POSTERS - BLAST X - University of Utah

BLAST X Poster #58
CHARACTERIZATION OF THE PERIPLASMIC DOMAIN OF THE SENSOR KINASE CpxA:
ROLE OF CONSERVED RESIDUES IN CpxA ACTIVITY
Malpica R and Raivio TL. Department of Biological Sciences, University of Alberta, CW405
Biological Sciences Building, Edmonton, Alberta, Canada T6G 2E9.
The Cpx two-component signal transduction system plays a major role in the
conservation of cell envelope integrity, as well in the regulation of surface structure assembly,
cellular attachment and pathogenesis in Escherichia coli. This system is comprised of the innermembrane
sensor kinase CpxA and the cytosolic response regulator CpxR. Envelope stress
caused by misfolded and mislocalized proteins activates the Cpx pathway, which also responds
to alkaline pH and overexpression of the outer membrane lipoprotein NlpE. In the presence of
these activating cues, CpxA autophosphorylates at a conserved His residue and then
transphosphorylates CpxR (CpxR-P). In turn, CpxR-P regulates the expression of several genes
such as periplasmic protein folding and degrading factors involved in envelope protein
manteinance under adverse conditions. It is known that in the absence of stress, the periplasmic
protein CpxP, whose expression is positively controlled by this pathway, inhibits the activation of
the Cpx system. The periplasmic domain of CpxA (CpxA-pd), which contains 133 residues, has
been proposed as the signal reception site and therefore as the regulatory element of the kinase
and phosphatase activities of CpxA.
To explore the role of CpxA-pd components in signal sensing and CpxA activity, we
generated mutants that carry substitution mutations in highly conserved residues of this domain.
The phenotypes of these mutants were evaluated, using alkaline pH or NlpE as inducing cues
and the activatable cpxP´-lacZ fusion as a reporter of Cpx pathway activity. Remarkably, the
substitution of Phe108, Gly130 and Pro164 by Ala lead to a constitutively active phenotype.
Also, mutants in other residues displayed a significant increase on pathway activity in the
absence of the inducing cues. Thus, we have identified relevant CpxA-pd elements for both
signalling and the overall enzymatic activity of CpxA.
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