Identification of inducers of the Yersinia enterocolitica phage shock protein system and comparison to the regulation of the RpoE and Cpx extracytoplasmic stress responses

Abstract

Known inducers of the phage shock protein (Psp) system suggest that it is an extracytoplasmic stress response, as are the well-studied RpoE and Cpx systems. However, a random approach to identify conditions and proteins that induce the Psp system has not been attempted. It is also unknown whether the proteins or mutations that induce Psp are specific or if they also activate the RpoE and Cpx systems. This study addressed these issues for the Yersinia enterocolitica Psp system. Random transposon mutagenesis identified null mutations and overexpression mutations that increase Phi(pspA-lacZ) operon fusion expression. The results suggest that Psp may respond exclusively to extracytoplasmic stress. Null mutations affected glucosamine-6-phosphate synthetase (glmS), which plays a role in cell envelope biosynthesis, and the F0F1 ATPase (atp operon). The screen also revealed that in addition to several secretins, the overexpression of three novel putative inner membrane proteins (IMPs) induced the Psp response. We also compared induction of the Y. enterocolitica Psp, RpoE, and Cpx responses. Overexpression of secretins or the three IMPs or the presence of an atpB null mutation only induced the Psp response. Similarly, known inducers of the RpoE and Cpx responses did not significantly induce the Psp response. Only the glmS null mutation induced all three responses. Therefore, Psp is induced distinctly from the RpoE and Cpx systems. The specific IMP inducers may be valuable tools to probe specific signal transduction events of the Psp response in future studies.

FIG. 1.

Overexpression mutations that induce the Psp response. A flag shows the approximate location of each transposon insertion, and the arrow shows the orientation of the tac promoter. Closed flags indicate the mutants used to determine the data shown in Table 3, and they are also listed in Table 1. Open reading frames are shown as horizontal arrows in the direction of their transcription. Genes labeled with numbers indicate the Y. enterocolitica ortholog of the annotated Y. pestis CO92 genome (e.g., 0432 = YPO0432 ortholog). Genes responsible for inducing Φ(pspA-lacZ) expression are boxed (see text).

FIG. 2.

Overexpression inducers are specific for the Psp, Cpx, and RpoE stress responses. Plasmid pVLT35 derivatives encoding the indicated genes expressed from the tac promoter were transferred into Φ(pspA-lacZ), Φ(cpxP-lacZ), and Φ(rpoE-lacZ) operon fusion strains YVM576, AJD243, and AJD242, respectively. Strains were grown as described in Materials and Methods, in the absence of IPTG (white bars) or with 0.2 mM IPTG (black bars). In most cases, cells were harvested for β-galactosidase assays 2 h after addition of IPTG. The one exception was the yggT overexpression strains, which were harvested in late stationary phase (22 h after addition of IPTG). For each fusion strain, the fold induction of β-galactosidase activity is with respect to the activity of the strain containing the pVLT35 vector plasmid alone, harvested at the same time point as the test strain. The basal expression level of each fusion (i.e., with pVLT35) in Miller units was 73 for Φ(pspA-lacZ), 190 for Φ(cpxP-lacZ), and 1,730 for Φ(rpoE-lacZ).

FIG. 3.

Detection of Psp response-inducing proteins. Strains containing plasmid pVLT35 (vector) or derivatives encoding the indicated genes were grown as described in the Fig. 2 legend. Total cell proteins were separated by SDS-12.5% PAGE, transferred to nitrocellulose, and detected with a penta-His antibody as described in Materials and Methods. The signals generated by the six-His-tagged proteins are underscored with asterisks. Molecular weights of the detected proteins were estimated by comparison to a prestained molecular weight marker (not shown). Note that the YPO0432 ortholog did not resolve from the dye front on the gel owing to its small size (approximately 6 kDa). −, no IPTG; +, 0.2 mM IPTG.

FIG. 4.

Null mutations that induce the Psp response. A flag represents the approximate location of each transposon insertion, and the arrow shows the orientation of the tac promoter. Open reading frames are shown as horizontal arrows in the direction of their transcription.

FIG. 5.

Effects of an atpB null mutation and alkaline pH on the Psp, Cpx, and RpoE systems. Φ(pspA-lacZ), Φ(cpxP-lacZ), and Φ(rpoE-lacZ) operon fusion strains YVM576, AJD243, and AJD242, respectively (WT [wild type]), or their ΔatpB derivatives (Table 1) were grown at 26°C after inoculation of cultures to an initial optical density at 600 nm of 0.05. Cultures were grown in LB medium buffered to either pH 7 or 8, and samples were taken after 8 h (white bars) or 24 h (black bars) for β-galactosidase assays (Sp. Act. = specific activity; see Materials and Methods).