Quorum sensing in Pseudomonas aeruginosa mediated by RhlR is regulated by a small RNA PhrD

Abstract

Pseudomonas aeruginosa is a highly invasive human pathogen in spite of the absence of classical host specific virulence factors. Virulence factors regulated by quorum sensing (QS) in P. aeruginosa cause acute infections to shift to chronic diseases. Several small regulatory RNAs (sRNAs) mediate fine-tuning of bacterial responses to environmental signals and regulate quorum sensing. In this study, we show that the quorum sensing regulator RhlR is positively influenced upon over expression of the Hfq dependent small RNA PhrD in Pseudomonas. RhlR transcripts starting from two of the four different promoters have same sequence predicted to base pair with PhrD. Over expression of PhrD increased RhlR transcript levels and production of the biosurfactant rhamnolipid and the redox active pyocyanin pigment. A rhlR::lacZ translational fusion from one of the four promoters showed 2.5-fold higher expression and, a 9-fold increase in overall rhlR transcription was seen in the wild type when compared to the isogenic phrD disruption mutant. Expression, in an E. coli host background, of a rhlR::lacZ fusion in comparison to a construct that harboured a scrambled interaction region resulted in a 10-fold increase under phrD over expression. The interaction of RhlR-5'UTR with PhrD in E. coli indicated that this regulation could function without the involvement of any Pseudomonas specific proteins. Overall, this study demonstrates that PhrD has a positive effect on RhlR and its associated physiology in P. aeruginosa.

Figure 1

Quorum sensing (QS) in P. aeruginosa. A pictorial representation of the interconnected las, rhl, pqs and iqs systems and the virulence genes regulated by them. Concept courtesy.

Figure 2

In silico analysis of PhrD. (a) Secondary structure of PhrD predicted by mfold. (b) RNA-RNA interaction between PhrD and RhlR, predicted using IntaRNA program. The arrow in the beginning of interaction region indicates the transcription start site of the P3 promoter of rhlR. The numbers on PhrD and RhlR are in reference to the nucleotides from the transcription start and the translational start respectively (c) Scrambled sequence of RhlR-PhrD interaction region incorporated in the mutant RhlR construct, B-pME6013.

Figure 3

Schematic representation of promoter region of rhlR gene. The upstream region of rhlR gene comprises of four promoters, P1 to P4 that are indicated by boxes. The Shine Dalgarno (SD sequence) of RhlR is indicated by a dotted box and the first 33 codons of RhlR are in bold. The PhrD interaction region is underlined by a solid line. The region comprising the P1 and P2 promoters is excluded from the translational fusion and is shown by a dotted underline. A 141 bp region comprising the P3 promoter and the interaction region was amplified by PCR and fused to the 139 bp region comprising the SD sequence and the first 33 codons to get the rhlR::lacZ translational fusion in the constructs A-pME6013, and B-pME6013 (with scrambled interaction sequence).

Figure 4

Expression analysis of phrD in P. aeruginosa strain PAO1. (a) Northern blots of PhrD in over expression and disruption. Lane 1-pHERDphrD: over expression; lane 2-pHERD30T: vector control; lane 3-phrDΩGm: disruption strain. Ethidium bromide stained 23S and 16S rRNA bands on agarose gels are shown as loading controls. (b) Time course qRT-PCR of phrD from the WT in LB, normalized with 16S rRNA and 0 h expression as the calibrator. (c) phrD expression in the WT in LB, phosphate deficient PPGAS and nitrogen limited MMP media. 5S rRNA band on agarose gel is shown as the loading control. The gel has loading differences with the RNA in the PPGAS lane being poorly loaded. The two lanes of 5S rRNA used as loading control for PPGAS medium are cropped from a different gel and merged with the existing gel. See original gels in Supplementary Fig. S3. (d) Comparative PhrD levels in WT under nitrogen limited MMP and phosphate limited PPGAS media compared to that in Luria broth.

Figure 5

Effect of PhrD on rhlR expression. qRT-PCR analysis of RhlR transcripts from cells grown in Luria broth. Two different vectors (pHERD30T, pUCP18) bearing phrD were separately used for measurement of RhlR in WT background (a), and in complemented disruption (b). (a) *Indicates statistically significant data between the strains determined by using Paired t test. (b) *Indicates statistically significant data between the strains determined by using one way ANOVA (**P < 0.01). The data is represented as the mean ± SD of three individual experiments and each sample was analyzed in triplicates.

Figure 6

PhrD positively regulates rhlR expression in P. aeruginosa PAO1. Time course measurement of RhlR levels by β-galactosidase activity of P3 rhlR::lacZ fusion (i), and qRT-PCR (ii), measured in WT and phrD disruption mutant under: (a) Phosphate limited PPGAS medium (b) Luria broth; (c) N-limited MMP medium. In case of MMP, the cells were first grown to the desired OD in N-rich medium and resuspended in N-limited MMP. There was no net growth owing to nitrogen starvation. β-galactosidase activity of the disruption strain complemented with phrD over expression plasmid is measured in Luria broth. The data is represented as the mean ± SD of three individual experiments and each sample was analyzed in triplicates.

Figure 7

PhrD regulates rhlR in a heterologous system. β-galactosidase activities from rhlR::lacZ fusion plasmids (continuous lines, filled symbols) and growth (dotted lines, open symbols) in E. coli cultures growing in LB at different time points. A-pME6013- fusion bearing intact PhrD interaction region; B-pME6013- fusion with scrambled interaction region; pHERDphrD- phrD over expression plasmid. The results are representative of three individual experiments.

Figure 8

PhrD positively influences rhamnolipid production (a) Spectrophotometric assay for rhamnolipid measurement from culture supernatants by methylene blue method. Increase in rhamnolipid production is represented as increase in the absorbance at 638 nm normalized to absorbance at 600 nm of the culture. (b,c) RhlR and PhrD levels respectively. All these measurements were carried out in M9 medium. **Indicates statistically significant data at P < 0.01 as analyzed by one way ANOVA. Each experiment was performed thrice.

Figure 9

Pyocyanin production under altered levels of PhrD. Pyocyanin levels in LB were measured as µg/ml/OD₆₀₀ of the culture. **and *indicate statistically significant data at P < 0.01 and P < 0.05, as analyzed by one way ANOVA performed on data obtained from three independent experiments.