FIG. 1.

Viability of E. coli cultures containing different levels of Hda protein. Fresh colonies of strain 1921 (a) and 1110 (b) were inoculated into 20 ml of Luria-Bertani broth (10 g of tryptone, 5 g of yeast extract [Difco], and 10 g of NaCl/liter) containing selective antibiotics (50 to150 μg/ml of penicillin and 30 to 50 μg/ml of chloramphenicol) and shaken at 37°C. At mid-logarithmic-phase growth (−3 h), IPTG was added as indicated, and viable cell counts were performed by serial dilution and plating over the indicated period. The values shown are representative of numerous experiments. For the analysis of Hda levels in strain 1921, induced for 30 to 60 min at mid-log phase by various concentrations of IPTG (c), extracts were prepared from cell precipitates (100-ml cultures) by sonication with the Fisher 50 sonic dismembrator and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Relative Hda concentrations were determined by densitometry, using the ZUV transilluminator with Kodak IP image analysis software. Actual protein concentrations were determined by Bio-Rad, using a purified T7-tagged Hda standard (8).