Fig 6.

Secretion stress reduces the stability of Ag43 mRNA. (A) MNY40 (MG1655 Ag43::His₆) transformed with plasmid pMY28 (encoding Ag43PD-HA) or pMY29 (encoding ΔSP-Ag43PD-HA) or the cloning vector (pTrc99a) were grown to an OD₆₀₀ of 0.4. Cultures were divided in half, and IPTG was added to one half to induce the expression of the plasmid-borne gene. After 30 min, steady-state levels of HA- and His-tagged proteins and SecA were determined by Western blotting. Analysis of one-tenth as much protein on a separate Western blot revealed the presence of precursor and mature forms of Ag43PD-HA. (B) Rifampin was added to cultures following the 30-min IPTG induction, and quantitative RT-PCR was performed to determine the percentage of the Ag43 gene transcripts present at t₀ that remained at the indicated time points. gapA mRNA served as an internal control. The averages from two independent experiments are shown.