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

<i>pvcA-D</i> gene cluster constitutes an operon in MPAO1.

<p>Confirmation that the <i>pvcA-D</i> gene cluster constitutes an operon was accomplished through a series of RT-PCR experiments using primers that correspond to regions within adjacent genes. <b>A.</b> Order and direction of transcription of <i>pvcABCD</i> on the MPAO1 chromosome is indicated by block arrows. The intergenic region between each pair of genes is shown above and the location of the primers for each pair of genes is marked by the inward-facing arrows below: (1) <i>pvcC-pvcD,</i> solid; (2) <i>pvcB-pvcC</i>, dashed; (3) <i>pvcA-pvcB</i>, dotted. Sizes of the expected products are indicated below the primer locations. <b>B.</b> RNA from MPAO1 grown in LB broth for 16 h was obtained, processed, and reverse transcribed to produce cDNA as described in Materials and Methods. PCR reactions using each pair of primers were run and the products separated on a 1% (w/v) agarose gel and stained with ethidium bromide. (1) 324-bp product from <i>pvcC-pvcD</i>; (2) 556-bp product from <i>pvcB-pvcC</i>; (3) 463-bp product from <i>pvcA-pvcB</i>; (NT) no cDNA template control (Std) molecular size standard.</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

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

Bacterial strains and plasmids used in this study.

<p>Cb, carbenicillin;</p><p>Km, kanamycin;</p>r<p>, resistant;</p><p>Tet, tetracycline.</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>pvcA</i> mutation reduces the expression of <i>cupB</i> and <i>cupC</i> gene clusters.

<p>The relative level of expression for each gene in PW4830 (Δ<i>pvcA</i>) compared to MPAO1 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><i>pvcA</i> mutation in MPAO1 significantly reduces the expression of all genes of the <i>cupB</i> gene cluster. Order and direction of transcription of <i>cupB1-6</i> on the MPAO1 chromosome is indicated by block arrows above the graph. <b>B.. </b><i>pvcA</i> mutation in MPAO1 significantly reduces the expression of all <i>cupC</i> genes. Order and direction of transcription of <i>cupC1-3</i> on the MPAO1 chromosome is indicated by block arrows above the graph. Values in A and B represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations ± SEM (n = 3); <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

<i>pvcA</i> mutation does not interfere with the expression of <i>pilA</i> or <i>flgK.</i><i>pvcA</i> mutation does not interfere with the expression of <i>pilA</i> or <i>flgK</i> but reduces <i>cup</i> gene expression.

<p>The relative level of expression for each gene in PW4830 (Δ<i>pvcA</i>) compared to MPAO1 was determined by RT-qPCR as described in Materials and Methods. The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (<i>rplS</i>) as an internal standard. A. Mutation in <i>pvcA</i> does not interfere with the expression of either <i>pilA</i> or <i>flgK</i> but enhanced expressions of <i>fliC</i> in MPAO1. B. Mutation in <i>pvcA</i> affects expression of <i>cupA1, cupB1</i> and <i>cupC1</i> in MPAO1. Values in A and B 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

Diagram of the proposed regulatory circuits through which the <i>pvc</i> operon enhances the expression of <i>cup</i> genes

<p>. (1) PtxR enhances <i>pvc</i> expression and the proteins encoded by the <i>pvc</i> operon synthesize paerucumarin. (2) As a molecular signal, paerucumarin may activate the roc system through histidine kinases RocS1 and RocS2. Alternatively, paerucumarin may activate a potential transcriptional activator which enhances the expression of <i>rocS1-rocA1</i> operon, <i>rocS2,</i> and <i>rocA2</i>. (3) RocS2 and RocS1 then activate RocA1, which in turn enhances <i>cupC1-3</i> expression. (4) Both RocS2 and RocS1 enhance <i>cupB1-6</i> expression by activating an unknown regulator (X) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062735#pone.0062735-Sivaneson1" target="_blank">[31]</a>. Y indicates potential paerucumarin activated transcriptional regulator. Direction of expression is indicated by placement of genes above or below the black lines.</p