title: Interactions between an anti-sigma protein and two sigma factors that regulate the pyoverdine signaling pathway in Pseudomonas aeruginosa.
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.
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. edgre961@student.otago.ac.nz.|School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand. edgre961@student.otago.ac.nz.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Edgar
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. xinxu9@gmail.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. barshirley1996@gmail.com.
authors.forename: Matt
authors.initials: M
authors.lastname: Shirley
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. afkonings@gmail.com.
authors.forename: Anna F
authors.initials: AF
authors.lastname: Konings
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. lois.martin@otago.ac.nz.
authors.forename: Lois W
authors.initials: LW
authors.lastname: Martin
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand. david.ackerley@vuw.ac.nz.
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. iain.lamont@otago.ac.nz.
authors.forename: Iain L
authors.initials: IL
authors.lastname: Lamont
mesh_terms: D004252:DNA Mutational Analysis; D015964:Gene Expression Regulation, Bacterial; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion; D012808:Sigma Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D009842:Oligopeptides; D012808:Sigma Factor; C042453:pyoverdin