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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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Co-purification of PvdS with FpvR1–89–TAP fromP. aeruginosa. Soluble protein was prepared from P. aeruginosa PAO1 fpvR expressing plasmid-borne (pUCP23) or chromosomally-integrated (ctx) FpvR1–89 fused to a C-terminal TAP tag. Protein was purified using the TAP protocol and the purified protein analyzed by Western blotting for FpvR1–89-CBP or PvdS. (A) anti-CBP; (B) anti-FpvR; (C) anti-PvdS. A mock purification was carried out with P. aeruginosa PAO1 fpvR carrying pUCP23 without the fpvR1–89-TAP fusion as a negative control for the TAP tag purification procedure. The positions of molecular weight markers are shown.
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Fig1: Co-purification of PvdS with FpvR1–89–TAP fromP. aeruginosa. Soluble protein was prepared from P. aeruginosa PAO1 fpvR expressing plasmid-borne (pUCP23) or chromosomally-integrated (ctx) FpvR1–89 fused to a C-terminal TAP tag. Protein was purified using the TAP protocol and the purified protein analyzed by Western blotting for FpvR1–89-CBP or PvdS. (A) anti-CBP; (B) anti-FpvR; (C) anti-PvdS. A mock purification was carried out with P. aeruginosa PAO1 fpvR carrying pUCP23 without the fpvR1–89-TAP fusion as a negative control for the TAP tag purification procedure. The positions of molecular weight markers are shown.

Mentions: We validated binding of PvdS by FpvR in P. aeruginosa by purifying the cytoplasmic portion of FpvR and determining whether PvdS was co-purified. P. aeruginosa (PAO1) was engineered to express the cytoplasmic portion and predicted sigma factor inhibitory region of FpvR (residues 1–89) [6,28] fused to a C-terminal tandem affinity purification (TAP) tag [29]. The FpvR1–89 -TAP fusion, expressed from the fpvR promoter, was either integrated into the bacterial chromosome using mini-CTX or was expressed from plasmid pUCP23. Chromosomal integration was used to demonstrate that FpvR1–89 and PvdS interact when expressed in physiological amounts. Higher plasmid-based expression was expected to titrate out any regulatory factors that may have limited FpvR1–89 expression, ensuring sufficient FpvR1–89 was present for visualisation and co-purification with PvdS. As expected, expression of FpvR from the chromosomally-integrated construct was repressed by the presence of iron in the King’s B medium (Additional file 1: Figure S1). FpvR1–89 fused to calmodulin binding protein (CBP) was purified using the TAP protocol. The purification resulted in a 15 kDa protein, the predicted size for FpvR1–89–CBP, which could be detected using antibodies against either CBP or FpvR1–89 (Figure 1). Fractions that contained purified FpvR1–89–CBP also contained co-purified PvdS. PvdS was not present in fractions obtained using the purification protocol with bacteria that did not contain the FpvR1–89–TAP construct, confirming that purification of PvdS was dependent on the presence of FpvR1–89–TAP. FpvI was not detected following FpvR1–89–TAP purification using a polyclonal FpvI antibody and a suitable monoclonal antibody was not available.Figure 1


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)

Co-purification of PvdS with FpvR1–89–TAP fromP. aeruginosa. Soluble protein was prepared from P. aeruginosa PAO1 fpvR expressing plasmid-borne (pUCP23) or chromosomally-integrated (ctx) FpvR1–89 fused to a C-terminal TAP tag. Protein was purified using the TAP protocol and the purified protein analyzed by Western blotting for FpvR1–89-CBP or PvdS. (A) anti-CBP; (B) anti-FpvR; (C) anti-PvdS. A mock purification was carried out with P. aeruginosa PAO1 fpvR carrying pUCP23 without the fpvR1–89-TAP fusion as a negative control for the TAP tag purification procedure. The positions of molecular weight markers are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256889&req=5

Fig1: Co-purification of PvdS with FpvR1–89–TAP fromP. aeruginosa. Soluble protein was prepared from P. aeruginosa PAO1 fpvR expressing plasmid-borne (pUCP23) or chromosomally-integrated (ctx) FpvR1–89 fused to a C-terminal TAP tag. Protein was purified using the TAP protocol and the purified protein analyzed by Western blotting for FpvR1–89-CBP or PvdS. (A) anti-CBP; (B) anti-FpvR; (C) anti-PvdS. A mock purification was carried out with P. aeruginosa PAO1 fpvR carrying pUCP23 without the fpvR1–89-TAP fusion as a negative control for the TAP tag purification procedure. The positions of molecular weight markers are shown.
Mentions: We validated binding of PvdS by FpvR in P. aeruginosa by purifying the cytoplasmic portion of FpvR and determining whether PvdS was co-purified. P. aeruginosa (PAO1) was engineered to express the cytoplasmic portion and predicted sigma factor inhibitory region of FpvR (residues 1–89) [6,28] fused to a C-terminal tandem affinity purification (TAP) tag [29]. The FpvR1–89 -TAP fusion, expressed from the fpvR promoter, was either integrated into the bacterial chromosome using mini-CTX or was expressed from plasmid pUCP23. Chromosomal integration was used to demonstrate that FpvR1–89 and PvdS interact when expressed in physiological amounts. Higher plasmid-based expression was expected to titrate out any regulatory factors that may have limited FpvR1–89 expression, ensuring sufficient FpvR1–89 was present for visualisation and co-purification with PvdS. As expected, expression of FpvR from the chromosomally-integrated construct was repressed by the presence of iron in the King’s B medium (Additional file 1: Figure S1). FpvR1–89 fused to calmodulin binding protein (CBP) was purified using the TAP protocol. The purification resulted in a 15 kDa protein, the predicted size for FpvR1–89–CBP, which could be detected using antibodies against either CBP or FpvR1–89 (Figure 1). Fractions that contained purified FpvR1–89–CBP also contained co-purified PvdS. PvdS was not present in fractions obtained using the purification protocol with bacteria that did not contain the FpvR1–89–TAP construct, confirming that purification of PvdS was dependent on the presence of FpvR1–89–TAP. FpvI was not detected following FpvR1–89–TAP purification using a polyclonal FpvI antibody and a suitable monoclonal antibody was not available.Figure 1

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