The main goal of this project is to understand how bacterial diversity is generated and maintained, and to improve the knowledge about the dynamics of phenotypic and genomic evolution. This knowledge is crucial for better understanding the emergence and evolution of pathogens because many of the difficulties encountered during efforts to prevent and control infections are due to the potential of bacteria to generate extensive variability within populations (e.g. antibiotic resistance).

This genome sequencing project makes use of evolving strains that have been isolated from the longest-running evolution experiment using Escherichia coli. A clone of E. coli B was used as the common ancestor to found twelve independent populations (Lenski, 2004). These populations have been propagated by daily serial transfer in the same defined glucose-limited environment for more than 40,000 generations (Lenski et al., 1991; Lenski and Travisano, 1994; Cooper and Lenski, 2000; Philippe et al., 2007). Over time, the populations diverged genetically from their ancestor and genetic diversity has accumulated within them (Papadopoulos et al., 1999). All of the populations achieved similar gains in competitive fitness, indicative of substantial adaptation, based on competition experiments with marked variants of the ancestor in the same glucose-limited environment (Lenski and Travisano, 1994; Cooper and Lenski, 2000). Several other phenotypic aspects also evolved in parallel including cell size (Lenski et al., 1998), growth parameters (Vasi et al., 1994), catabolic functions (Cooper and Lenski, 2000; Pelosi et al., 2006), DNA topology (Crozat et al., 2005), and global gene expression (Cooper et al., 2003).

In one of the populations, a cross-feeding polymorphism evolved where two morphs, called large (L) and small (S) based on their colony morphology, coexist in a frequency-dependent manner (Rozen and Lenski, 2000). Two factors enabled their coexistence, despite the fact that L grows exponentially about 20% faster than S. First, L excretes a metabolite that differentially promotes the growth of S. Second, L experiences increased death during stationary phase (i.e., after glucose is depleted) when S is present. The S and L morphs are both present from at least as early as generation 6,000 and they have continued to coexist for tens of thousands of generations. Interestingly, however, the relationship between the morphs is dynamic through time, with the frequency of S rising and falling several times from about 10-20% to about 50-90% of the total population. Restriction Fragment Length Polymorphism analyses, using Insertion Sequence elements to resolve the phylogenetic history of L and S, demonstrated that all of the S clones are monophyletic, indicating a long history of coexistence with L (Rozen et al., 2005). Moreover, competition assays using clones of both morphs from different generations, indicated that both lineages continued to adapt, and their continued evolution contributed to fluctuations in their relative abundance over evolutionary time. Based on their phylogenetic history and independent evolutionary trajectories, S and L fulfill one set of criteria for being different asexual species (Cohan, 2002).

This genome sequencing project aims to identify all of the mutations responsible for the divergence and maintenance of both asexual morphs, and therefore to understand mechanisms of microbial diversification and speciation. Moreover, the population from which these two morphs arose evolved a mutator phenotype, leading to an increase of about 100-fold in mutation rate (Sniegowski et al., 1997).

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