A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa - PubMed (original) (raw)

A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa

Sherry L Kuchma et al. J Bacteriol. 2005 Feb.

Abstract

Biofilms are structured communities found associated with a wide range of surfaces. Here we report the identification of a three-component regulatory system required for biofilm maturation by Pseudomonas aeruginosa strain PA14. A transposon mutation that altered biofilm formation in a 96-well dish assay originally defined this locus, which is comprised of genes for a putative sensor histidine kinase and two response regulators and has been designated sadARS. Nonpolar mutations in any of the sadARS genes result in biofilms with an altered mature structure but do not confer defects in growth or early biofilm formation, swimming, or twitching motility. After 2 days of growth under flowing conditions, biofilms formed by the mutants are indistinguishable from those formed by the wild-type (WT) strain. However, by 5 days, mutant biofilms appear to be more homogeneous than the WT in that they fail to form large and distinct macrocolonies and show a drastic reduction in water channels. We propose that the sadARS three-component system is required for later events in biofilm formation on an abiotic surface. Semiquantitative reverse transcription-PCR analysis showed that there is no detectable change in expression of the sadARS genes when cells are grown in a planktonic culture versus a biofilm, indicating that this locus is not itself induced during or in response to biofilm formation. DNA microarray studies were used to identify downstream targets of the SadARS system. Among the genes regulated by the SadARS system are those required for type III secretion. Mutations in type III secretion genes result in strains with enhanced biofilm formation. We propose a possible mechanism for the role that the SadARS system plays in biofilm formation.

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Figures

FIG. 1.

Microtiter dish phenotypes of WT strain PA14 and isogenic mutant strains. (A) Biofilm formation phenotypes of the WT and sad_-160::Tn_5 mutant strains at 8 h in the microtiter dish assay. Cells were grown for 8 h in minimal M63 medium supplemented with MgSO4, glucose, and CAA. The arrowhead indicates crystal violet staining of the biofilm at the ALI. The bracket indicates the increased CV staining below the ALI in the Tn_5_ mutant. (B) Biofilm formation by the WT, the sad_-160::Tn_5 transposon mutant, and the Δ_sadRS_::Gmr mutant strain observed at 4 and 8 h. In panels A and B, the microtiter wells are inverted. (C) Quantification of the biofilm formed by the WT and the sad_-160::Tn_5 and Δ_sadRS_::Gmr mutants in microtiter dishes. Biofilms were quantified at 4, 8, and 24 h by solubilization in 30% glacial acetic acid, and the _A_550 of the resulting solution was measured. See the Materials and Methods for experimental details. Error bars indicate standard deviations.

FIG. 2.

The sadARS locus. (A) The top of the panel shows the genomic organization of the sadARS region. The inverted triangle labeled Tn_5_ indicates the insertion site of the original sad_-160::Tn_5 mutant in the sadR_-sadA intergenic region. The arrow within the Tn_5 transposon indicates the direction of transcription of a putative promoter associated with the Tn_5_ insertion (see text for details). Each open reading frame is labeled with the annotation number (PA3946 to PA3948) from the P. aeruginosa genome project (

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) and the corresponding sad gene designation. (B) Predicted structures of the SHK (SadS) and RRs (SadR and SadA). Abbreviations for the SHK are as follows: PBP, periplasmic binding protein domain; TM, transmembrane domain; PAS, PAS domain. The kinase, receiver (R), and HPT domains comprise the catalytic functions required for autophosphorylation and phosphotransfer activities. RR abbreviations: EAL, EAL family; R-Che, CheY-like phospho-receiver domain; HTH, HTH DNA binding domain.

FIG. 3.

Biofilm formation under continuous flow. Biofilm formation at day 2 (A) and day 5 (B) is shown for modified EPRI-grown bacteria in a flow cell. (A) Top-down phase-contrast micrographs at a magnification of ×1,400 are shown for the WT and three representative mutants. (B) Top-down fluorescence images of 5-day-old biofilms at a magnification of ×630. (C) CSLM images of 5-day old biofilms, at a magnification of ×600, of the WT and the ΔsadRS::Gmr mutant, showing the xy and xz planes. Flow cell experiments were performed as described in Materials and Methods.

FIG. 4.

Expression of the sadARS locus. (A) Semiquantitative RT-PCR analysis of sadR expression in planktonically grown cells of WT strain PA14 and various isogenic sadARS mutants. (B) Relative expression levels of the sadR, sadA, and sadS genes in the WT and various sadARS mutants in planktonic cultures analyzed as for panel A. Expression levels are measured in fluorescence units (emitted at 537 nm), using Image Quant software and normalized to the rplU gene. (C) Semiquantitative RT-PCR analysis of sadR, sadA, and sadS gene expression in planktonic (P)- versus biofilm (B)-grown populations of the WT and the sad_-160::Tn_5 mutant. (D) Expression levels from panel C, measured in fluorescence units (as described above) and normalized to the rplU gene. Error bars indicate standard deviations.

FIG. 5.

Characterization of TTSS gene expression and TTSS mutant biofilm formation. (A) QRT-PCR analysis of TTSS gene expression of WT strain PA14 and isogenic sadARS mutants. Relative expression levels of PA1706 (pcrV), PA1707 (pcrH), and PA1708 (popB) in the WT (grey bars), sad_-160::Tn_5 (white bars), Δ_sadRS_ (black bars), Δ_sadR_ (hatched bars), and Δ_sadA_ (stippled bars) strains are shown. Expression levels were quantified as picograms of input cDNA and normalized to rplU levels. Relative expression levels are plotted, with WT levels set equal to 1. (B) Quantification of the biofilm formed by WT PAO1 and the pcrV::IS_phoA_/hah, pcrH::IS_phoA_/hah, and popB::IS_phoA_/hah mutants in microtiter dishes. Biofilms were quantified at 6 h after inoculation into minimal glucose medium plus MgSO4 and CAA. Error bars indicate standard deviations. (C) Biofilm formation at the ALI in 24-well flat-bottomed plates. Top-down phase-contrast micrographs at a magnification of ×400 are shown for the WT PAO1 strain and two representative TTSS mutants. Bar, 35 μm. The lower panels are centered at 140 μm below the ALI, as indicated. Strains were grown in minimal glucose medium plus MgSO4 and CAA for 6 h. ALI assays were performed as described in Materials and Methods.

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A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa - PubMed (original) (raw)