Membrane association of PspA depends on activation of the phage‐shock‐protein response in Yersinia enterocolitica

Abstract

SummaryRegulation of the bacterial phage-shock-protein (Psp) system involves communication between integral (PspBC) and peripheral (PspA) cytoplasmic membrane proteins and a soluble transcriptional activator (PspF). In this study protein subcellular localization studies were used to distinguish between spatial models for this putative signal transduction pathway in Yersinia enterocolitica. In non-inducing conditions PspA and PspF were almost exclusively in the soluble fraction, consistent with them forming an inhibitory complex in the cytoplasm. However, upon induction PspA, but not PspF, mainly associated with the membrane fraction. This membrane association was dependent on PspBC but independent of increased PspA concentration. Analysis of psp null, overexpression and altered function mutants further supported a model where PspA is predominantly membrane associated only when the system is induced. Activation of the Psp system normally leads to a large increase in PspA concentration and we found that this provided a second mechanism for its membrane association, which did not require PspBC. These data suggest that basal PspFABC protein levels constitute a regulatory switch that moves some PspA to the membrane when an inducing trigger is encountered. Once this switch is activated PspA concentration increases, which might then allow it to directly contact the membrane for its physiological function.

Identification of YsaP, the Pilotin of the Yersinia enterocolitica Ysa Type III Secretion System

“…For construction of AJD4991, an araC-araBp fragment was amplified from pBAD33 by using primers 321 and 433, digested with SpeI-XbaI, and cloned into the unique XbaI site of a previously described sacB ϩ allelic exchange plasmid that integrates into the yenR deletion site in all our Y. enterocolitica strains (30). The araC-araBp cassette was then introduced into the chromosomal yenR deletion site of ⌬ysaC strain YVM1178 (9) by plasmid integration, followed by selection for sucrose-resistant segregants and confirmation by colony PCR.…”

Phage Shock Protein C (PspC) of Yersinia enterocolitica Is a Polytopic Membrane Protein with Implications for Regulation of the Psp Stress Response

“…Induction of pspA operon expression is thought to be mediated by interactions between Psp proteins and the relocalization of PspA from the cytoplasm to the cytoplasmic membrane (58).…”

Protecting from Envelope Stress: Variations on the Phage-Shock-Protein Theme

“…Recent work raises the possibility that PspF activity can be altered without dissociation of the PspA-F complex [28]. Moreover, PspA can associate with the membrane independent of PspBC, when expressed at high levels [23]. The independent association of PspA and PspBC with the cell membrane is consistent with the idea that PspA and PspBC may respond to different stress signals and/or relieve the membrane from damage caused by different stressors (reviewed in [7]).…”

“…This idea currently lacks experimental support. Third, since increased production of PspA circumvents the requirement for membrane-targeting partners in Y. enterocolitica [23], production of PspA from multiple genes may result in protein levels that render PspA partitioning to the membrane independent of membrane-targeting partners. Functional characterization of the individual paralogs and identification of the genes that regulate the orphan pspA copies are necessary to test the above scenarios.…”

“…PspA establishes interactions with membranebound PspBC and also directly with the membrane (membrane-stabilizing, 'effector' PspA). Thus, the Psp response revolves around two stress-induced, partner-switching mechanisms: (i) PspA transitions from the regulatory PspA-F complex to the effector, membrane-bound state [23], and (ii) the C-terminal portion of PspC is released from the PspB interaction and can bind PspA [24,25] ( Figure 1A). …”