Genetic variation in biofilms and the insurance effects of diversity

Blaise R. Boles, Matthew Thoendel and Pradeep K. Singh

Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

Correspondence
Pradeep K. Singh
(pradeep-singh{at}uiowa.edu)

In the paper referenced above we presented evidence that P. aeruginosa undergoes extensive genetic diversification during growth in biofilm communities (Boles et al., 2004). The genetic changes arise via a recA-dependent mechanism and affect multiple traits. We also showed that biofilms that had genetically diversified were better able to withstand an applied physiological stress as compared to biofilms that were unable to diversify. We related these findings to the insurance hypothesis, an ecological hypothesis which posits that diversity generally increases the ability of biological communities to withstand changes in environmental conditions (Yachi & Loreau, 1999). Cooper et al. have raised questions about our interpretation of the data, and have objected to a portion of the discussion in which we discuss mechanisms that could generate the observed diversity. Here we respond to the points they have raised.

(1) Cooper et al. acknowledge that we demonstrate increased stress resistance in high (as compared to low) diversity biofilms. However, they state, ‘What also needs to be demonstrated (to claim an insurance effect) is a trade-off over different environments such that some members are better in some environments while others are better in others’. We agree that the differential responses of community members to environmental conditions are fundamental to the insurance hypothesis. This point is made in our manuscript. On p. 16634 we state:

‘Indeed, the fitness of each specialized population is likely to depend on prevailing conditions; a given phenotype may be advantageous in certain circumstances and detrimental or neutral in others. The insurance hypothesis relates to the effects of diversity on the community as a whole. It predicts that a community composed of functionally diverse populations is likely to perform better in general because of the likelihood that some subpopulation will thrive as prevailing conditions change.’

We considered this point intuitive and thus did not highlight it further; it is generally thought that most specialized phenotypes will have advantages in some conditions and disadvantages in others (MacLean et al., 2004; Wilson & Yoshimura, 1994).

Though this point was not stressed, data presented in our paper did provide evidence for such a ‘trade-off’. To emphasize this point we have combined data found in Figs 2(b) and 5(a) of our paper in the accompanying Fig. 1. To use the terminology of Cooper et al., we call our standard biofilm medium Environment #1, and medium with peroxide added after 3 days of growth, Environment #2. In Environment #1 ‘typical’ cells were most prevalent in the biofilm community, whereas in Environment #2 (assayed after the same period of growth), wrinkly variants dominated. These results demonstrate a fitness ‘trade-off’ in different environments and suggest that the wrinkly variant is not ‘simply a superior competitor...regardless of environment’ as Cooper et al. suggest.



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Fig. 1. Effect of environmental conditions on community composition. In Environment #1 (standard biofilm medium), the typical and mini subpopulations dominate. In Environment #2 (peroxide added after 3 days of growth) the wrinkly subpopulation is the most successful. Data from Environment #1 are mean values from three experiments; data in Environment #2 are mean values of four experiments; error bars show SEM. Data taken from Boles et al. (2004).

 
The data in Fig. 4 of our paper shows another ‘trade-off’ attributable to the wrinkly variant phenotype. There we show that whereas wrinkly variants have an increased capacity to adhere to surfaces and form biofilms that are more resistant to oxidant stress, they also manifest a decreased capacity for biofilm detachment. While these experiments address the ‘trade-off’ point raised by Cooper et al. in a less direct manner than the data discussed above, they also suggest that the wrinkly phenotype will not be advantageous under all conditions. The capacity for biofilm detachment is thought to be critical for the survival of organisms when local conditions deteriorate.

As with most experimental studies, there are limitations to our data. First, it could be argued that the wrinkly variant could dominate ‘regardless of environment’ if the growth period was extended for a longer time. While we cannot exclude this possibility with certainty, we have grown wild-type biofilms (which are proficient in diversification) for 30 days in standard medium and colonies with the wrinkly phenotype remain a minority population.

Second, as has been done in other studies (MacLean et al., 2004; Rainey & Travisano, 1998), we used the observable trait of colony morphology to identify and follow bacterial subpopulations. It is possible that colony morphology may not always track with the traits actually responsible for a population's differential fitness. If this were the case, error could exist in the subpopulation counts that we performed. We think this source of error is unlikely to confound our finding that wrinkly variants dominate under oxidative stress conditions. The fact that wrinkly variants were repeatedly found in the highest numbers in communities exposed to oxidative stress suggests a strong linkage between the wrinkly morphology and traits responsible for increased fitness in these conditions (see Fig. 5 in Boles et al., 2004).

Another factor introducing complexity is that community members have the capacity to switch types. This is obviously not the case for experiments with plant or animal communities that relate to the insurance hypothesis. Thus, while we contend that the experiments in this paper do demonstrate an insurance effect, we agree that more work is required to further investigate the effects of this diversity on biofilm community functioning.

(2) Cooper et al. object to our use of oxidative stress in a test of community functioning because other experiments in our paper showed that biofilms formed by pure-culture wrinkly variants were more resistant to this stress than the wild-type. We disagree that this introduces bias to our experiments.

We think the results of landmark field experiments by Tilman and colleagues are instructive in this regard (Tilman et al., 1997). They investigated the effects of diversity on ecosystem productivity using mixtures of savanna plants and found that functional diversity increased community productivity. The plant species used were known to differ in phenotypes that affected their performance at the field site where the experiments were conducted. For example, some of the plant species grew best in the cool season, others in the warm season. The plants also differed in their use of nitrogen, the major limiting nutrient at the site.

In our view, foreknowledge of the susceptibility of community members to particular stresses in the environment does not bias their findings. This is because the purpose of these experiments was not to ascertain how particular community members respond to novel stresses, but rather to determine how functional diversity impacts community performance overall.

Though our study differed from Tilman's work in many ways, we believe that the same principle applies; foreknowledge of key phenotypic characteristics of community members (or of the experimental environment) does not negate the results. We would also like to point out that this is an initial paper on the subject of biofilm-induced genetic diversity. We agree that additional work examining a greater range of stress conditions and other types of insurance effects will extend and broaden these findings.

(3) Finally, Cooper et al. object that we raised the possibility that genetic variation could somehow be increased in biofilms. We do not discuss ‘why’ diversity arises in biofilms, nor do we state that we favour this view (as Cooper et al. assert). Cooper et al. favour the view that the biofilm-generated diversity results from ‘random variation and strong selection’. We agree that this is a possibility; indeed, it is the first explanation we raise in our discussion (p. 16634):

‘Whereas our experiments show that diversity is rapidly produced in biofilms, we do not yet know how it is generated. One possibility is that the rate of genetic variation is similar in the biofilm and planktonic cultures, and the diversity we observed is caused by powerful selective pressures inherent to biofilms’. We further state:

‘Although further work will be required, we do not favour this as the sole explanation for our findings because it seems unlikely that strong selective pressures for auxotrophy would exist within biofilms, and recA gene function is not typically required for spontaneous growth-dependent mutation caused by replication errors.’

Cooper et al. suggest other explanations. They raise the possibility that auxotrophs may have some adaptive benefit in our growth conditions. This is an interesting point and we agree that this could be a factor in the emergence of auxotrophs in our system. They also suggest that the reduced genetic diversity in recA biofilms may be due to a growth defect. While this is possible, we think this highly unlikely in view of the data we presented in our paper. As Fig. 5 in the paper shows, recA biofilms produce similar cell yields as the wild-type, in side-by-side experiments.

Finally, we would like to point out that we raised these mechanistic possibilities in the discussion section of our paper. In our view it is appropriate (and often valuable) to discuss alternative explanations for findings in the Discussion. Even explanations that run contrary to accepted thinking can be constructive and thought-provoking if they are consistent with the data and are presented in a responsible manner.

REFERENCES

Boles, B. R., Thoendel, M. & Singh, P. K. (2004). Self-generated diversity produces ‘insurance effects’. Proc Natl Acad Sci U S A 101, 16630–16635.[Abstract/Free Full Text]

MacLean, R. C., Bell, G. & Rainey, P. B. (2004). The evolution of a pleiotropic fitness tradeoff in Pseudomonas fluorescens. Proc Natl Acad Sci U S A 101, 8072–8077.[Abstract/Free Full Text]

Rainey, P. B. & Travisano, M. (1998). Adaptive radiation in a heterogeneous environment. Nature 394, 69–72.[CrossRef][Medline]

Tilman, D., Knops, J., Wedin, D., Reich, P., Ritchie, M. & Siemann, E. (1997). The influence of functional diversity and composition on ecosystem processes. Science 277, 1300–1302.[Abstract/Free Full Text]

Wilson, D. S. & Yoshimura, J. (1994). On the coexistence of specialists and generalists. Am Nat 144, 692–707.[CrossRef]

Yachi, S. & Loreau, M. (1999). Biodiversity and ecosystem productivity in a fluctuating environment: the insurance hypothesis. Proc Natl Acad Sci U S A 96, 1463–1468.[Abstract/Free Full Text]





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