The <i>pvc</i> Operon Regulates the Expression of the <i>Pseudomonas aeruginosa</i> Fimbrial Chaperone/Usher Pathway (<i>Cup</i>) Genes

<div><p></p><p>The <i>Pseudomonas aeruginosa</i> fimbrial structures encoded by the <i>cup</i> gene clusters (<i>cupB</i> and <i>cupC</i>) contribute to its attachment to abiotic surfaces and biofilm formation. The <i>P. aeruginosa pvcABCD</i> gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the <i>pvc</i> operon and the <i>cup</i> gene clusters in the <i>P. aeruginosa</i> strain MPAO1. Mutations within the <i>pvc</i> genes compromised biofilm development and significantly reduced the expression of <i>cupB1-6</i> and <i>cupC1-3</i>, as well as different genes of the <i>cupB</i>/<i>cupC</i> two-component regulatory systems, <i>roc1/roc2</i>. Adjacent to <i>pvc</i> is the transcriptional regulator <i>ptxR</i>. A <i>ptxR</i> mutation in MPAO1 significantly reduced the expression of the <i>pvc</i> genes, the <i>cupB/cupC</i> genes, and the <i>roc1/roc2</i> genes. Overexpression of the intact chromosomally-encoded <i>pvc</i> operon by a <i>ptxR</i> plasmid significantly enhanced <i>cupB2</i>, <i>cupC2</i>, <i>rocS1</i>, and <i>rocS2</i> expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of <i>cupB2</i>, <i>cupC2</i>, <i>rocS1</i> and <i>rocS2</i> in the <i>pvcA</i> mutant. Our results suggest that <i>pvc</i> influences <i>P. aeruginosa</i> biofilm development through the <i>cup</i> gene clusters in a pathway that involves paerucumarin, PtxR, and different <i>cup</i> regulators.</p></div

Exogenous paerucumarin enhances <i>cupB2</i> expression in the absence of <i>rocS1</i> and <i>rocS2</i>.

<p>Overnight cultures of PW7672 (Δ<i>rocS1</i>) and PW6105 (Δ<i>rocS2</i>) were subcultured into fresh LB broth to an OD₆₀₀ of 0.02. A 15-µl aliquot of either ethyl acetate (mock) or purified paerucumarin (600 µM) was added at the time of subculturing. Cells were grown at 37°C for 16 h. The levels of <i>cupB2</i> gene expression were compared between mock treated and paerucumarin containing PW7672 and PW6105. The relative level of expression for <i>cupB2</i> in each pair of strains was determined by RT-qPCR. The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (<i>rplS</i>) as an internal standard. Values represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations ± SEM (n = 3); <i>P</i><0.001 (***).</p

Quantification of the effect of <i>pvcA</i> mutation on biofilm formation.

a<p>Strains, both carrying pMRP9-1, were grown for 16 h at 37°C without shaking.</p>b<p>Image stacks were acquired in triplicate at 10X magnification and analyzed using the COMSTAT program; values represent the mean ± SEM.</p>c<p>Biomass of the biofilm.</p>d<p>Spatial size of the biofilm.</p>e<p>Variation in the thickness of the biofilm, or heterogeneity.</p>f<p>Total of the area occupied in each image stack.</p>g<p>Portion of the biofilm exposed to nutrients; biovolume divided by the surface area of the substratum.</p

<i>pvcA</i> regulates <i>cup</i> genes through <i>roc1/roc2</i> systems.

<p>The relative level of expression for each gene in MPAO1 compared to PW4830 or PW4830/pLL4 compared to PW4830/pCR2.1-1.8 (vector control) was determined by RT-qPCR. The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (<i>rplS</i>) as an internal standard. <b>A.</b> The <i>pvcA</i> mutation (PW4830) significantly reduces the expression of <i>rocS1</i>, <i>rocS2</i>, <i>rocA2</i>, and <i>rocR</i>, while expression of <i>rocA1</i> was less affected. <b>B.</b> Plasmid pLL4 carrying intact <i>rocS1</i> constitutively expressed from the <i>lac</i> promoter significantly increases the expression of <i>rocS1</i> and <i>rocA1</i> in PW4830 (Δ<i>pvcA</i>). PW4830 was transformed with pLL4 or pCR2.1-1.8 (vector control). <b>C.</b> Overexpression of <i>rocS1</i> from the <i>lac</i> promoter in pLL4 complements the defect of PW4830 in <i>cupB2</i> and <i>cupC2</i> expression. Values in A–C represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations ± SEM (n = 3); <i>P</i><0.05 (*); <i>P</i><0.01 (**); <i>P</i><0.001 (***).</p

<i>pvc</i> genes affect biofilm development in MPAO1.

<p><b>A.</b> Mutation in <i>pvcA</i> reduces biofilm development in the MPAO1 isogenic mutant PW4830. Strains were transformed with pMRP9-1, which expresses GFP. Overnight cultures were subcultured into tryptone broth as described in Materials and Methods. Bacterial biofilms formed in a ring at the air-liquid interface were washed to remove planktonic cells and the biofilm cells were removed by vortexing in PBS, diluted tenfold, and plated to quantify the viable microorganisms within the biomass (CFU ml⁻¹). <b>B and C.</b> Representative photomicrographs of the biofilms formed by (<b>B</b>) MPAO1 and (<b>C</b>) PW4830 (Δ<i>pvcA</i>) visualized with CLSM at 40X magnification. Z slices of 0.5 µm were generated; the <i>zy</i> and <i>zx</i> planes of the Z images are shown to the left and below the flat field, respectively. Bars equal 50 nm. <b>D–G.</b> Biofilms were developed and CFU assayed as described in A. <b>D.</b> Plasmid pLL1 carrying intact <i>pvcA</i> constitutively expressed from the <i>lac</i> promoter complements the defect of PW4830 in biofilm formation. Strains were transformed with pLL1 or pCR2.1-1.8 (vector control). <b>E.</b> Mutation in <i>pvcB</i> also reduces biofilm development in the MPAO1 isogenic mutant PW4832. <b>F.</b> Plasmid pLL2 carrying intact <i>pvcB</i> constitutively expressed from the <i>lac</i> promoter complements the defect of PW4832 (Δ<i>pvcB</i>) in biofilm formation. Strains were transformed with pLL1 or pCR2.1-1.8 (vector control). <b>G.</b> Mutation in <i>pvcC-D</i> reduces biofilm development in the MPAO1 isogenic mutant MPAO1Δ<i>pvcC-D</i>. Values in A and D-G represent the average of three independent experiments ± standard error of the mean (SEM). Statistical significance in viable biomass between the strains was calculated by Student’s unpaired <i>t-</i>test. <i>P</i><0.05 (*); <i>P</i><0.01 (**).</p