagram for mucoid conversion in CF149 caused by competition between two sigma factors, RpoD and AlgU.Stress or starvation can affect the expression of anti-σ70 factors resulting in a decrease in the activity of RpoD, which in turn increases the chances for the alternative sigma factor AlgU to bind to the core RNAP to initiate transcription for alginate biosynthesis. MucA is an anti-sigma factor that has the ability to inhibit the activity of AlgU in alginate production. Competition for RNAP between RpoD and AlgU can determine which promoter is activated. RpoD binds to core RNAP to promote the transcription of housekeeping genes, while AlgU binds to core RNAP at the PalgD promoter to activate alginate production.
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Related In: Results  -  Collection

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pone-0072329-g007: Schematic diagram for mucoid conversion in CF149 caused by competition between two sigma factors, RpoD and AlgU.Stress or starvation can affect the expression of anti-σ70 factors resulting in a decrease in the activity of RpoD, which in turn increases the chances for the alternative sigma factor AlgU to bind to the core RNAP to initiate transcription for alginate biosynthesis. MucA is an anti-sigma factor that has the ability to inhibit the activity of AlgU in alginate production. Competition for RNAP between RpoD and AlgU can determine which promoter is activated. RpoD binds to core RNAP to promote the transcription of housekeeping genes, while AlgU binds to core RNAP at the PalgD promoter to activate alginate production.
Mentions: Individuals with CF are thought to acquire initial colonization of P. aeruginosa from environmental sources [9]. These early colonizing strains display a non-mucoid phenotype with a wild-type MucA [17]. Due to strong selective pressure in CF lungs, mucoid mucA mutants eventually become a dominant population [14], [40]. However, secondary mutations that suppress alginate overproduction have been reported [8], [19]. One presumed advantage with non-mucoid suppressors is the loss of mucoid status is coupled with the presence of the flagella, which may promote the colonization of new niches in the lungs [41], [42]. Through screening a transposon library, we found that overexpression of sspA and CF149 algU, and reduced expression of rpoD, are functionally equivalent in causing mucoid conversion in the non-mucoid clinical isolate CF149. This mucoid phenotype can be suppressed by overexpression of the anti-sigma factor MucA [12]. We propose that the mechanism for mucoid conversion mediated by AlgU, SspA and RpoD in CF149 may be related to the competition between sigma factors RpoD and AlgU for the core RNAP binding site (Figure 7). Because of the differential binding ability among sigma factors for core RNAP [43], σ factor competition exists within a cell at any given time [44]. We investigated whether this competition is also present in CF149 and CF149(−rpoD), by measuring the promoter activity of Palgw whose activation depends on RpoN [45], and PalgD which is driven by AlgU [11]. As seen in Figures 3C and S3, activities of both PalgD and PalgW were increased in CF149 (−rpoD). Furthermore, the mucoid suppression resulting from the overexpression of RpoD can be attributed to the competition between two sigma factors (Table 2). Table S2 illustrates that sigma factors besides RpoD can also exert the same effect on mucoid suppression in mucA plus and minus mucoid strains. Thus, any major shift in the intracellular level of sigma factors can potentially affect mucoid conversion, because the pool of core RNAP, which is made up of five different subunits, must be in a limiting amount inside bacterial cells. However, data in Figure S1 demonstrate that induction of FlgM, which is the anti-sigma factor for RpoF responsible for the transcription initiation of flagella biosynthesis [46], failed to induce mucoidy in PAO1 and CF149. This may be due to the fact that the impact on the pool of RNAP is somewhat different between a minor sigma factor RpoF and a major sigma factor RpoD. Therefore not all anti-sigma factors are functionally equal in terms of alginate induction.

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