Abstract
PspA is a key component of the bacterial Psp membrane-stress response system. The biochemical and functional characterization of PspA is impeded by its oligomerization and aggregation properties. It was recently possible to solve the coiled coil structure of a completely soluble PspA fragment, PspA(1–144), that associates with the σ54 enhancer binding protein PspF at its W56-loop and thereby down-regulates the Psp response. We now found that the C-terminal part of PspA, PspA(145–222), also interacts with PspF and inhibits its activity in the absence of full-length PspA. Surprisingly, PspA(145–222) effects changed completely in the presence of full-length PspA, as promoter activity was triggered instead of being inhibited under this condition. PspA(145–222) thus interfered with the inhibitory effect of full-length PspA on PspF, most likely by interacting with full-length PspA that remained bound to PspF. In support of this view, a comprehensive bacterial-2-hybrid screen as well as co-purification analyses indicated a self-interaction of PspA(145–222) and an interaction with full-length PspA. This is the first direct demonstration of PspA/PspA and PspA/PspF interactions in vivo that are mediated by the C-terminus of PspA. The data indicate that regulatory binding sites on PspF do not only exist for the N-terminal coiled coil domain but also for the C-terminal domain of PspA. The inhibition of PspF by PspA-(145–222) was reduced upon membrane stress, whereas the inhibition of PspF by PspA(1–144) did not respond to membrane stress. We therefore propose that the C-terminal domain of PspA is crucial for the regulation of PspF in response to Psp system stimuli.
Highlights
Many proteobacteria possess the phage shock protein (Psp) system that is upregulated under various stress conditions that can harm the cytoplasmic membrane [1,2]
The regulatory interactions of the Psp components PspA, PspB, PspC, and PspF in response to membrane stress are in the focus of most current research that is done in the Psp field
E. coli, it was recently shown that six PspA(1–144) domains stably associate with hexameric PspF, exhibiting a slow dissociation kinetics [13], and that PspF can be regulated by PspA and PspA(1–144) in vivo and in vitro when it is bound to it [13]
Summary
Many proteobacteria possess the phage shock protein (Psp) system that is upregulated under various stress conditions that can harm the cytoplasmic membrane [1,2]. In Escherichia coli and other enterobacteria, the Psp components are encoded by the pspABCDE operon and the monocistronic pspG gene. The expression of these genes depends on σ54 that is regulated by the PspF component, an enhancer binding protein divergently encoded upstream of the pspABCDE operon [3,4]. PspF in turn is regulated by PspA, the first product of the pspABCDE operon [5,6]. It is believed that PspA is a key regulator and a membrane-.
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