Limits...
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: 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.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

The activity of PvdS and FpvI in the presence and absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing either (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing FpvR1–89 and either (A) FpvI or (B) PvdS. An empty pETDuet control is also shown to control for background expression of lacZ from pMP190::PfpvA_lacZ or pMP190::PpvdE_lacZ. Averages were obtained from three biological replicates. Error bars are ±1 SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: The activity of PvdS and FpvI in the presence and absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing either (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing FpvR1–89 and either (A) FpvI or (B) PvdS. An empty pETDuet control is also shown to control for background expression of lacZ from pMP190::PfpvA_lacZ or pMP190::PpvdE_lacZ. Averages were obtained from three biological replicates. Error bars are ±1 SD.

Mentions: To investigate the inhibition of FpvI and PvdS by FpvR1–89, a system was established for detecting the activity of FpvI or PvdS when co-expressed with FpvR1–89 in E. coli. The pETDuet vector allows a 1:1 molar ratio of expression from two multiple cloning sites (MCS). DNA encoding fpvR1–89 was inserted into MCS2 and either fpvI or pvdS was inserted into MCS1, giving plasmids pETDuetfpvI_fpvR1–89 and pETDuetpvdS_fpvR1–89 respectively (Table 1). Reporter plasmids (Table 1) carrying fpvA or pvdE promoters fused upstream of lacZ were used to detect FpvI or PvdS activity respectively [6,30]. In the absence of FpvR1–89, FpvI induced fpvA promoter activity and PvdS induced pvdE promoter activity (Figure 2). The activity of FpvI and PvdS was strongly repressed in the presence of FpvR1–89. These results demonstrated the inhibitory function of the cytoplasmic region of FpvR on both FpvI and PvdS activity in E. coli.Table 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)

The activity of PvdS and FpvI in the presence and absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing either (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing FpvR1–89 and either (A) FpvI or (B) PvdS. An empty pETDuet control is also shown to control for background expression of lacZ from pMP190::PfpvA_lacZ or pMP190::PpvdE_lacZ. Averages were obtained from three biological replicates. Error bars are ±1 SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: The activity of PvdS and FpvI in the presence and absence of FpvR1–89. β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing either (A) pMP190::PfpvA_lacZ or (B) pMP190::PpvdE_lacZ, along with pETDuet expressing FpvR1–89 and either (A) FpvI or (B) PvdS. An empty pETDuet control is also shown to control for background expression of lacZ from pMP190::PfpvA_lacZ or pMP190::PpvdE_lacZ. Averages were obtained from three biological replicates. Error bars are ±1 SD.
Mentions: To investigate the inhibition of FpvI and PvdS by FpvR1–89, a system was established for detecting the activity of FpvI or PvdS when co-expressed with FpvR1–89 in E. coli. The pETDuet vector allows a 1:1 molar ratio of expression from two multiple cloning sites (MCS). DNA encoding fpvR1–89 was inserted into MCS2 and either fpvI or pvdS was inserted into MCS1, giving plasmids pETDuetfpvI_fpvR1–89 and pETDuetpvdS_fpvR1–89 respectively (Table 1). Reporter plasmids (Table 1) carrying fpvA or pvdE promoters fused upstream of lacZ were used to detect FpvI or PvdS activity respectively [6,30]. In the absence of FpvR1–89, FpvI induced fpvA promoter activity and PvdS induced pvdE promoter activity (Figure 2). The activity of FpvI and PvdS was strongly repressed in the presence of FpvR1–89. These results demonstrated the inhibitory function of the cytoplasmic region of FpvR on both FpvI and PvdS activity in E. coli.Table 1

Bottom Line: 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.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