Self-generated diversity produces "insurance effects" in biofilm communities

Abstract

Diversity generally protects communities from unstable environmental conditions. This principle, known as the "insurance hypothesis," has been tested in many different ecosystems. Here we show that the opportunistic pathogen Pseudomonas aeruginosa undergoes extensive genetic diversification during short-term growth in biofilm communities. The induced genetic changes are produced by a recA-dependent mechanism and affect multiple traits, including the behavior of the bacteria in biofilms. Some biofilm-derived variants exhibit an increased ability to disseminate, whereas others manifest accelerated biofilm formation. Furthermore, the presence of these functionally diverse bacteria increases the ability of biofilms to resist an environmental stress. These findings suggest that self-generated diversity in biofilms provides a form of biological insurance that can safeguard the community in the face of adverse conditions.

Fig. 1.

Variant colonies produced by biofilm growth. (a) Micrograph of variant colonies on agar produced by a 5-day-old P. aeruginosa biofilm. (b) Time course at which variants arise from biofilms. A simultaneous growth curve shows rate of cell accumulation. (c) Production of variants and growth curve in batch planktonic culture. Data in b and c are means of three experiments and representative of four others. Error bars show SEM.

Fig. 2.

Role of recA in biofilm-induced diversity. (a) Micrographs of colonies produced by 5-day-old wild-type and recA^– biofilms. (b) Proportion of bacteria with variant colony morphology arising from biofilms after 5 days of growth. Biofilms were grown with isogenic wild-type, recA^–, recA^–-complemented, and dinP^– strains. Data are means of three experiments; error bars show SEM. (c) Variance in swimming distance induced by biofilm growth. The swimming capability of bacteria from typical colonies from biofilms was compared with the capability of bacteria from the inoculum. The biofilm-induced variation required recA. Data are the variance of 50 randomly picked wild-type and recA^– colonies. (d) Generation of auxotrophs by biofilms. Data are means of four experiments. Error bars show SEM. (e) Generation of strains overproducing pyomelanin by biofilms. Agar plates show pyomelanin-overproducing colonies from wild-type but not from recA^– biofilms. Data in the graph are the mean of four experiments; error bars show SEM.

Fig. 3.

Behavior of wild type and variants grown in biofilms. Confocal images of wild type (a) and mini (b) and wrinkly (c) variants expressing GFP; day 1 images are x–y views; scale, 10 μm. Day 2 and day 4 images, x–z views; dashed line represents biofilm attachment surface; scale, 50 μm. Results are representative of six experiments with each strain.

Fig. 4.

Biofilm phenotypes of variants. (a) Quantitative detachment rates of wild type and the mini- and wrinkly-variant biofilms. Data are means of three experiments; error bars show SEM. (b) Adherence of the wild type and wrinkly variant to the biofilm growth surface. Data are means of three experiments; error bars show SEM. (c) Susceptibility of pure-culture wild-type and wrinkly-variant biofilms to H₂O₂. Data are three replicates from one experiment and are representative of three others. Error bars show SEM.

Fig. 5.

The presence of the wrinkly-variant subpopulation enhances biofilm resistance. (a) Number and types of bacteria in wild-type biofilms before and after exposure to H₂O₂.(b) Number and types of bacteria in recA^– biofilms. No bacteria withstood H₂O₂ treatment. Data in a and b are means of four experiments; error bars indicating SEM are hidden by data points.