Evidence for a second regulatory binding site on PspF that is occupied by the C-terminal domain of PspA

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.

Evidence for an Adaptation of a Phage-Derived Holin/Endolysin System to Toxin Transport in Clostridioides difficile

“…Site specific mutagenesis of pHIS1522- tcdB D286/288N ( Wohlan et al, 2014 ) was used to introduce a silent mutation in tcdB at postion A473 with the primer pair tcdB -sil-NdeI-F and tcdB -sil-NdeI-R, which eliminated an intrinsic NdeI-site, resulting in the vector pHIS1522- tcdB D286/288N silent NdeI. To analyze protein–protein interactions by the B2H screen, tcdE -M1, tcdL , and tcdB were amplified with the primer pairs 2H- tcdE -BamHI-F/2H- tcdE -KpnI-R, 2H- tcdB -BamHI-F/2H- tcdB -KpnI-R, and 2H- tcdL -BamHI-F/2H- tcdL -KpnI-R, and cloned into the corresponding sites of pU-strep- pspA (25–144)-T18, pUT18C- pspA (25–144)-strep, pK-strep- pspA (25–144)-T25, and pKT25- pspA (25–144)-strep ( Heidrich and Brüser, 2018 ). Single amino acid exchanges in TcdE for I151V were done as described above.…”