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Regulation 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.

The Yersinia enterocolitica phage-shock-protein (Psp) stress response system is activated by mislocalized outer-membrane secretin components of protein export systems and is essential for virulence. The cytoplasmic membrane proteins PspB and PspC were proposed to be dual function components of the system, acting both as positive regulators of psp gene expression and to support survival during secretin-induced stress. In this study we have uncoupled the regulatory and physiological functions of PspBC and discovered unexpected new roles, functional domains and essential amino acids. First, we showed that PspB controls PspC concentration by both pre- and post-transcriptional mechanisms. We then screened for PspBC mutants with altered transcriptional regulatory function. Unexpectedly, we identified PspB and PspC mutants that activated psp gene expression in the absence of secretin-induced stress. Together with a subsequent truncation analysis, this revealed that the PspC cytoplasmic domain plays an unforeseen role in negatively regulating psp gene expression. Conversely, mutations within the PspC periplasmic domain abolished its ability to activate psp gene expression. Significantly, PspC mutants unable to activate psp gene expression retained their ability to support survival during secretin-induced stress. These data provide compelling support for the proposal that these two functions are independent.

IS911 naturally produces transposase (OrfAB) derivatives truncated at the C-terminal end (OrfAB-CTF) and devoid of the catalytic domain. A majority species, OrfAB*, was produced at higher levels at 42 degrees C than at 30 degrees C suggesting that it is at least partly responsible for the innate reduction in IS911 transposition activity at higher temperatures. An engineered equivalent of similar length, OrfAB[1-149], inhibited transposition activity in vivo or in vitro when produced along with full-length transposase. We isolated several point mutants showing higher activity than the wild-type IS911 at 42 degrees C. These fall into two regions of the transposase. One, located in the N-terminal segment of OrfAB, lies between or within two regions involved in protein multimerization. The other is located within the C-terminal catalytic domain. The N-terminal mutations resulted in reduced levels of OrfAB* while the C-terminal mutation alone appeared not to affect OrfAB* levels. Combination of N- and C-terminal mutations greatly reduced OrfAB* levels and transposition was concomitantly high even at 42 degrees C. The mechanism by which truncated transposase species are generated and how they intervene to reduce transposition activity is discussed. While transposition activity of these multiply mutated derivatives in vivo was resistant to temperature, the purified OrfAB derivatives retained an inherent temperature-sensitive phenotype in vitro. This clearly demonstrates that temperature sensitivity of IS911 transposition is a complex phenomenon with several mechanistic components. These results have important implications for the several other transposons and insertion sequences whose transposition has also been shown to be temperature-sensitive.