(A) HtrA and LppZ expression decrease Ami3 levelsto varying degrees. Strains in which the only copy of htrA or lppZ was under an aTc-inducible promoter were constructed and grown with or without 100 ng/mL aTc for 12 hr. HtrA(S354A) is an allele of htrA presumed to exhibit reduced catalytic activity in HtrA homologs due to a mutation in its catalytic serine (see Figure S3). Whole-cell lysate was analyzed using western blotting using anti-Strep and anti-RpoB as a loading control.
(B) Higher expression of HtrA(S354A) further decreases Ami3. A second copy of aTc-inducible htrA(S354A) was transformed into the mutant strain described in (A), and all strains were grown with or without 100 ng/mL aTc for 8 hr. Whole-cell lysate was analyzed using western blotting using anti-Strep and anti-HtrA and anti-RpoB as a loading control. Western blot images were cropped but display all relevant lanes and reactive bands.