Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development

Abstract

An analysis of the Pseudomonas aeruginosa genomic sequence revealed three gene clusters, PA1381-1393, PA2231-2240, and PA3552-3558, in addition to the alginate biosynthesis gene cluster, which appeared to encode functions for exopolysaccharide (EPS) biosynthesis. Recent evidence indicates that alginate is not a significant component of the extracellular matrix in biofilms of the sequenced P. aeruginosa strain PAO1. We hypothesized that at least one of the three potential EPS gene clusters revealed by genomic sequencing is an important component of P. aeruginosa PAO1 biofilms. Thus, we constructed mutants with chromosomal insertions in PA1383, PA2231, and PA3552. The mutant with a PA2231 defect formed thin unstructured abnormal biofilms. The PA3552 mutant formed structured biofilms that appeared different from those formed by the parent, and the PA1383 mutant formed structured biofilms that were indistinguishable from those formed by the parent. Consistent with a previous report, we found that polysaccharides were one component of the extracellular matrix, which also contained DNA. We suggest that the genes that were inactivated in our PA2231 mutant are required for the production of an EPS, which, although it may be a minor constituent of the matrix, is critical for the formation of P. aeruginosa PAO1 biofilms.

FIG. 1.

Growth of the parent and mutant strains in static biofilm cultures in microtiter dishes (A) and in nonbiofilm broth cultures (B). (A) Bars indicate the standard deviations around the means from five independent experiments, with each sample assayed in triplicate. *, P < 0.001 (Mann-Whitney U test). (B) Growth curves for P. aeruginosa PAO1 (open circles), ΔpslAGm (open squares), Δ1383Sm (filled squares), and Δ3552Gm (filled circles).

FIG. 2.

Influence of putative EPS gene mutations on P. aeruginosa biofilm development. The parent and mutants (indicated on the left) were grown in flow chambers, and SCLM images were acquired at the times shown at the top. Horizontal sections are shown for each time, and vertical reconstructions are shown underneath many of the horizontal images. Bars, 50 μm (note that the magnification for images acquired on days 3 to 7 was different from that for images acquired during earlier stages of development).

FIG. 3.

Images from SCLM of a 7-day P. aeruginosa biofilm stained with the double-stranded DNA stain PicoGreen. (A) Reconstructed three-dimensional image of a mushroom-like structure. Bar, 50 μm. (B) Horizontal section of the biofilm at the glass surface showing that the material between cells is stained as well as the cells in the biofilm. Bar, 10 μm.

FIG. 4.

Analysis of the PA2231-2245 gene cluster by RT-PCR of intergenic regions. Lane 1, size markers; lane 2, PCR amplification product of PA2230-2231 cluster with genomic DNA as the template (this serves as a control for RT-PCR of this region); lane 3, RT-PCR of PA2230 and -2231 (the lack of product indicates that PA2230 and -2231 are not cotranscribed); lane 4, size marker; lane 5, RT-PCR of PA2231 and -2232; lane 6, PA2232 and -2233; lane 7, PA2233 and -2234; lane 8, PA2234 and -2235; lane 9, PA2235 and -2236; lane 10, PA2236 and -2237; lane 11, PA2237 and -2238; lane 12, PA2238 and -2239; lane 13, PA2239 and -2240; lane 14, PA2240 and -2241; lane 15, PA2241 and -2242; lane 16, PA2242 and -2243; lane 17, PA2243 and -2244; lane 18, PA2244 and -2245. Control experiments without reverse transcriptase reactions prior to PCR were negative.