Abstract
The phage shock protein (Psp) system is a widely conserved cell envelope stress response that is essential for the virulence of some bacteria, including Yersinia enterocolitica Recruitment of PspA by the inner membrane PspB-PspC complex characterizes the activated state of this response. The PspB-PspC complex has been proposed to be a stress-responsive switch, changing from an OFF to an ON state in response to an inducing stimulus. In the OFF state, PspA cannot access its binding site in the C-terminal cytoplasmic domain of PspC (PspC(CT)), because this site is bound to PspB. PspC has another cytoplasmic domain at its N-terminal end (PspC(NT)), which has been thought to play a role in maintaining the OFF state, because its removal causes constitutive activation. However, until now, this role has proved recalcitrant to experimental investigation. Here, we developed a combination of approaches to investigate the role of PspC(NT) in Y. enterocolitica Pulldown assays provided evidence that PspC(NT) mediates the interaction of PspC with the C-terminal cytoplasmic domain of PspB (PspB(CT)) in vitro Furthermore, site-specific oxidative cross-linking suggested that a PspC(NT)-PspB(CT) interaction occurs only under noninducing conditions in vivo Additional experiments indicated that mutations in pspC might cause constitutive activation by compromising this PspC(NT) binding site or by causing a conformational disturbance that repositions PspC(NT) in vivo These findings have provided the first insight into the regulatory function of the N-terminal cytoplasmic domain of PspC, revealing that its ability to participate in an inhibitory complex is essential to silencing the Psp response. IMPORTANCE: The phage shock protein (Psp) response has generated widespread interest because it is linked to important phenotypes, including antibiotic resistance, biofilm formation, and virulence in a diverse group of bacteria. Therefore, achieving a comprehensive understanding of how this response is controlled at the molecular level has obvious significance. An integral inner membrane protein complex is believed to be a critical regulatory component that acts as a stress-responsive switch, but some essential characteristics of the switch states are poorly understood. This study provides an important advance by uncovering a new protein interaction domain within this membrane protein complex that is essential to silencing the Psp response in the absence of an inducing stimulus.