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Science 338 (6104): 264-266

Copyright © 2012 by the American Association for the Advancement of Science

Bacterial Quorum Sensing and Metabolic Incentives to Cooperate

Ajai A. Dandekar1, Sudha Chugani2, and E. Peter Greenberg2,*

1 Division of Pulmonary and Critical Care Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA.
2 Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195, USA.

Figure 1
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Fig. 1. Adenosine restrains emergence of social cheaters in casein medium. (A) P. aeruginosa PAO1 in minimal medium containing casein alone or casein and adenosine at increasing levels of adenosine (total of casein and adenosine is 1% w/v). Protease-negative cheater abundance was determined at 5-day intervals and was essentially identical to the cheater abundance determined by absence of growth on adenosine agar. (B) Growth yields at 24-hour intervals measured as optical density at 600 nm (OD600). The cell densities (OD600) after 30 days were 1.84 in casein, 1.93 in 0.75% casein with 0.25% adenosine, 1.99 in 0.5% casein with 0.5% adenosine, 1.81 in 0.25% casein with 0.75% adenosine, and 1.89 in 0.1% casein with 0.9% adenosine. The total number of generations in all the experimental groups was nearly equal after 30 days. As discussed in the text, variants with improved growth on adenosine emerged and were isolated from day 25 cultures. (C) The reduction in LasR mutants is not a consequence of resource denial or substitution. Cheaters emerge when strain PAO1 is grown on 0.25% casein with or without 0.75% glucose in place of adenosine. (D) Cheater emergence is restrained when casein is at a fixed concentration and adenosine concentration exceeds 0.5%. (E) The clinical isolate CI27 was grown in minimal medium containing casein or casein and adenosine [as in (A) for strain PAO1]. (F) Quorum sensing is required for growth of most, but not all, P. aeruginosa isolates tested. We grew six different P. aeruginosa strains (left) and their respective lasI, rhlI mutants (right) on adenosine agar as described in the methods. In all cases, the wild-type strains grew well. With the exception of BE177, the mutants did not show appreciable growth on adenosine. Except for panels (B) and (F), which are representative experiments, panels show means ± SEM for three independent experiments.


Figure 2
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Fig. 2. Adenosine reduces the relative fitness of LasR mutants. (A) Cheaters are rapidly eliminated from adenosine-enriched medium. A mixture of cheaters and wild-type bacteria derived from either strain PAO1 (squares) or CI27 (circles) growing on casein alone was transferred to 0.9% adenosine and 0.1% casein and subcultured daily. The abundance of protease-negative bacteria was assessed on skim milk agar. (B) There is no selection against cheaters in LB broth. A mixture of cheaters and wild-type bacteria derived from strain PAO1 was grown in LB broth buffered with MOPS, pH 6.8, with daily subculture, and protease-negative bacteria were counted daily. (C) The addition of glucose, which is metabolized by a quorum sensing–independent pathway, does not prevent adenosine suppression of social cheaters. A wild-type and cheater mixture derived from strain PAO1 was placed into medium containing 1% glucose, 0.9% adenosine, and 0.1% casein, with daily subculture, and protease-negative bacteria were enumerated daily. In all panels, data are displayed as a competitive index or the end point cheater:wild type ratio compared with the initial ratio on a logarithmic scale. Data are shown as means ± SEM and reflect the combined results of three independent experiments.


Figure 3
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Fig. 3. Quorum-sensing regulation of a private good can prevent a tragedy of the commons. (A) Pyocyanin in cultures of P. aeruginosa PAO1 grown with casein as the sole nitrogen, carbon, and energy source (solid lines, three independent experiments). By day 10, there is three to four times as much pyocyanin at the time of transfer as pyocyanin in a culture grown with NH4Cl as a nitrogen source (dotted line). Pyocyanin was measured as described elsewhere (19). (B) Hyperproduction of the blue phenazine pigment pyocyanin is evident in NH4Cl-free medium by day 20. (C) Overexpression of quorum sensing–controlled factors correlates with the emergence of LasR mutants, which reach about 80% of the total population, after which cultures do not grow upon transfer. The tragedy of the commons does not occur in a medium containing adenosine (0.75%) and casein (0.25%) (inverted triangles) although LasR mutants are detectable (mean of three experiments; SEM at all points is <3%).


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