PspA(145–222) is necessary for keeping PspF activity at the basal level in a natively regulated Psp system.

<p>(A) LacZ activities of a <i>pspFABCDE</i> deletion P_<i>pspA</i> reporter strain MC3 <i>pspFABCDE</i>::<i>kan</i> with an in trans complemented minimal regulatory cascade using vector pUL-<i>pspF</i>strep-<i>pspABC</i>-H6 or pUL-<i>pspF</i>strep-<i>pspA</i>(1–144)BC-H6. Membrane stress was induced by rhamnose-dependent production of TatA(NT)-Hip. As negative controls, strains with the empty pBW22 vector and without the pBW22 vector (w/o) were used. (B) SDS-PAGE/Western blot analysis of crude extract, soluble and membrane fractions of the control strains (w/o) used in (A). PspA fragments were detected using PspA-specific antibodies (* = cross reaction of the PspA antibody). His-tagged PspC was detected via His-tag specific antibodies, and BCCP was detected as fractionation control with the Strep-tactin-AP conjugate. (C) LacZ activites of MC3 <i>pspFABCDE</i>::<i>kan</i> transformed with either pUL-<i>pspF</i>strep-<i>pspABC</i>-H6, pUL-<i>pspF</i>strep-<i>pspA</i>(1–144)BC-H6, or pUL-<i>pspF</i>strep-<i>pspA</i>(1–144)BC(V105D)-H6 for determination of PspC dependence of the results obtained in (B). (D) LacZ activites and detection of protein levels of MC3 <i>pspFABCDE</i>::<i>kan</i> transformed with either pUL-<i>pspF</i>strep-<i>pspABC</i>-H6, pUL-<i>pspF</i>(W56A)strep-<i>pspABC</i>-H6, pUL-<i>pspF</i>strep-<i>pspA</i>(1–144)BC-H6, pUL-<i>pspF</i>(W56A)strep-<i>pspA</i>(1–144)<i>BC</i>-H6. PspA detection in cellular fractions by SDS-PAGE/Western blot analysis, using PspA-specific antibodies.