Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 β-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.

TEM-1 β-lactamase is one of the most well-known antibiotic resistance determinants around. It confers resistance to penicillins and early cephalosporins and has shown an astonishing functional plasticity in response to the introduction of novel drugs derived from these antibiotics. Since its discovery in the 1960s, over 170 variants of TEM-1 - with different amino acid sequences and often resistance phenotypes - have been isolated in hospitals and clinics worldwide. Next to this well-documented 'natural' evolution, the in vitro evolution of TEM-1 has been the focus of attention of many experimental studies. In this review, we compare the natural and laboratory evolution of TEM-1 in order to address the question to what extent the evolution of antibiotic resistance can be repeated, and hence might have been predicted, under laboratory conditions. We also use the comparison to gain an insight into the adaptive relevance of hitherto uncharacterized substitutions present in clinical isolates and to predict substitutions not yet observed in nature. Based on new structural insights, we review what is known about substitutions in TEM-1 that contribute to the extension of its resistance phenotype. Finally, we address the clinical relevance of TEM alleles during the past decade, which has been dominated by the emergence of another β-lactamase, CTX-M.

Understanding epistasis is central to biology. For instance, epistatic interactions determine the topography of the fitness landscape and affect the dynamics and determinism of adaptation. However, few empirical data are available, and comparing results is complicated by confounding variation in the system and the type of mutations used. Here, we take a systematic approach by quantifying epistasis in two sets of four beneficial mutations in the antibiotic resistance enzyme TEM-1 β-lactamase. Mutations in these sets have either large or small effects on cefotaxime resistance when present as single mutations. By quantifying the epistasis and ruggedness in both landscapes, we find two general patterns. First, resistance is maximal for combinations of two mutations in both fitness landscapes and declines when more mutations are added due to abundant sign epistasis and a pattern of diminishing returns with genotype resistance. Second, large-effect mutations interact more strongly than small-effect mutations, suggesting that the effect size of mutations may be an organizing principle in understanding patterns of epistasis. By fitting the data to simple phenotype resistance models, we show that this pattern may be explained by the nonlinear dependence of resistance on enzyme stability and an unknown phenotype when mutations have antagonistically pleiotropic effects. The comparison to a previously published set of mutations in the same gene with a joint benefit further shows that the enzyme's fitness landscape is locally rugged but does contain adaptive pathways that lead to high resistance.

Epistasis is a key factor in evolution, since it determines which combinations of mutations provide adaptive solutions and which mutational pathways towards these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis – a complete reversion of mutational effects, particularly in protein evolution, yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in the antibiotic-resistance enzyme TEM-1 β-lactamase. Separated by 10Å, these mutations initiate two separate trajectories towards increased hydrolysis rates and resistance towards second and third-cephalosporins antibiotics. Both mutations allow the enzyme’s active-site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures we found that whereas G238S induces discrete conformations, R164S causes local disorder. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme’s catalytic pre-organization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing, global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of proteins structures and functions, but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.

We present a novel millifluidic droplet analyser (MDA) for precisely monitoring the dynamics of microbial populations over multiple generations in numerous (≥10(3)) aqueous emulsion droplets (~100 nL). As a first application, we measure the growth rate of a bacterial strain and determine the minimal inhibitory concentration (MIC) for the antibiotic cefotaxime by incubating bacteria in a fine gradient of antibiotic concentrations. The detection of cell activity is based on the automated detection of an epifluorescent signal that allows the monitoring of microbial populations up to a size of ~10(6) cells. We believe that this device is helpful for the study of population dynamic consequences of microbe-environment interactions and of individual cell differences. Moreover, the fluidic machine may improve clinical tests, as it simplifies, automates and miniaturizes the screening of numerous microbial populations that grow and evolve in compartments with a finely tuned composition.