Abstract
Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.
Highlights
Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators
PupR C-terminal CSS domain (CCSSD) truncations indicate that the C-terminal juxta-membrane subdomain (CJM) and STN are both required for binding the PupB N-terminal signaling domain (NTSD) in vitro
In contrast to the information provided here, a recent NMR study investigating the interaction of the C-terminal domain (CTD) of sigma regulator HasS with the NTSD of its cognate transducer HasR, members of the heme acquisition system (Has) of Serratia marcescens, suggests that the HasS CTD is partially disordered and contains a region that may interact with the inner membrane [24]
Summary
Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. We present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. CSS systems are associated with biofilm formation, intercellular interactions, and release of virulence factors, in addition to metabolite transfer and regulation [2] One such CSS pathway involves iron import in Gram-negative bacteria. The best characterized CSS iron import systems are the ferric citrate (fec) transport system from Escherichia coli, the ferric pyoverdine (fpv) import system from Pseudomonas aeruginosa, and the ferric pseudobactin BN7/BN8 (pup) system from Pseudomonas capeferrum (formerly Pseudomonas putida WCS358) Each of these homologous pathways involves a TonB-dependent transporter/transducer, an inner membrane sigma regulator, and an extra-cytoplasmic function sigma factor (Table 1). The structure of the periplasmic domain of sigma regulators has not been described
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