Effects of <i>lys</i> box mutations on <i>pltLp</i> expression and the interaction between PltR and <i>pltLp</i>.

<p>(<b>A</b>) Construction map of the mutagenized <i>pltLp</i> with <i>lys</i> box mutations. Three <i>pltLp</i> mutant fragments, <i>pltLp-M2</i>, <i>-M4</i>, and <i>-M6</i>, respectively carried 2 bp, 4 bp, and 6 bp replacements in the <i>lys</i> box. The <i>pltLp</i> mutant M4-M was constructed by introducing a 4 bp complementary replacement into the <i>pltLp-M4</i> mutant. The above four <i>pltLp</i> mutagenized fragments were respectively cloned into pME6522. (<b>B</b>) β-galactosidase expression (Miller Units) from the above <i>lacZ</i> fusion plasmids was measured in the M18 strain. The <i>pltLp</i> expression gradually and significantly decreased, even entirely inhibited, with the opening of the <i>lys</i> box formed stem loop (Fig. 4A) by base substitutions. (<b>C</b>) The binding of PltR to <i>pltLp</i> was significantly weakened and even eliminated by the <i>lys</i> box mutations. <i>pltLp</i> and its derivative mutated fragments (0.5 nM) were respectively incubated with increasing amounts of PltR protein. The concentration of PltR was 0, 10, and 60 μM, respectively.</p

PltR transcriptionally activated <i>pltLABCDEFG</i> operon expression <i>via</i> direct binding to the <i>pltLp</i> region from −84 bp to +1.

<p>(<b>A</b>) Transcriptional activation of <i>pltLp</i> by PltR. The β-galactosidase expression (Miller Units) from <i>pltLp–lacZ</i> transcriptional fusion (p6522–pltLp) was assayed in M18, M18pltR, M18pltR/pBBR–pltR, and DH5α. The <i>pltLp</i>–<i>lacZ</i> expression was entirely inhibited in the <i>pltR</i> mutant M18pltR, which, in turn, was reversed and even enormously enhanced by the exogenous <i>pltR</i> overexpression from the plasmid pBBR–pltR. (<b>B</b>) Direct binding of PltR to <i>pltLp</i>, assessed by EMSA. 0.5 nM biotin-labelled <i>pltLp</i> promoter fragment (−84 bp to +1) were respectively incubated with increasing amounts of His–PltR protein (0 to 70 μM, Lane 1 to 13). For competition reactions, 0.5 μM unlabeled <i>pltLp</i> fragment was included in the binding reaction in molar as shown in Lane 14. The Hfq protein was used as a negative control (Lane 15).</p

Exogenous Plt specifically induced the <i>pltLp</i> promoter expression and thus enhanced the <i>plt</i> operon transcription.

<p>(<b>A</b> and <b>B</b>) β-galactosidase expression (Miller Units) from p6522–pltLp and p6522–L6-1, which respectively carry <i>pltLp</i> (−84 bp to +1) and its upstream P_x promoter (+66 bp to +124 bp), was assayed in the KMB without or with 10 μg mL⁻¹ exogenous Plt. The addition of 10 μg ml⁻¹ exogenous Plt significantly enhanced the <i>pltLp–lacZ</i> expression (A), but did not influence <i>P_X–lacZ</i> fusion expression (B) in the Plt-defective mutant M18pltB. (<b>C</b>) qRT-PCR analysis of the <i>pltA</i> transcript level in the strain M18pltB grown in the KMB without or with 10 μg ml⁻¹ exogenous Plt. The <i>pltA</i> transcript level was significantly enhanced by the addition of exogenous Plt.</p

The 22 bp lys box (−74 to −53 bp) neighboring upstream of pltLp was indispensable to the transcriptional activation of PltR on the plt operon and the direct binding of PltR to pltLp.

<p>(<b>A</b>) The putative DNA secondary structure (by MEME) of the pltLp region (−84 bp to +1). (<b>B</b>) The <i>pltLp</i> fragment (<i>pltLp</i>, −84 bp to +1) and two truncated fragments with a half or entire deletion of the palindrome motif (<i>pltLp-2</i> and <i>pltLp-3</i>) were respectively fused with the <i>lacZ</i> reporter gene in pME6522. The palindrome <i>lys</i> box is shown with two thick arrows. (<b>C</b>) β-Galactosidase expression (Miller Units) from three <i>lacZ</i> fusion plasmids (p6522–pltLp, pltLp-2, pltLp-3) was assayed in the M18 strain. A half or entire deletion of the palindrome motif resulted in complete inhibition of the <i>pltLp</i>–<i>lacZ</i> expression in the M18 strain. (<b>D</b>) Direct binding of PltR to <i>pltLp</i> was eliminated by a half or entire deletion in the palindrome <i>lys</i> box. 0.5 nM <i>pltLp</i> and two truncated fragments (<i>pltLp-2</i> and <i>pltLp-3</i>) were respectively incubated with increasing amounts of His–PltR protein.</p

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.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Mapping the TSSs and promoters of <i>pltR</i> and <i>pltL</i> genes through 5′RACE and the above <i>lacZ</i> reporter analysis (Fig. 1).

<p>P_R, the <i>pltR</i> promoter; P_L, the <i>pltLp</i> which needs to be specifically activated by PltR; P_X, a non-PltR controlled promoter closely neighboring the upstream of P_L; P_Y, another potential promoter silent in <i>P. aeruginosa</i> M18, but active in <i>E. coli</i> DH5α. The TSS (+1) of the <i>pltR</i> transcript was located at 63 bp upstream of the <i>pltR</i> translational start codon ATG, which is identical to the predicted +1 site. The TSS of the <i>plt</i> operon was located at 46 bp upstream of the <i>pltL</i> ATG. Closely linked to the <i>pltLp</i> with the P_x promoter, a palindromic <i>lys</i> box, including two 9 bp inverted complementary sequences separated by 4 bases, is boxed and highlighted by two head-to-head arrows.</p

Proposed model for the transcriptional activation of PltR and autoinduction of pyoluteorin on Plt biosynthetic operon in <i>P. aeruginosa</i> M18.

<p>(<b>A</b>)<b> </b> Thick arrows indicate activation, autoinduction, and positive regulation; thin arrows indicate biogenesis. P_L and P_R denote the promoters of <i>pltL</i> and <i>pltR</i>. P_X and P_Y are two uncharacterized promoters. The TSS is shown with +1. The LysR-type regulator PltR transcriptionally activates the <i>plt</i> operon expression by directly binding to the <i>pltLp</i> region at the indispensable palindromic <i>lys</i> box. PltR also functions as a candidate receptor of Plt. Plt, as a potential and nonessential cofactor of PltR, specifically induces the expression of the <i>pltL</i> promoter and thus the <i>plt</i> operon. (<b>B</b>) <b>The PltR protein comprise two domains:</b> the N-terminal LysR-family HTH (helix-turn-helix) DNA-binding domain and the C-terminal co-inducer binding domain (predicted by Pfam 25.0). aa, amino acids.</p

5′-UTR sequence of the two <i>phz</i> gene clusters.

<p>The 5′-untranslated nucleotide sequences between the transcriptional start site (TSS) and the translation start codon (ATG) in the <i>phzA1</i> (A) and <i>phzA2</i> (B) gene. B_1–7 and S_1–4 (GGA motifs): potential RsmA binding sites.</p

Functional analysis of the putative RsmA-binding region and its flanking sequences of <i>phz2</i> transcripts.

<p>(A) Construction of a WT <i>phzA2′-‘lacZ</i> translational fusion and two mutant fusions for pMP2L-M1 and pMP2L-M2 in which the RsmA-binding site (RABS) and both RABS along with its flanking sequences for the <i>phz2</i> leader region were replaced, respectively. (B) β-gal activities of the three fusions were determined in strain M18 and mutant M18ΔRA in PPM broth at 28°C. Symbols: pMP2L, translational fusion of WT <i>phz2</i> cluster; pMP2L-M1 fusion, containing a substitution of the RsmA binding site; pMP2L-M2 fusion, containing a substitution of the paired-base region. Values are the means ± standard deviations of triplicate cultures.</p