FIG. 5.
Susceptibility of periplasmic BlaM and cytoplasmic H-NS to extracellular protease. BS103 derivatives expressing either MxiM, MxiJ, or MxiD or various combinations of these proteins (indicated to the right of each panel) were incubated with proteinase K (PK) and/or Congo red (CR) for 20 min. Whole-cell protein extracts of each strain were resolved on SDS–10% polyacrylamide gels and examined by immunoblotting with either anti-BlaM (A to G) or anti-H-NS (H) antibodies. The arrow to the left of each panel indicates the position of BlaM (∼29 kDa) or H-NS (∼15 kDa). In these backgrounds, MxiD was expressed from the high-copy-number vector pBAD18, while MxiJ was expressed from pBAD33. MxiM was expressed from either pBAD33 (when it was used alone or with MxiD) or from pWSK129. Protein from 5 × 108 bacteria was used in each lane of panels A to G; protein from 5 × 107 bacteria was used in each lane of panel H. The minor higher-Mr band present in some panels probably corresponds to unprocessesed β-lactamase.