The putative enoyl-coenzyme A hydratase DspI is required for production of the Pseudomonas aeruginosa biofilm dispersion autoinducer cis-2-decenoic acid

Abstract

In the present study, we report the identification of a putative enoyl-coenzyme A (CoA) hydratase/isomerase that is required for synthesis of the biofilm dispersion autoinducer cis-2-decenoic acid in the human pathogen Pseudomonas aeruginosa. The protein is encoded by PA14_54640 (PA0745), named dspI for dispersion inducer. The gene sequence for this protein shows significant homology to RpfF in Xanthomonas campestris. Inactivation of dspI was shown to abolish biofilm dispersion autoinduction in continuous cultures of P. aeruginosa and resulted in biofilms that were significantly greater in thickness and biomass than those of the parental wild-type strain. Dispersion was shown to be inducible in dspI mutants by the exogenous addition of synthetic cis-2-decenoic acid or by complementation of ΔdspI in trans under the control of an arabinose-inducible promoter. Mutation of dspI was also shown to abolish cis-2-decenoic acid production, as revealed by gas chromatography-mass spectrometry (GC-MS) analysis of cell-free spent culture medium. The transcript abundance of dspI correlated with cell density, as determined by quantitative reverse transcriptase (RT) PCR. This regulation is consistent with the characterization of cis-2-decenoic acid as a cell-to-cell communication molecule that regulates biofilm dispersion in a cell density-dependent manner.

Fig 1

Microcolonies of P. aeruginosa PA14 biofilms grown in 24-well cell culture plates demonstrating the native dispersion response. (A) Transmitted-light images showing the presence and absence of interior voids formed within microcolonies of wild-type PA14 and 8 putative enoyl-CoA hydratase mutants. Biofilms of the dspI (PA14_54640) mutant showed no evidence of void formation. All images are shown at the same relative size at ×200 magnification. Scale bars, 50 μm. (B) Quantification of microcolony voids formed as a percentage of the total number of microcolonies observed for biofilms of PA14 and ΔdspI strains.

Fig 2

dspI is required for native biofilm dispersion. Transmitted-light images (A and B) and confocal laser scanning microscopy images (C) at a magnification of ×500 of P. aeruginosa wild-type and dspI mutant biofilms. The photomicrographs show microcolonies of biofilms grown in modified EPRI medium (A) or 5% LB medium (B and C) for 6 days, with continuous dspI induction in the complemented dspI mutant strain. Microcolonies of the dspI mutant remained solid, whereas wild-type and complemented mutant biofilms showed dispersion. Experiments were completed in triplicate. Scale bars, 50 μm.

Fig 3

dspI mutant biofilms disperse in the presence of exogenous cis-DA. (A) Biofilms of wild-type PA14, dspI mutants, or complemented dspI mutants were grown in continuous-culture tube reactors for 6 days and switched to fresh medium or cis-DA for 2 h under static conditions. The numbers of released cells in the bulk liquid of each tube and of the remaining biofilm cells in each tube were determined by viable count (CFU). Percent dispersion was calculated as a function of released cells (CFU) divided by the total number of CFU from each tube (released cells plus remaining biofilm cells). Error bars indicate one standard deviation. (B) CLSM images of mature dspI mutant biofilm microcolonies grown in continuous culture in a microscope-mounted flow cell before and after the addition of cis-DA. Microcolony disaggregation is shown following treatment under static conditions for 1 h. Control biofilms treated with fresh medium showed no disaggregation (not shown). The images are the same relative size at ×500 magnification. Scale bars, 50 μm. Experiments were completed in triplicate. *, values significantly different from the respective negative control (P < 0.01).

Fig 4

dspI is required for synthesis of cis-2-decenoic acid in P. aeruginosa. (A) DspI contains a conserved domain (gray) belonging to the crotonase/enoyl-CoA hydratase family, which includes many diverse enzymes involved in fatty acid metabolism. (B) The predicted enzymatic reaction performed by the enoyl-CoA hydratase dspI includes the formation of a double bond at the β-carbon of small fatty acids. (C) Spectral analysis of synthetic cis-DA and CSM prepared from the P. aeruginosa PA14 wild type and mutants with dspI inactivated or complemented was performed using gas chromatography-mass spectrometry. The y axes indicate intensity; the x axes indicate time in minutes. (D) MS-MS fragmentation patterns of the 7.0-min peak from the GC-MS spectra of cis-DA, PA14 CSM, and CSM of the complemented dspI mutant. The y axes indicate intensity; the x axes indicate m/z.

Fig 5

Expression and transcript abundance levels of dspI in planktonic and biofilm cells. (A) Growth curve of P. aeruginosa PA14 in LB medium. The curve represents the average of 3 replicates. The error bars indicate standard deviations. (B) Fold change in dspI mRNA levels in P. aeruginosa planktonic and biofilm cells compared to lag phase planktonic cells. Experiments were performed in triplicate. (C) Transcriptional reporter fusion assay for dspI expression in P. aeruginosa wild-type 6.5-h-, 10-h-, 12.5-h-, 15-h-, and 24-h-old planktonic cells and 1-day-, 3-day-, and 5-day-old biofilm cells. The values indicated by asterisks differ significantly from the values of the preceding growth phase (P < 0.05).

Fig 6

Microscopic analysis of dspI transcriptional-reporter activity during planktonic and biofilm growth. P. aeruginosa PA14 harboring a dspI-lacZ reporter construct was grown in batch or continuous culture in medium supplemented with MUG. The indicated planktonic (A to E) and biofilm (F to J) conditions are shown (bright-field images [left] and fluorescent cells displaying β-galactosidase activity [right]). Scale bars, 20 μm.

Fig 7

Multiple-sequence alignment of DspI, RpfF homologs, and rat mitochondrial enoyl-CoA hydratase. The sequences were obtained from the NCBI (http://www.ncbi.nlm.nih.gov/) and were aligned using ClustalW. Fully conserved (*), strongly conserved (:), and weakly conserved (.) amino acid residues are indicated. (A) The 29 amino acid residues of the predicted ligand binding site for RpfF in X. oryzae pv. oryzae are boxed (47). DspI contains 15 out of 29 conserved amino acid residues of the predicted DSF ligand binding site of RpfF. (B) Conserved glutamate residues at the enoyl-CoA active site of rat mitochondrial enoyl-CoA hydratase, Glu¹⁴⁴ and Glu¹⁶⁴, align with Glu¹²⁶ and Glu¹⁴⁶ of DspI (shaded in red and indicated by triangles).