The purification of the σFpvI/FpvR20 and σPvdS/FpvR20 protein complexes is facilitated at room temperature☆
Introduction
Pseudomonas (P.) aeruginosa is an opportunistic pathogen and one of the most common bacteria causing chronic infections in immunocompromised individuals [[1], [2], [3], [4]]. Iron is an essential nutrient required for P. aeruginosa virulence and growth and has limited bioavailability in the human body [5,6]. The production of the siderophore pyoverdine contributes to iron acquisition by P. aeruginosa. Pyoverdine chelates Fe3+ ions from the environment for cellular import (as Fe3+-pyoverdine complexes) via the cell surface receptor FpvA (ferric-pyoverdine receptor) [7,8]. The σPvdS (pyoverdine synthesis) and σFpvI (FpvA synthesis) proteins are alternative sigma factors, which act to regulate gene expression in response to environmental stimuli [[9], [10], [11], [12]]. In the absence of ferripyoverdine the activities of these sigma factors are inhibited by the 20 kDa antisigma protein FpvR20, through the formation of protein complexes [9,11,13,14]. The σFpvI and σPvdS proteins are unique in that their activities are regulated by the same antisigma factor.
In a positive feedback loop, upon ferric-pyoverdine binding to FpvA, the signaling domain of FpvA (which binds to TonB in the absence of ferri-pyoverdine) undergoes structural reorganization, allowing the FpvA signaling domain to be available to interact with the periplasmic domain of FpvR [9,15,16]. Ferric-pyoverdine binding to its receptor FpvA triggers complete proteolysis of the FpvR20 protein, resulting in σFpvI and σPvdS becoming active [15,17]. The release of σFpvI promotes the expression of the pyoverdine receptor FpvA [10], while σPvdS is required for the expression of pyoverdine synthesis genes [[18], [19], [20], [21], [22]]. σPvdS is also required for maximal expression of the virulence factors exotoxin A [23] and the PrpL protease [11].
The interactions between the sigma factors σPvdS and σFpvI and the antisigma protein FpvR20 have been difficult to study experimentally due to the insolubility of the recombinantly produced σPvdS and σFpvI proteins in solution [24]. This is a common observation for sigma factor proteins [[25], [26], [27]]. For example, the σ70 from Escherichia coli [27], and the σD, σK, σF and σL proteins from Mycobacterium tuberculosis [28], have all been described as predominantly insoluble or of low solubility when produced recombinantly. In contrast, the cytoplasmic regions of the antisigma factor proteins PupR from Pseudomonas putida [29], RsbW, RskA and RslA from M. tuberculosis [30] have been shown to be soluble and stable in solution. This has, for example, facilitated the three-dimensional structure solution of the cytoplasmic region of PupR from P. putida [29]. Critically, the production and structural elucidation of the sigma factors σ70 from E. coli and σK and σL from M. tuberculosis have been facilitated by their co-production and purification with their respective antisigma proteins Rsd [31] RskA [32] and RslA [30].
The structural and biophysical characterizations of the σFpvI/FpvR20 and σPvdS/FpvR20 protein complexes, which are crucial for understanding their mechanism of action, have been hampered by the difficulty in isolating soluble and stable proteins of significant purity. This study describes a protocol that enabled the co-expression and purification of the two sigma factor proteins σFpvI and σPvdS in complex with the antisigma factor FpvR20 (specifically N-terminal constructs FpvR1-67 (residues 1–67) and FpvR1-89 (residues1-89)). FpvR20 residues 1–89, having been previously demonstrated as the anti-sigma inhibitory region required for the formation of stable, soluble sigma/antisigma protein complexes [13,14]. Additionally, the biophysical characterization of the His6-σFpvI/FpvR1-67 and His6-σPvdS/FpvR1-67 protein complexes and comparisons with those of the His6-σFpvI/FpvR1-89 and His6-σPvdS/FpvR1-89 complexes [33] is described.
Section snippets
Overexpression and purification of the sigma/antisigma factor protein complexes
The plasmids pETDuetfpvI/fpvR1-67, pETDuetfpvI/fpvR1-89, pETDuetpvdS/fpvR1-67 and pETDuetpvdS/fpvR1-89 were generated as previously described for the expression of the His6-σFpvI/FpvR1-67, His6-σFpvI/FpvR1-89, His6-σPvdS/FpvR1-67 and His6-σPvdS/FpvR1-89 protein complexes, respectively in E. coli BL21 (DE3) [14]. Transformed cells were grown at 37 °C in LB media supplemented with ampicillin (50 μg/mL) until the cell density reached an OD600 of 0.5–0.6. The growth temperature was lowered to
Purification of protein complexes
The biochemical, biophysical and structural characterization of sigma/antisigma factor protein complexes has been limited by their insolubility in aqueous solution [[25], [26], [27]]. To date, the crystal structures of the σ70/Rsd, σ70/Asia and σE/RseA complexes from E. coli, the σCnrH/CnrY complex from Cupriavidus matallidurans, the σE/ChrR from Rhodobacter sphaeroides, the σK/RskA, σD4/RsdA and σL/RslA complexes from M. tuberculosis and the σW/RsiW from Bacillus subtilis, have been reported
Discussion
The success of previous studies aimed at characterizing protein complexes between the alternative sigma factors σFpvI or σPvdS and the antisigma protein FpvR20 from P. aeruginosa has been limited by the poor solubility of the sigma factor proteins when produced recombinantly in isolation. The co-expression and co-purification strategies, successfully implemented for a number of other sigma/antisigma factor protein complexes [28,31,46], were applied here to obtain soluble preparations of the σ
Conclusion
The σPvdS, σFpvI and FpvR20 proteins, which act to moderate pyoverdine-mediated iron uptake in P. aeruginosa, are an important target for biochemical and structural studies. The study of these proteins has been hampered by difficulties in purifying soluble proteins. In this study, we developed an approach where the recombinant production of the His6-σFpvI/FpvR1-67, His6-σFpvI/FpvR1-89, His6-σPvdS/FpvR1-67 and His6-σPvdS/FpvR1-89 protein complexes was facilitated by their purification at room
Author contributions
G.P.C.G performed the experiments, analysed the experimental data and wrote the manuscript, M.A.P planned the AUC experiments, analysed the AUC data and wrote the manuscript, I.L.L. provided the protein constructs and wrote the manuscript, M.J.M directed the project, planned the experiments and wrote the manuscript. We are grateful to Lois Martin for her assistance in preparing plasmid constructs.
Acknowledgments
We would like to acknowledge the La Trobe University Comprehensive Proteomics Platform for providing infrastructure for the analytical ultracentrifugation experiments and circular dichroism spectroscopy. G.P.C.G acknowledges La Trobe University Full Fee Research scholarship support.
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The authors have no conflict of interest to declare.