Regulatory Feedback Loop of Two phz Gene Clusters through 5′-Untranslated Regions in Pseudomonas sp. M18

BACKGROUND: Phenazines are important compounds produced by pseudomonads and other bacteria. Two phz gene clusters called phzA1-G1 and phzA2-G2, respectively, were found in the genome of Pseudomonas sp. M18, an effective biocontrol agent, which is highly homologous to the opportunistic human pathogen P. aeruginosa PAO1, however little is known about the correlation between the expressions of two phz gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: Two chromosomal insertion inactivated mutants for the two gene clusters were constructed respectively and the correlation between the expressions of two phz gene clusters was investigated in strain M18. Phenazine-1-carboxylic acid (PCA) molecules produced from phzA2-G2 gene cluster are able to auto-regulate expression itself and activate the expression of phzA1-G1 gene cluster in a circulated amplification pattern. However, the post-transcriptional expression of phzA1-G1 transcript was blocked principally through 5'-untranslated region (UTR). In contrast, the phzA2-G2 gene cluster was transcribed to a lesser extent and translated efficiently and was negatively regulated by the GacA signal transduction pathway, mainly at a post-transcriptional level. CONCLUSIONS/SIGNIFICANCE: A single molecule, PCA, produced in different quantities by the two phz gene clusters acted as the functional mediator and the two phz gene clusters developed a specific regulatory mechanism which acts through 5'-UTR to transfer a single, but complex bacterial signaling event in Pseudomonas sp. strain M18

Bacterial strains and plasmids used in this study.

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

The PCA production was negatively regulated by GacA via <i>phzA2-G2</i> cluster mainly at post-transcriptional level.

<p>PCA production and cell growth curves in single <i>gacA</i> inactivated mutant M18ΔG (square), double <i>gacA</i> and <i>phzA1-G1</i> mutant M18ΔGΔP1 (triangle) or double <i>gacA</i> and <i>phzA2-G2</i> mutant M18ΔGΔP2 (diamond) (A). The cell growth and expression of transcriptional fusion pMP2C (B) or translational fusion pMP2L (C) in wild type strain M18 (circle) and mutant M18ΔG (square). Symbols: solid, growth curves; empty, PCA production or β-Galactosidase activities. All experiments were performed in triplicate, and each value is presented as the average ± standard deviation.</p

Secondary structures in the 5′-UTRs of two <i>phz</i> gene clusters were predicted by RNA fold.

<p>The predicted secondary structures in 5′-UTR of <i>phzA1-G1</i> (1–337 nt) and <i>phzA2-G2</i> (1–198 nt) gene clusters (A). The conserved RNA secondary structures were predicted from the alignment of <i>Pseudomonas</i> sp. M18 and <i>P. aeruginosa</i> PA7. The base pairing probabilities were annotated with colors. Three suboptimal secondary structures were also predicted for a portion of the 5′-UTR of the <i>phzA1-G1</i> gene cluster in <i>Pseudomonas</i> sp. M18 (B). Gibbs free energies (ΔG) of the three suboptimal local structures from left to right are −39.3 kcal/mol, −38.1 kcal/mol and −36.7 kcal/mol, respectively.</p

Integrative relationship of the Gac/Rsm signal transduction pathway and the expressions of two <i>phz</i> clusters.

<p>Diagrammatic representation of the integrative relationship of the Gac/Rsm signal transduction pathway and the expressions of two <i>phz</i> clusters in <i>Pseudomonas</i> sp. M18. Less efficiency of expression of <i>phzA2-G2</i> gene cluster produce a small amount of PCA signal molecule to auto-induce transcriptional activity itself and the expression of <i>phzA1-G1</i> gene cluster (red fine lines). The high efficiency expression of <i>phzA1-G1</i> at transcriptional level were blocked by its 5′-UTR region and could be relieved partial in post-transcriptional level by PCA or some unknown factor(s) (red thick lines). The interactions of induction and self-induced among two <i>phz</i> clusters resulted in a large amount of PCA production as antibiotics for bio-control. The <i>phzA2-G2</i> expression was negatively controlled mainly at the post-transcriptional level by regulator GacA in respond to environmental signals at overall level. Symbols: solid circle, inhibition; solid arrow, activation; diamond, environmental signals; X, unknown factor(s).</p

The physical map, growth and PCA production of strain M18 and its two <i>phz</i> mutants.

<p>The two inactivated <i>phz</i> gene clusters with their flanking genes in <i>Pseudomonas</i> sp. M18 (A) and the growth curves (B) and PCA production (C) in wild type strain and the two mutants M18ΔP1 and M18ΔP2 with or without exogenous PCA molecules. Symbols in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019413#pone-0019413-g001" target="_blank">figure 1A</a>: red solid arrows denote the two <i>phz</i> core gene clusters; empty arrows, the <i>phz</i> flanking genes; fine line, Gm, genamycin resistence gene; +1, indicates the transcriptional start site (TSS) and the numbers indicate the relative length from TSS; black lines, intergenic regions between <i>phz</i> gene clusters and flanking genes; black solid lines denote the 88 bp sequence homologous to that in <i>P. aeruginosa</i> PAO1; grey solid line denotes additional 56 bp sequence in strain M18. Symbols in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019413#pone-0019413-g001" target="_blank">figure 1B and 1C</a>: square, wild type strain M18; triangle, mutant M18ΔP1; circle, mutant M18ΔP2; red solid and blue empty denote presence or absence of exogenous PCA in culture, respectively. All experiments were performed in triplicate, and each value is presented as the average ± standard deviation.</p

Effects of exogenous PCA on the transcription of two <i>phz</i> gene clusters in <i>Pseudomonas</i> sp. M18 and its two mutants M18ΔP1 and M18ΔP2.

a<p>Values were measured during exponential phase at an OD₆₀₀ of 2.0 to 2.5. CT, cycle threshold.</p>b<p>Values were measured at stationary phase at OD₆₀₀ of 3.5 to 4.0.</p>c<p>ΔCT = CT<i>phz</i>−CT<i>rpod</i>, CT<i>phz</i> denotes cycle threshold of <i>phzC</i>, <i>phzA2</i> and <i>phzA1</i> transcripts in strain M18, M18ΔP1 and M18ΔP2, respectively.</p>d<p>Z1 or Z2 = 2^{−Δ(ΔCT<i>phz</i>C-ΔCT<i>phzA2</i>)} (in M18); 2^{−Δ(ΔCT<i>phzA1</i>-ΔCT<i>phzA2</i>)} (in M18ΔP2); 2^{−Δ(ΔCT<i>phzA1</i>-ΔCT<i>phzA2</i>)} (in M18ΔP1), ΔΔCT = ΔCT_low−ΔCT_high (The relative high ΔCT value was defined as 1).</p>e<p>M, the increased ratio of expression 2^−ΔΔCT without and with the addition of exogenous PCA in culture. ΔΔCT = ΔCT_with–ΔCT_without.</p

Primers designed for this study.

a<p>These oligonucleotides were from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019413#pone.0019413-Yan1" target="_blank">[28]</a>.</p

Activities of various <i>phz1‘-’ lacZ</i> transcriptional fusions during logarithmic phase in strain M18 and its mutants.

<p>The various <i>phz1‘-’lacZ</i> transcriptional fusions in plasmid pME6522 (A) and their β-Galactosidase activities (B) in wild type strain <i>Pseudomonas</i> sp. M18 (black), <i>phzA1-G1</i> inactivated mutant M18ΔP1 (red) and <i>phzA2-G2</i> inactivated mutant M18ΔP2 (blue). All experiments were performed in triplicate, and each value is presented as the average ± standard deviation.</p