Detection of promoter activity in the intergenic region between divergently transcribed <i>pltR</i> and <i>pltLABCDEFG</i> genes using the <i>lacZ</i> reporter gene.

<p>(<b>A</b> and <b>B</b>) Promoter detection in the <i>pltR</i> direction. (A) Four fragments, R1 to R4, containing five putative promoters in the <i>pltR</i> direction (predicted by the NNPP promoter online prediction with a score cutoff 0.80) were fused with the promoterless <i>lacZ</i> gene on the plasmid pME6522. The R3 fragment includes two putative promoters. The putative +1 site indicates the putative TSS of the fifth predicted promoter. (B) β-Galactosidase expression (Miller Units) from above these <i>lacZ</i> fusion plasmids was assayed in both <i>E. coli</i> DH5α and <i>P. aeruginosa</i> M18 after 15 h of growth in KMB. The R4 fragment was detected to include a true promoter (shown by an asterisk). (<b>C</b> and <b>D</b>) Promoter detection in the <i>pltLABCDEFG</i> operon direction. (C) Five fragments, L1 to L5, which contain five putative promoters in the <i>pltL</i> direction, were respectively fused into the upstream of the promoterless <i>lacZ</i> gene on pME6522. Putative +1 indicates the putative TSS of the third predicted promoter. (D) β-Galactosidase expression from the above <i>lacZ</i> fusion recombined plasmids was measured in both DH5α and M18. The L1 fragment (marked with a triangle) exhibited a certain degree of promoter activity in DH5α, but not in M18. The other four fragments, L2 to L5, did not show any evident promoter activity. (<b>E</b> and <b>F</b>) Detection of promoter or its elements by prolongation analysis within the DNA fragment spanning from +10 to the <i>pltL</i> ATG, which was not predicted to carry promoters. (E) Four prolonging fragments based on L3, namely, L3–1 to L3–4, were respectively fused with the <i>lacZ</i> gene in pME6522. (F) The resulting <i>lacZ</i> fusion plasmids were assayed for β-galactosidase expressions in both DH5α and M18. The region covering +66 to +124 bp (relative to the putative +1 site) contained either a stronger promoter or its elements (marked with an asterisk). The <i>L3–3–lacZ</i> fusion expression significantly decreased, compared with that in the <i>L3–2–lacZ</i> fusion expression in <i>E. coli</i> DH5α, which implies that the promoter located at +66 to +124bp is not likely <i>pltLp</i> activated by PltR. Moreover, the region from +124 to +205 bp (marked with a triangle) possibly contains another promoter under the transcriptional activation of PltR. (<b>G</b> and <b>H</b>) Distinguishing <i>pltLp</i> activated by PltR from other promoters by deletion analysis. (G) Four fragments (L6 and its shortened derivative fragments, namely, L6–1 to L6–3), four continuously prolonged fragments based on L6–3 (L6-3-1 to L6-3-4), and the L6–4 fragment, were respectively cloned into pME6522. The L6 fragment was not predicted (score cutoff 0.60) to contain putative promoter regions. (H) The β-galactosidase expression from these recombinant <i>lacZ</i> reporter plasmids was analyzed in <i>E. coli</i> DH5α, <i>P. aeruginosa</i> M18, and the <i>pltR</i> mutant M18pltR. An intact promoter contained in the region from +66 to +124 (marked with an asterisk) was not controlled by PltR. The <i>pltLp</i> activated by PltR was located from +124 to +205 bp (marked with a triangle), relative to the putative +1 site.