Fig. 6.
Characterization of a strain with a tac promoter-controlled pspA operon.
A. Diagrams of the wild type pspA locus (pspAp-pspA) and the derivative with the pspA operon expressed from the tac promoter (tacp-pspA). In the tacp-psp strain, the region encompassing pspF and the pspA promoter was replaced by the E. coli lacIq gene and the tac promoter.
B. Growth of pspAp-pspA and tacp-pspA operon strains. A derivative of the pspAp-pspA strain with all psp genes deleted was included as a secretin-sensitivity control (Δpsp; strain AJD1171). Strains contained pBAD33 (− YsaC) or pAJD935 (+ YsaC) and were grown in the presence of 0.02% arabinose. 10 μM IPTG was included in all cultures except to one set of the tacp-pspA strains as indicated (− IPTG). Optical density was measured hourly.
C. Anti-PspA, -PspB and –PspC immunoblot analysis of samples taken at the 4 h time point from the cultures in panel B. Ponceau S stain of the PspA-PspC region of the nitrocellulose membrane is shown as a loading control.
D. ONPG hydrolysis by derivatives of the pspAp-pspA and tacp-pspA operon strains with a Φ(catp-lacZ) operon fusion on the chromosome. Strains contained either pBAD33 (− YsaC) or pAJD935 (+ YsaC). Arabinose was added to all cultures at 0.02% and 10 μM IPTG was also included where indicated. ONPG hydrolysis was monitored by increased absorbance at 420 nm. Readings were normalized so absorbance at 2 min was zero for all samples. Total β-galactosidase activities of cells permeabilized with SDS and chloroform were not significantly different, indicating no differences in β-galactosidase enzyme content (data not shown).