Fig 3. The qseEGF operon is not subject to transcriptional autoregulation.

A. Expression of transcriptional qseE’-lacZ reporter fusions is not affected by deletion of qseF or qseG. The glmY locus and the adjacent qseEGF operon are schematically depicted at the top. Experimentally verified promoters directing expression of glmY and qseEGF, respectively [9], are indicated by arrows. For reporter gene studies, the regions indicated by horizontal lines and roman numerals were fused to lacZ. Positions are relative to the first nucleotide of the qseE start codon. The lacZ fusions were placed on plasmids pBGG273 (fusion I), pBGG274 (fusion II), pBGG354 (fusion III), pBGG355 (fusion IV) and subsequently introduced into strains R1279 (wild-type), Z179 (ΔqseF) and Z1117 (ΔqseG), respectively. The β-galactosidase activities of these transformants were determined from exponentially growing cells (bottom) as well as from stationary cells (S6 Fig). B. Plasmid-driven over-expression of qseF-D56E, qseG, qseE or glmY does not affect the level of chromosomally encoded QseG. Strain Z951 was addressed, which carries the sequence coding for the 3×FLAG epitope fused in frame to the 3’ end of qseG encoded at its natural locus in the chromosome. In addition, strain Z951 carried the following plasmids overproducing the indicated genes, respectively: pKESK23 (empty vector control for qse plasmids; lanes 1, 7), pYG90 (qseF-D56E; lanes 2, 8), pYG220 (qseG; lanes 3, 9), pYG221 (qseE; lanes 4, 10), pBR-plac (empty vector control for glmY plasmid; lanes 5, 11), pYG83 (glmY; lanes 6, 12). As a negative control, strain R1279 lacking a FLAG epitope was tested in lane 13. The various transformants were grown in LB and total protein extracts, prepared from cells harvested in the exponential as well as in the stationary growth phase, were analyzed by Western blotting using α-FLAG antiserum (top panel). As a loading control, the Coomassie blue stained PAA gel is shown in the bottom panel.