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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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Increased alginate production in three mutants of CF149 with an algU-suppressor mutation.(A) Schematic diagram showing the transposon insertions of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD), respectively. (B) Alginate production of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD) in comparison to other strains of P. aeruginosa. Three mucoid mutants were identified as a result of a transposon library screen. Alginate production was measured on PIA plates after incubation at 37°C for 24 hrs. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. M and NM, represent mucoidy and nonmucoidy, respectively.
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pone-0072329-g001: Increased alginate production in three mutants of CF149 with an algU-suppressor mutation.(A) Schematic diagram showing the transposon insertions of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD), respectively. (B) Alginate production of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD) in comparison to other strains of P. aeruginosa. Three mucoid mutants were identified as a result of a transposon library screen. Alginate production was measured on PIA plates after incubation at 37°C for 24 hrs. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. M and NM, represent mucoidy and nonmucoidy, respectively.

Mentions: CF149 is a clinical isolate from a patient with CF [24]. CF149 displays a nonmucoid phenotype on PIA and PIA plus ammonium metavanadate (PIA-AMV) plates [29]. Previously, we reported CF149 have two mutations resulting in abrogation of an AlgU-dependent transcription of lipotoxin LptF [21]. First, a frameshift mutation in mucA is predicted to produce a truncated MucA protein with 128 amino acids in contrast to wild type MucA with 194 amino acids. Second, CF149 carries a missense mutation in algU predicted to result in a substitution of alanine for valine at position 61 of the primary amino acid sequence of AlgU (AlgUA61V) [21]. To determine whether the alginate suppressor strain CF149 still had the ability to restore mucoidy, a transposon library was constructed and screened. As seen in Figure 1A, we identified three insertions that promote alginate production [17]. Southern blot analysis showed that only one copy of the transposon was inserted on the chromosome in these mucoid strains (data not shown). We mapped the insertion site using inverse PCR as previously described [17], [23]. Two insertions were identified in intergenic regions between PA0762 (algU) and PA0761 (nadB), and PA4428 (sspA) and PA4429 (probable cytochrome c1 precursor) (Figure 1A). In these two mutants, the transposon was upstream of algU and sspA, and was oriented in the same direction as the previously-observed insertion causing an overexpression of mucE[17]. These two mutants likely had an overexpression of algU and sspA from read through of the gentamicin-resistance gene (aacC1) promoter (PGm) [30]. These strains were named CF149 (+algU) and CF149 (+sspA). The third mutant had an insertion site of 78 base pairs behind the stop codon of the rpoD gene (Figure 1A). The rpoD gene (PA0576) encodes the major housekeeping sigma factor (σ70). RpoD is essential for the growth and viability of cells, and no rpoD mutant has been reported in the P. aeruginosa mutant bank [31]. Because the orientation of the pFAC PGm is opposite to the direction of rpoD gene, the insertion is predicted to reduce the expression of rpoD. This strain was named CF149 (−rpoD). To verify the decreased transcription of rpoD in CF149 (−rpoD), we compared the transcript level by real-time PCR, and the activity of an RpoD-dependent promoter PssrA-lacZ in two isogenic strains. The results showed that the transcript level of rpoD in CF149 (−rpoD) was 60% of the value of CF149. The Miller assay results also showed that the PssrA-lacZ activity reduced by 68% in CF149 (−rpoD) compared to CF149. The alginate production by these mucoid strains was also measured and shown to be more than 2-fold higher than the parent strain (Figure 1B).


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)

Increased alginate production in three mutants of CF149 with an algU-suppressor mutation.(A) Schematic diagram showing the transposon insertions of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD), respectively. (B) Alginate production of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD) in comparison to other strains of P. aeruginosa. Three mucoid mutants were identified as a result of a transposon library screen. Alginate production was measured on PIA plates after incubation at 37°C for 24 hrs. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. M and NM, represent mucoidy and nonmucoidy, respectively.
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Related In: Results  -  Collection

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pone-0072329-g001: Increased alginate production in three mutants of CF149 with an algU-suppressor mutation.(A) Schematic diagram showing the transposon insertions of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD), respectively. (B) Alginate production of CF149 (+algU), CF149 (+sspA), and CF149 (−rpoD) in comparison to other strains of P. aeruginosa. Three mucoid mutants were identified as a result of a transposon library screen. Alginate production was measured on PIA plates after incubation at 37°C for 24 hrs. Alginate production (µg/ml/OD600) was measured as described in Materials and Methods. M and NM, represent mucoidy and nonmucoidy, respectively.
Mentions: CF149 is a clinical isolate from a patient with CF [24]. CF149 displays a nonmucoid phenotype on PIA and PIA plus ammonium metavanadate (PIA-AMV) plates [29]. Previously, we reported CF149 have two mutations resulting in abrogation of an AlgU-dependent transcription of lipotoxin LptF [21]. First, a frameshift mutation in mucA is predicted to produce a truncated MucA protein with 128 amino acids in contrast to wild type MucA with 194 amino acids. Second, CF149 carries a missense mutation in algU predicted to result in a substitution of alanine for valine at position 61 of the primary amino acid sequence of AlgU (AlgUA61V) [21]. To determine whether the alginate suppressor strain CF149 still had the ability to restore mucoidy, a transposon library was constructed and screened. As seen in Figure 1A, we identified three insertions that promote alginate production [17]. Southern blot analysis showed that only one copy of the transposon was inserted on the chromosome in these mucoid strains (data not shown). We mapped the insertion site using inverse PCR as previously described [17], [23]. Two insertions were identified in intergenic regions between PA0762 (algU) and PA0761 (nadB), and PA4428 (sspA) and PA4429 (probable cytochrome c1 precursor) (Figure 1A). In these two mutants, the transposon was upstream of algU and sspA, and was oriented in the same direction as the previously-observed insertion causing an overexpression of mucE[17]. These two mutants likely had an overexpression of algU and sspA from read through of the gentamicin-resistance gene (aacC1) promoter (PGm) [30]. These strains were named CF149 (+algU) and CF149 (+sspA). The third mutant had an insertion site of 78 base pairs behind the stop codon of the rpoD gene (Figure 1A). The rpoD gene (PA0576) encodes the major housekeeping sigma factor (σ70). RpoD is essential for the growth and viability of cells, and no rpoD mutant has been reported in the P. aeruginosa mutant bank [31]. Because the orientation of the pFAC PGm is opposite to the direction of rpoD gene, the insertion is predicted to reduce the expression of rpoD. This strain was named CF149 (−rpoD). To verify the decreased transcription of rpoD in CF149 (−rpoD), we compared the transcript level by real-time PCR, and the activity of an RpoD-dependent promoter PssrA-lacZ in two isogenic strains. The results showed that the transcript level of rpoD in CF149 (−rpoD) was 60% of the value of CF149. The Miller assay results also showed that the PssrA-lacZ activity reduced by 68% in CF149 (−rpoD) compared to CF149. The alginate production by these mucoid strains was also measured and shown to be more than 2-fold higher than the parent strain (Figure 1B).

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