ADAPTATION & ATOMS:
exploring the biophysical origins of molecular evolution
We are interested in the underlying biophysical principles of adaptation within bacterial populations during protein evolution. Our interest in this field is stimulated by the rise in drug resistant pathogens as well as our own curiosity about the physical basis for molecular evolution. By combining approaches from biophysics and experimental evolution we are able to identify and characterize intermediates along the mutational pathways of adaptation and then link those intermediates to the overall evolutionary trajectory of the bacterial populations. Adaptive changes in protein sequence and expression impact organismal fitness and, consequently, dictate population dynamics. We have used a “weak link” method to force changes in a particular gene, allowing those changes to be studied in physicochemical detail. The essential adenylate kinase gene (adk) of the thermophilic bacterium Geobacillus stearothermophilus was replaced by a copy from the mesophile Bacillus subtilis.
We continue to apply experimental evolution to study the rise of antibiotic resistance. Each year the CDC estimates that there are approximately two million cases of nosocomial infection that result in over 80,000 patient deaths. Antibiotic resistance is an evolutionary consequence of successful drug therapies and highlights the role of natural selection in shaping the molecular mechanisms leading to resistance. By taking an interdisciplinary approach combining genomic, biophysical and population strategies, changes at the level of atoms can be linked quantitatively to their consequences for the organism in its environment and vice versa. In addition to studying specific genes such as B. fragilis Tn4400 tetX and E. faecalis OG1RF with (Tn925) tetM we are expanding our studies to include changes to the entire genome. Our work has been designed to test and validate specific attributes of quantitative molecular evolution so that they might be used to build the integrative links necessary for a list of genes to become a useful model for adaptation. An important aspect of this work is the incorporation of an inclusive approach to embedding the list of candidate genes identified by selection into an adaptive network for the identification of the most important nodes for drug discovery and intervention.
Is adaptation to antibiotics too complex for study using in vitro approaches? Adaptation is complex, and the role of in vitro biochemistry in predicting clinical outcomes is still a fair question, but it is precisely because antimicrobial resistance is complex that we should craft careful and well thought out studies using in vitro or animal models. The next five years will see an exciting and profound change to the way in which we can study complex phenomena such as bacterial adaptation. We have entered a new genomic era in which previously unapproachable and highly complex phenomena such as drug resistance can be examined both in toto and in detail. At present there are 758 fully assembled microbial genomes, with another 1242 in progress within the public domain (ncbi.nlm.nih.gov/genomes). This number will continue to increase as the cost of sequencing a microbial genome falls to less than $1,000 in the coming years. Soon it will be the norm to sequence the genome of a strain of interest from any number of patients, animals, tissues and drug regimens. The initial output of these studies will be lists of candidate genes that are associated with adaptation. If we are merely satisfied with deriving long lists of genes that heritably change as a consequence of adaptation, we will miss the richest opportunity to understand and thereby limit adaptation. To facilitate the development of novel therapies that limit the general ability of bacteria to adapt to a drug or environment, we must understand these mechanisms at the molecular level to knockout the most critical paths to resistance. We are in a position to begin tackling adaptation to drugs in a manner that was hard to imagine just a few years ago.
Another primary emphasis of our lab is in structural biology using X-ray crystallography. In addition to being a powerful analytical method for our work in molecular evolution, we have many collaborations to study nucleic acid and nucleotide binding proteins. We are particularly interested in mechanisms of sequence-specific RNA recognition and the replisome. I have studied these proteins since I was an undergraduate at Carnegie-Mellon and have always had a soft spot for these fascinating proteins. Many years later it is nice to continue the work I began then.