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. 2010 Dec 21;6:452. doi: 10.1038/msb.2010.105

Figure 2.

Figure 2

Combining substitutions at sites I–V gives rise to signal response, bifunctional behavior, and robustness. (A) CpxAmut1 was constructed by combining substitutions selected from libraries at sites I–V. CpxAmut2 was isolated in a screen for increased signal response. (B) CpxAmut1 and CpxAmut2 decrease OmpR-regulated transcription in a strain expressing a kinase+ phosphatase− EnvZ mutant. The bars denote averages and standard deviations of three independent experiments. Cultures of the ompC-cfp reporter strains AFS225 (EnvZ−) or AFS226 (EnvZ kinase+ phosphatase−) containing empty vector or plasmid that expresses wild-type CpxA, CpxAmut1, or CpxAmut2 were grown in minimal glucose medium at pH 5.5. (C) CpxAmut1 and CpxAmut2 modulate cross-talk to OmpR in response to input stimulus (over-expression of the lipoprotein NlpE (Snyder et al, 1995)). For comparison, the effects of EnvZ stimulation with 10 mM procaine are also shown. The bars denote averages and standard deviations of three independent experiments. Cultures of the ompC-cfp reporter strain AFS29 (envZ−cpxRA−) containing plasmid that expresses wild-type CpxA, CpxAmut1, or CpxAmut2 and either empty vector or plasmid expressing NlpE and cultures of the ompC-cfp reporter strain EPB62 (cpxRA−) containing empty vectors were grown in glycerol minimal medium supplemented with 10 mM arabinose. (D) CpxAmut2 dephosphorylates OmpR-P in vitro. OmpR was phosphorylated in vitro with a kinase+ phosphatase− EnvZ mutant and subsequently added to lysates of cells expressing cytoplasmic fragments of the indicated sensor kinases or a lysate of cells containing an empty control plasmid. (E) OmpR-regulated transcription is robust to changes in CpxAmut2 and EnvZ expression level. Note that there is a break in the y-axis scale. Cultures of the ompC-cfp reporter strain AFS29 (envZ−cpxRA−) containing plasmids that express wild-type CpxA or CpxAmut2 and plasmid containing PBAD-nlpE and cultures of the strain AFS29 containing only plasmid that expresses EnvZ were grown in glycerol minimal medium supplemented with 0.1 mM arabinose. See Supplementary information for a detailed description of the strains and growth conditions.