Adaptation to life in biofilms (supported by NIH 1R15AI082528, DOE JGI Community Sequencing Award).
We are studying the causes and dynamics of evolutionary adaptation to the biofilm lifestyle, which is strongly correlated with the ability to form chronic infections. This is most clearly the case in pulmonary infections of persons with cystic fibrosis (CF). Two of the most worrisome bacteria that cause these infections are Burkholderia cepacia and Pseudomonas aeruginosa.
- Does adaptation to the biofilm lifestyle occur at the expense of planktonic growth?
- Is biofilm ecological complexity evolutionarily stable?
- Is biofilm diversity more a product of physiological variability or heritable evolutionary change?
- Is the biofilm whole greater than the sum of its parts?
How does biodiversity influence productivity? This question applies to bacterial biofilms, in which evolved diversity may increase or decrease productivity. Ph.D. student Steffen Poltak experimentally evolved biofilm populations from a single clone to study how diversification affects productivity and to identify the ecological and genetic mechanisms of adaptation. We devised a novel method of selection by transferring only bacterial cells that adhered to a floating plastic bead. Populations of Burkholderia cenocepacia, known to cause severe infections in patients with cystic fibrosis, underwent more than 1,500 generations of biofilm selection. Each of six replicate biofilm populations underwent a common dynamic of adaptive diversification into three ecologically distinct genotypes. Diverse communities produced more cells than any monoculture and the relative yield of each variant was greater than expected in mixture. We demonstrated that biofilm synergy was caused by nutrient cross-feeding and the partitioning of biofilm space, such that interactions among mutant pairs are mutualistic. More generally, the colony morphologies and their associated mutations that evolved in each population demonstrate substantial convergent evolution with isolates of Burkholderia and Pseudomonas from chronic lung infections and suggest that altered metabolism of cyclic-di-GMP and iron and mutant-specific patterns of polysaccharide synthesis underlie biofilm adaptation. In summary, we demonstrated that synergistic diversity evolves in long-term, biofilm selection in the laboratory along similar pathways to those found in chronic infections, which sheds light on the specific forms of selection in infectious biofilms and implies that biofilm adaptive radiations, either in vitro or in vivo, may inevitably enhance productivity (Poltak and Cooper, submitted).
We are now examining how each adaptive mutation contributes to biofilm fitness. Master’s student Laura Benton has found that complementing a mutation affecting cyclic-di-GMP turnover causes mutant colonies to regress towards the ancestral form, reduces their biofilm fitness, and alters their quorum sensing function. Another example of convergent adaptation between our system and infectious biofilms is a mutation in one morphotype in the cation/multidrug efflux pump mexD. In infections, this mutation has previously been assumed to result from antimicrobial selection, but no antibiotics were added to our model biofilms. In our system, this mutation indeed also increases tobramycin resistance (a commonly used therapeutic in Burkholderia lung infections) that in turn benefits the entire biofilm community. Thus, while the biofilm itself can increase resistance by producing a barrier, adaptation to the biofilm lifestyle also apparently favors mutations that directly increase resistance even when antibiotics are absent. These two examples are the beginning of several promising years of studying the molecular basis of biofilm evolution, a subject that undergraduates Charles Traverse and AJ Troiano are addressing.
We have also been studying the evolution of biofilm diversity in P. aeruginosa using our novel bead-selection model and find similar patterns of niche differentiation, though master’s student Kenny Flynn finds that this occurs more rapidly and produces greater diversity than in Burkholderia populations. We will soon begin to study whether P. aeruginosa and B. cenocepacia mutants of the same morphology are functionally interchangeable, how in vitro diversification may explain the exceptional diversity seen in chronic infections of CF patients, and ultimately, whether biofilm adaptation may follow a conserved genetic and ecological program of diversification.
We are grateful to be collaborating with the DOE Joint Genome Institute to identify all adaptive mutations in all six Burkholderia biofilm populations by sequencing individual clones and complete population metagenomes. We will seek support to do the same with our Pseudomonas populations. These results will open a new frontier for understanding microbial evolution in structured environments and will define a large fraction of our laboratory’s focus for years to come. Our overarching aims are:
1) Quantify the effects of biofilm diversity on the resilience and pathogenic potential of the community. a. Do serial isolates from Burkholderia and Pseudomonas infections diversify similarly, and how does biofilm diversity affect patient outcomes? b. How does diversity influence pathogenic potential in tissue or animal models? c. How does adaptation in the presence of antibiotic affect biofilm diversity and pathogenic potential? (Potential funding: NIH, Evol. Mechanisms in Infectious Disease; Cystic Fibrosis Foundation)
2) How does biofilm diversity originate and how does it persist? Ph.D. student Crystal Ellis has found evidence of coevolution and character displacement in biofilm communities that enhances community synergy over time. She also has studied ecological release by evolving biofilm variants in isolation, demonstrating how these mutants expand their niches, reduce biofilm yield when reintroduced to their original community, and in some cases, re-diversify. She and undergraduate Rachel Staples are now addressing effects of ecological specialization on the evolvability of biofilm variants. These and other studies contribute to our ultimate goal to model the evolution of ecological complexity (speciation) from perspectives ranging from the molecule to the community.
See: http://www.cff.org/LivingWithCF/StayingHealthy/Germs/Bcepacia/ to learn more about how B. cepacia infections affect the CF community.
