Center for Microbial Ecology

Contributors:

STC Faculty -- F. De Bruijn, D. Fulbright, A. Jarosz, R. Lenski, T. Marsh, T. Schmidt, M. Thomashow, J. Tiedje, G. Velicer

Collaborators -- C. Adami (Caltech), A. Bennett (Univ. California, Irvine), J. Blanchard (Promega Corp.), M. Blot (Grenoble, France), B. Bohannan (Stanford Univ.), A. De Visser (Wageningen, The Netherlands), S. Elena (Valencia, Spain), L. Forney (Groningen, The Netherlands), P. Gerrish (Los Alamos National Lab.), D. Kaiser (Stanford Univ.), K. Kersters (Gent, Belgium), R. Korona (Krakow, Poland), L. Kroos (Biochem., MSU), J. Mongold (Urbana, Illinois), C. Nakatsu (Purdue Univ.), M. Radman (Paris, France), D. Schneider (Grenoble, France), P. Sniegowski (Univ. Pennsylvania), V. Souza (Mexico City), S. Strauss (Univ. California, Davis); M. Travisano (Univ. Houston), P. Turner (Valencia, Spain), M. Vulic (Harvard Univ.), C. Zeyl (Wake Forest Univ.)

Postdocs -- F. Crocker, A. Cullum, F. Moore, C. Ofria, S. Remold, M. Roberts

Graduate Students -- V. Cooper, E. Ostrowski, M. Riley, K. Ritalahti, D. Rozen, M. Stanek, K. Stredwick, F. Vasi

Undergraduate Students -- K. Breining, C. Duskin H. Jaworski, M. Petersen, K. Sproull, E. Victory, C. Westbrook, R. Woods

1999 Research Accomplishments:

During 1999, this thrust group has focused on characterizing the phenotypic and genetic changes that occur in evolving populations of bacteria. The overarching goal is to better understand the processes that promote adaptation and diversification of bacteria. This work has focused on three model systems: Escherichia coli populations that are evolving for in a glucose-limited medium; populations of Ralstonia strain TFD41 that have adapted to growth on 2,4-D; and populations of Myxococcus xanthus adapting under various conditions that favor asocial or social behaviors.

1. Dynamics of phenotypic and molecular evolution in E. coli. We continued multifaceted analyses of the phenotypic and genetic changes that have taken place in 12 populations of E. coli during 20,000 generations of experimental evolution. In one study, we compared the dynamics of phenotypic and molecular evolution, with the latter based on DNA fingerprints using IS elements as probes (Papadopoulos et al. 1999). We saw significant discrepancies between rates of phenotypic and molecular evolution, which have often been postulated based on comparative studies but not directly observed as in our experiment. In particular, rates of phenotypic change in competitive fitness and cell size decelerated over the course of the evolution experiment, whereas the rate of molecular divergence from the ancestor continued apace. In another study, we are quantifying the extent of specialization via losses of diverse catabolic functions that have experienced relaxed selection in the glucose-limited environment. Moreover, we are taking advantage of two previous findings - the deceleration over time in the rate of adaptation in all 12 populations, and the evolution of hypermutable genotypes in some of them - to disentangle the contribution of two distinct population-genetic processes to the diminished catabolic breadth. Antagonistic pleiotropy involves tradeoffs, whereby the same mutations that promote adaptation to the glucose regime cause reduced capacity to use other substrates. By contrast, mutation accumulation is based on the substitution of secondary mutations in genes that harm growth capacities on other substrates, but which are inconsequential for fitness in the glucose regime.

A third focus has been examining the short- and long-term dynamics of a stable polymorphism that emerged in one of the populations (Rozen and Lenski, in press). This polymorphism is stable over the short-term because one genotype has a competitive advantage for glucose but secretes a metabolic byproduct into the medium that the other morph is better able to utilize. Yet, over thousands of generations, the relative abundance of these two types has shown pronounced fluctuations, the basis of which we are currently investigating. Another study seeks to identify and track particular mutations that contribute to the adaptation of these experimental populations. One such mutation is a 1-bp insertion in an upstream regulatory region of glmUS. We have shown that this mutation swept through one population between generations 500 and 1500, yet it was not substituted into the other evolving populations even after 20,000 generations. We are presently transducing this mutation into the other derived backgrounds to determine whether its benefit was usurped by other beneficial mutations that preceded it in most populations; if so, that would indicate that the order in which mutations occur is an important evolutionary factor.

2. Genetic and phenotypic determinants of competitiveness in Ralstonia TFD41. Eighteen populations of Ralstonia strain TFD41 were propagated for 1000 generations on either liquid or solid medium with 2,4-D as their primary carbon source. We reported previously that all 18 lines improved substantially in their competitive fitness relative to their common ancestor, and that they all exhibited parallel deletions in the same region of their chromosome. Despite these similarities, we have now also shown that the replicate evolved lines show remarkable diversity in many other respects including large changes to the outer envelope, loss of encapsulation, substantial variation in carbon utilization profiles, as well as significant variation in cell length and shape (see figure). Recent measurements of cell length from scanning electron microscopy indicate that the length varies over almost an order of magnitude, from 1.27 �m up to 10.52 �m in length (0.73 to 6.07 times the average length of the ancestral strain). Future work will focus on the molecular mechanisms of such rapid formation of genotypic and phenotypic variation in this organism. Of particular interest is the ability to induce deletion mutations by imposing an environmental stress during cultivation, which we have documented in Ralstonia. The degree to which this process provides an important mechanism for the generation of diversity and subsequent evolution will be explored.

3. Changes in Myxococcus xanthus under social and asocial growth conditions. Like many species of Myxobacteria, M. xanthus exhibits a variety of social traits including social motility, swarming and fruiting-body formation during nutritional stress, and collective attacks against prey organisms. We have begun a series of studies to examine the evolution of these social traits under different selective regimes, and to identify the genetic changes responsible for observed changes in their behavior. Our eventual goal is to understand conditions that promote cooperation versus conflict in primitive social systems. In our first experiment, we allowed 12 populations to evolve in an unstructured liquid environment. All of the populations lost one or more social functions to some degree, although the quantitative extent of their adaptation to the liquid environment was not correlated with the degree of loss of their social phenotypes. Gene replacement experiments indicate that mutations in genes that encode pili used in social motility are responsible for some, but not all, of the observed adaptation to the liquid regime. Also, in at least some cases, we can partially restore social traits by selecting for enhanced motility on agar plates.

We have recently begun experiments to examine whether these asocial selected lines might behave as 'cheaters' when mixed with their socially competent ancestors in fruiting-body developmental assays. That is, are asocial genotypes over-represented among spores when they are initially rare, and do they harm the group's overall performance? Preliminary experiments indicate that these criteria may indeed be fulfilled; we are presently engaged in more definitive experiments.

Future Plans:

Even as the core NSF support is winding down, the Population Dynamics and Evolution Thrust Group anticipates continuing its research along several fronts. Some projects have already been successfully funded, including research on the dynamics of the chestnut blight fungus, led by Andrew Jarosz and Dennis Fulbright. Other projects have new homes with former graduate students and postdoctoral associates, who are continuing CME-related research as new faculty elsewhere; for example, Paul Sniegowski (Univ. Pennsylvania) has obtained funding to continue research on the evolution of mutation rates. Another project that will certainly continue is the long-term experimental evolution with E. coli. One emphasis in that project will be the coupling between phenotypic and genetic changes, which involves collaborations with Peg Riley (Yale Univ.) and Michel Blot (Grenoble, France). Another emphasis will be extending this approach to evolution under more complex regimes, including the presence of competing species as well as multiple resources. An exciting new direction will be to compare and contrast the results of evolutionary studies in bacteria with parallel studies of 'digital organisms' - programs that self-replicate, mutate, compete for limiting resources, and evolve (Lenski et al. 1999).

In addition to primary research papers, another important legacy will be reviews that synthesize some of our important findings and place them in a broader context. Several synthetic reviews have already been published on the role of theory and experiments in understanding microbial evolution (Lenski, in press), the uses of experimental evolution in physiology (Bennett and Lenski 1999), the complex underpinnings of competitive fitness (Lenski et al. 1998), and evolutionary causes and consequences of mutation (Sniegowski and Lenski 1995). Former graduate student Brendan Bohannan (Stanford Univ.) and Richard Lenski are currently writing a review article on the effects of molecular evolution in bacteria-bacteriophage interactions on community structure; former graduate student Michael Travisano (Univ. Houston) will edit a book on experimental evolution; and Richard Lenski has started writing a monograph that summarizes findings from 20,000 generations of experimental evolution of E. coli.