RpoN-Dependent Direct Regulation of Quorum Sensing and the Type VI Secretion System in Pseudomonas aeruginosa PAO1

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, particularly those with cystic fibrosis. As a global regulator, RpoN controls a group of virulence-related factors and quorum-sensing (QS) genes in P. aeruginosa To gain further insights into the direct targets of RpoN in vivo, the present study focused on identifying the direct targets of RpoN regulation in QS and the type VI secretion system (T6SS). We performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) that identified 1,068 binding sites of RpoN, mostly including metabolic genes, a group of genes in QS (lasI, rhlI, and pqsR) and the T6SS (hcpA and hcpB). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA), lux reporter assay, reverse transcription-quantitative PCR, and phenotypic detection. The ΔrpoN::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in the ΔrpoN::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS.IMPORTANCE As a global regulator, RpoN controls a wide range of biological pathways, including virulence in P. aeruginosa PAO1. This work shows that RpoN plays critical and global roles in the regulation of bacterial pathogenicity and fitness. ChIP-seq provided a useful database to characterize additional functions and targets of RpoN in the future. The functional characterization of RpoN-mediated regulation will improve the current understanding of the regulatory network of quorum sensing and virulence in P. aeruginosa and other bacteria.

Keywords: Pseudomonas aeruginosa; T6SS; quorum-sensing system.

FIG 1

ChIP-seq assay revealed in vivo binding sites of RpoN in the P. aeruginosa genome. (A) Pie chart of 1,068 RpoN-binding peaks. (B) Pie chart displaying the percentage of RpoN targets with functional categories defined in the Pseudomonas database (http://pseudomonas.com). (C) Most significant motif derived from ChIP-seq binding sequence returned by the MEME tool. The height of each letter represents the relative frequency of each base at different positions in the consensus sequence.

FIG 2

Functional profiling of the RNA-seq in P. aeruginosa. DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/home.jsp) was used to categorize members with gene transcription reduced in the ΔrpoN::Tc strain compared with the wild type (A) and gene transcription elevated in the ΔrpoN::Tc strain compared with the wild type (B). The enrichment of specific gene classes is displayed. The P values of the enriched categories are provided in Table S4.

FIG 3

RpoN positively regulated the las-QS system by lasI. (A to C) EMSA showed that RpoN directly bound to the rsaL-lasI intergenic region (A) and the promoter of algD (B) but did not bind to the promoter of wzx (C). PCR products containing the lasI, algD, and wzx promoter regions were added to the reaction mixtures at 30 ng for each well. RpoN protein was added to reaction buffer in lanes with 0, 0.5, 1.25, 2.0, and 2.5 μM. (D) The relative expression of lasI was downregulated in the rpoN mutant. The relative gene expression level in the wild-type PAO1 was set to 1, and the other values were adjusted accordingly. (E) Expression of lasI-lux in PAO1 and its derivatives. Bacteria were grown in LB at 37°C for 12 h with shaking (220 rpm), and then the lasI-lux activity was measured. CPS (counts per second) values represent relative promoter-lux activities. All experiments were independently repeated at least three times, and the data shown represent comparable results. Values represent means ± standard errors of the means (SEM). (F) P. aeruginosa PAO1 and its derivatives were grown in LB medium at 37°C for 16 h with shaking (220 rpm); the presence of the blue-green pigment indicates pyocyanin production. (G) Proteolytic activity of three P. aeruginosa strains on milk plates. (H) Effect of RpoN mutation on motility. Overnight cultures were spotted onto swarming plates (2-μl aliquots), and the plates were incubated at 37°C. The images were captured after 20 h of growth. The experiments were repeated at least three times, and similar results were observed. (I and J) The quantitative analysis (diameter of motility locus) of swarming (I) and swimming (J). P < 0.01 (**) and P < 0.001 (***) compared to the wild-type or complemented strain by Student's t test. Results represent means ± standard deviations (SD), and data are representative of at least three independent experiments. EV represents empty vector pAK1900; p-RpoN represents pAK1900-rpoN.

FIG 4

RpoN negatively regulated the rhl-QS system. (A to C) EMSA showed that RpoN directly bound to the rhlI (A), rhlA (B), and rhlR (C) promoter regions. PCR products containing the rhlI, rhlA, and rhlR promoter regions were added to the reaction mixtures at 30 ng in each well. RpoN protein was added to reaction buffer in lanes at 0, 0.5, 1.25, 2.0, and 2.5 μM. (D) The relative expression of rhlA and rhlR upregulated in ΔrpoN::Tc strain. The relative gene expression level in wild-type PAO1 was set to 1, and the other values were adjusted accordingly. (E) Relative amount of C₄-HSL measured by pKD-rhlI plus pMCSG19-rhlR in the DH5α system. E. coli DH5α containing pKD-rhlI and pMCSG19-rhlR plasmids, PAO1, and its derivatives were grown in LB medium at 37°C for 12 h with shaking (220 rpm). The E. coli DH5α culture, PAO1, and its derivative cultures then were mixed at 37°C for 4 h with shaking (220 rpm). The mixed cultures were subsequently measured for their relative C₄-HSL contents. Plasmid pKD-rhlI carries the C₄-HSL-responsive rhlI promoter fused to luxCDABE, so CPS values become an indirect measure of supernatant C₄-HSL. (F) Bacterial strains were inoculated onto cetyltrimethylammonium bromide (CTAB) plates and incubated at 37°C for 24 h and then for 36 h at room temperature. The presence of a halo surrounding the colonies indicates the production of rhamnolipids. P < 0.01 (**) and P < 0.001 (***) compared to the wild-type or complemented strain by Student's t test. Results represent means ± SD, and data are representative of at least three independent experiments.

FIG 5

RpoN positively regulated the pqs-QS system. (A to C) EMSA showed that RpoN directly bound to pqsA (A), pqsH (B), and pqsR (C) promoter regions. PCR products containing the pqsA, pqsH, and pqsR promoter regions were added to the reaction mixtures at 30 ng for each well. RpoN protein was added to reaction buffer in lanes at 0, 0.5, 1.25, 2.0, 2.5 μM. (D) The production of PQS was decreased in the ΔrpoN::Tc strain. Lanes 1 to 3 represent wild-type PAO1, the ΔRpoN strain, and the ΔRpoN complemented strain, respectively. (E) The relative quantitative analysis (percentage of value for grey-shaded bar) of PQS was also compared among three strains. (F to H) Expression of pqsA-lux (F), pqsH-lux (G), and pqsR-lux (H) in PAO1 and its derivatives. Bacteria were grown in LB at 37°C for 12 h with shaking (220 rpm), and then pqsA-lux, pqsH-lux, and pqsR-lux activity was measured. P < 0.05 (*) and P < 0.01 (**) compared to the wild-type or complemented strain by Student's t test. Results represent means ± SD, all experiments were independently repeated at least three times, and the data shown represent comparable results. EV, empty vector.

FIG 6

RpoN positively regulated hcpA and hcpB in T6SS. (A and B) EMSA showed that RpoN directly bound to hcpA (A) and hcpB (B) promoter regions. PCR products containing the hcpA and hcpB promoter regions were added to the reaction mixtures at 30 ng for each well. RpoN protein was added to reaction buffer in lanes at 0, 0.5, 1.25, 2.0, and 2.5 μM. (C) The relative expression of hcpA and hcpB downregulated in the RpoN mutant. The relative gene expression level in the wild-type PAO1 was set to 1, and the other values were adjusted accordingly. (D and E) Expression of hcpA-lux (D) and hcpB-lux (E) in PAO1 and its derivatives. Bacteria were grown in LB 37°C for 12 h with shaking (220 rpm), and then the promoter-lux activity was measured. (F) The expression of Hcp1 was downregulated in the ΔrpoN::Tc strain. The relative Hcp1-Flag expression level in wild-type PAO1 (OD₆₀₀) was set to 1, and the other values were adjusted accordingly. P < 0.01 (**) and P < 0.001 (***) compared to the wild-type or complemented strain by Student's t test. Results represent means ± SD, all experiments were independently repeated at least three times, and the data shown represent comparable results. EV, empty vector.

FIG 7

RpoN affected biofilm formation. (A) Quantification of CV staining of biofilm grown in borosilicate tubes at 24 h after standing incubation at 30°C. Photos of the tubes from the binding assay were taken. (B) The deletion of rpoN changed the production of exopolysaccharides (EPS) in P. aeruginosa. Different levels of red of colony morphology on the Congo red plate represent the relative amounts of EPS. Results represent means ± SEM, and data are representative of three independent experiments.

FIG 8

Schematic of the proposed RpoN regulatory mechanism on QS, T6SS, and major metabolic pathways. The potential regulatory pathways and interplays of RpoN are proposed according to our observations and previous studies. RpoN affects the expression of nitrogen-regulated genes (glnK-amtB, nirBD, nasA, nasST, and nosRZDFYL) (16) and regulates dicarboxylates transport, amino acid catabolism, ethanolamine catabolism, (R)-3-hydroxybutyrate, methylarginines, and acetoin catabolism (24–29). The typical QS systems, including the las, rhl, and pqs systems and their interactions, were summarized based on previous reports (8). In the present study, we showed that RpoN directly bound to the lasI and pqsR promoter regions and positively regulated the expression of las and pqs QS systems, while it negatively regulated the rhl QS system and biofilm formation. Moreover, RpoN directly bound to the hcpA and hcpB promoter regions. In addition, RpoN regulated the major metabolic pathways in P. aeruginosa.