FpvIR Control of fpvA Ferric Pyoverdine Receptor Gene Expression in Pseudomonas aeruginosa : Demonstration of an Interaction between FpvI and FpvR and Identification of Mutations in Each Compromising This Interaction

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Abstract
FpvR is a presumed cytoplasmic membrane-associated anti-sigma factor that controls the activities of extracytoplasmic function sigma factors PvdS and FpvI responsible for transcription of pyoverdine biosynthetic genes and the ferric pyoverdine receptor gene, fpvA , respectively. Using deletion analysis and an in vivo bacterial two-hybrid system, FpvR interaction with these σ factors was confirmed and shown to involve the cytoplasmic N-terminal 67 amino acid resides of FpvR. FpvR bound specifically to a C-terminal region of FpvI corresponding to region 4 of the σ 70 family of sigma factors. FpvR and FpvI mutant proteins compromised for this interaction were generated by random and site-directed PCR mutagenesis and invariably contained secondary structure-altering proline substitution in predicted α-helices within the FpvR N terminus or FpvI region 4. PvdS was shown to bind to the same N-terminal region of FpvR, and FpvR mutations compromising FpvI binding also compromised PvdS binding, although some mutations had a markedly greater impact on PvdS binding. Apparently, these two σ factors bind to FpvR in a substantially similar but not identical fashion. Intriguingly, defects in FpvR binding correlated with a substantial drop in yields of the FpvI and to a lesser extent PvdS σ factors, suggesting that FpvR-bound FpvI and PvdS are stable while free and active sigma factor is prone to turnover.

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