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

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 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 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 ␣-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 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 factors, suggesting that FpvR-bound FpvI and PvdS are stable while free and active sigma factor is prone to turnover.Iron is an essential nutrient whose acquisition by Pseudomonas aeruginosa is often facilitated by high-affinity iron-chelating molecules termed siderophores that, together with cell surface receptors specific for the iron-siderophore complexes, serve to provide the organism with iron under the most nutritionally dilute conditions (35). A major siderophore produced by P. aeruginosa and, indeed, all fluorescent pseudomonads is pyoverdine, a mixed catecholate-hydroxamate siderophore characterized by a conserved dihydroxyquinoline chromophore to which is attached a peptide chain of variable length and composition (5, 30). This variation likely explains the noted specificity vis-à-vis pyoverdine utilization by Pseudomonas spp., where a given strain will often use only its own pyoverdine and not that of other Pseudomonas strains (6, 14), and suggests that the peptide moiety is involved in receptor recognition and binding. A number of genes for the biosynthesis of pyoverdine have been identified to date in P. aeruginosa (19, 20, 26-28, 32, 48-50) and cluster within a region of the chromosome referred to as the pvd locus (47), although an operon implicated in synthesis of the chromophore, pvcABCD (45, 46), occurs elsewhere. Still, a pvd gene (pvdL) has also been reported to function in chromophore synthesis (31), raising questions about the necessity of the pvc genes in this.The ferric pyoverdine receptor of P. aeruginosa PAO1 (FpvA or FpvAI) is a ca. 90-kDa outer membrane protein inducible under conditions of iron limitation (29, 36) and encoded by the fpvA gene (37) that is also found in the pvd locus (32). FpvA exhibits features typical of...

Role of Cell Surface Signaling in Proteolysis of an Alternative Sigma Factor inPseudomonas aeruginosa

“…Our data suggest that a likely reason for reduced amounts of PvdS is increased proteolysis following binding by FpvR. Genetic evidence has shown that the cytoplasmic part of FpvR interacts with PvdS (32). Collectively, these data are consistent with a model in which, in the absence of pyoverdine or FpvA, FpvR binds to PvdS and renders it susceptible to proteolytic degradation, with the subfragment observed in Fig.…”

“…Strains of P. aeruginosa secrete pyoverdines, yellowgreen fluorescent siderophores that have a high affinity (ϳ10 32 ) for Fe 3ϩ ions, with different strains secreting different pyoverdines (25). Following iron chelation, ferripyoverdines are taken up by the bacteria via high-affinity cell surface receptors and the iron is released for incorporation into bacterial proteins (30).…”

“…FpvR is an antisigma factor that binds PvdS (32), with the antisigma activity of FpvR being controlled by the concentration of pyoverdine (22). In the absence of pyoverdine, FpvR inhibits the activity of PvdS.…”

Long-term social dynamics drive loss of function in pathogenic bacteria

“…In 1973, DK2 acquired a mutation in the σ-factor fpvI gene, canceling up-regulation of receptor expression in the presence of pyoverdine ( Fig. 1) and also, lowering background receptor expression (18,27). This mutation is, however, followed by no fewer than 17 unique nonsynonymous fpvA mutations in nine independent lines across patients, suggesting that even limited background expression of fpvA is costly.…”

Mutational Analysis of an Extracytoplasmic-Function Sigma Factor To Investigate Its Interactions with RNA Polymerase and DNA

“…By analogy with 70 , regions 4.2 and 2.4 of PvdS are most likely to be responsible for the recognition of these motifs. The activity of PvdS is controlled posttranslationally by an anti-sigma factor, FpvR, that can bind to PvdS, inhibiting its activity (31,48).…”