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Interactions between an anti-sigma protein and two sigma factors that regulate the pyoverdine signaling pathway in Pseudomonas aeruginosa.

Edgar RJ, Xu X, Shirley M, Konings AF, Martin LW, Ackerley DF, Lamont IL - BMC Microbiol. (2014)

Bottom Line: Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase.These data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity.FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. edgre961@student.otago.ac.nz.

ABSTRACT

Background: Synthesis and uptake of pyoverdine, the primary siderophore of the opportunistic pathogen Pseudomonas aeruginosa, is dependent on two extra-cytoplasmic function (ECF) sigma factors, FpvI and PvdS. FpvI and PvdS are required for expression of the ferri-pyoverdine receptor gene fpvA and of pyoverdine synthesis genes respectively. In the absence of pyoverdine the anti-sigma factor FpvR that spans the cytoplasmic membrane inhibits the activities of both FpvI and PvdS, despite the two sigma factors having low sequence identity.

Results: To investigate the interactions of FpvR with FpvI and PvdS, we first used a tandem affinity purification system to demonstrate binding of PvdS by the cytoplasmic region of FpvR in P. aeruginosa at physiological levels. The cytoplasmic region of FpvR bound to and inhibited both FpvI and PvdS when the proteins were co-expressed in Escherichia coli. Each sigma factor was then subjected to error prone PCR and site-directed mutagenesis to identify mutations that increased sigma factor activity in the presence of FpvR. In FpvI, the amino acid changes clustered around conserved region four of the protein and are likely to disrupt interactions with FpvR. Deletion of five amino acids from the C-terminal end of FpvI also disrupted interactions with FpvR. Mutations in PvdS were present in conserved regions two and four. Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase.

Conclusions: These data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity. FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.

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The activity of FpvI and PvdS mutants in the absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.
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Fig5: The activity of FpvI and PvdS mutants in the absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.

Mentions: The activity of FpvI and PvdS mutants in the presence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing FpvR1–89 and (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Mutants that were further investigated in Figure 5 are indicated in bold. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.


Interactions between an anti-sigma protein and two sigma factors that regulate the pyoverdine signaling pathway in Pseudomonas aeruginosa.

Edgar RJ, Xu X, Shirley M, Konings AF, Martin LW, Ackerley DF, Lamont IL - BMC Microbiol. (2014)

The activity of FpvI and PvdS mutants in the absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4256889&req=5

Fig5: The activity of FpvI and PvdS mutants in the absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.
Mentions: The activity of FpvI and PvdS mutants in the presence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing FpvR1–89 and (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Mutants that were further investigated in Figure 5 are indicated in bold. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.

Bottom Line: Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase.These data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity.FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. edgre961@student.otago.ac.nz.

ABSTRACT

Background: Synthesis and uptake of pyoverdine, the primary siderophore of the opportunistic pathogen Pseudomonas aeruginosa, is dependent on two extra-cytoplasmic function (ECF) sigma factors, FpvI and PvdS. FpvI and PvdS are required for expression of the ferri-pyoverdine receptor gene fpvA and of pyoverdine synthesis genes respectively. In the absence of pyoverdine the anti-sigma factor FpvR that spans the cytoplasmic membrane inhibits the activities of both FpvI and PvdS, despite the two sigma factors having low sequence identity.

Results: To investigate the interactions of FpvR with FpvI and PvdS, we first used a tandem affinity purification system to demonstrate binding of PvdS by the cytoplasmic region of FpvR in P. aeruginosa at physiological levels. The cytoplasmic region of FpvR bound to and inhibited both FpvI and PvdS when the proteins were co-expressed in Escherichia coli. Each sigma factor was then subjected to error prone PCR and site-directed mutagenesis to identify mutations that increased sigma factor activity in the presence of FpvR. In FpvI, the amino acid changes clustered around conserved region four of the protein and are likely to disrupt interactions with FpvR. Deletion of five amino acids from the C-terminal end of FpvI also disrupted interactions with FpvR. Mutations in PvdS were present in conserved regions two and four. Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase.

Conclusions: These data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity. FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.

Show MeSH
Related in: MedlinePlus