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The importance of the human microbiome in health may be the single most valuable development in our conception of the microbial world since Pasteur’s germ theory of the 1860s. Its implications for our understanding of health and pathogenesis are profound. Coupled with the revolution in diagnostics that we are now witnessing – a revolution that changes medicine from a science of symptoms to a science of causes – we cannot continue to develop antibiotics as we have for the past 80 years. Instead, we need to usher in a new conception of the role of antibiotics in treatment: away from single molecules that target broad phylogenetic spectra and towards targeted molecules that cripple the pathogen while leaving the rest of the microbiome largely intact.

Pathogenic bacteria resistant to many or all antibiotics already exist. With the decline in microbiological research at pharmaceutical companies, the high rate at which resistance has evolved and spread has demanded a novel approach to addressing this critical human health issue. In the present paper, we propose a new paradigm in antibiotic discovery and development, one that applies ecological and evolutionary theory to design antimicrobial drugs that are more difficult and/or more costly to resist. In essence, we propose to simply adopt the strategies invented and applied by bacteria for hundreds of millions of years. Our research focuses on bacteriocins, powerful biological weapons, and their use as alternative therapeutics in human health.

In an effort to develop derivatives of the Escherichia coli antimicrobial protein colicin E9 that exhibit novel interactions with a target cell, we mutagenized a 10-amino acid region located at the C terminus of the colicin receptor-binding domain. We subsequently selected for those colicin molecules that retain the antimicrobial phenotype and found that, despite a mutagenic strategy that alters every amino acid in the targeted domain, more than 70% of the engineered colicins retained antimicrobial activity. This result is all the more surprising given the extensive phylogenetic conservation of this receptor-binding domain, which originally suggested the operation of strong selective constraints on the amino acid sequence of this region. This apparent contradiction between our experimental results and the comparative data is resolved by exploring the fitness consequences of the experimentally induced amino acid substitutions. In 17 of 52 cases we examined, the fitness of cells harboring the functional engineered colicins was lower than that of our control line (containing wild-type colicin E9), and in 33 of 52 cases, equal to it. Paradoxically, two of the engineered colicins appear to confer a higher fitness to the producer cell lines. While the mechanism linking changes in the amino acid sequence of the colicin receptor-binding domain and the growth rate of the cells remains unclear, these results illustrate the surprising versatility of the colicin/receptor interaction and underscore the importance of distinguishing molecular function from organismal fitness.

Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant ( k cat ) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step. This increase is offset by a reduction in substrate binding, as measured by K M . We trace the catalytic enhancement to two ubiquitous A → U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein. The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels. In contrast, improved DNA cleavage activity is accompanied by a 500-fold decrease in pre-tRNA cleavage efficiency ( k cat / K M ). The presence of the C5 component does not buffer this tradeoff in catalytic activities, despite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P. The change at position 136, located in the J11/12 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff. These results thus shed light both on structure/function relations in E. coli RNase P and on the crucial role of proteins in enhancing the catalytic repertoire of RNA.

The rise of antibiotic-resistant pathogens focuses our attention on the source of antibiotic resistance genes, on the existence of these genes in environments exposed to little or no antibiotics, and on the relationship between resistance genes found in the clinic and those encountered in non-clinical settings. Here, we address the evolutionary history of a class of resistance genes, the SHV β-lactamases. We focus on bla SHV genes isolated both from clinical and non-clinical sources and show that clinically important resistance determinants arise repeatedly from within a diverse pool of bla SHV genes present in the environment. While our results argue against the notion of a single common origin for all clinically derived bla SHV genes, we detect a characteristic selective signature shaping this protein in clinical environments. This clinical signature reveals the joint action of purifying and positive selection on specific residues, including those known to confer extended-spectrum activity. Surprisingly, antibiotic resistance genes isolated from non-clinical—and presumably antibiotic-free—settings also experience the joint action of purifying and positive selection. The picture that emerges undercuts the notion of a separate reservoir of antibiotic resistance genes confined only to clinical settings. Instead, we argue for the presence of a single extensive and variable pool of antibiotic resistance genes present in the environment.

Last year, some 50 million pounds of antibiotics were used in the United States, an amount that would correspond to roughly 5 ta blespoons?or 75 doses?of antibiotics per person. In fact, much of this antibi otic?as much as 70 percent by some estimates?is being used not to treat infections, but instead to promote food production, as antibiotics have become a key ingredient in the American food chain. We are, in short, marinating the living world in antibiotics. Against this backdrop, the emergence of antibiotic-resistant pathogenic bacte ria can hardly come as a surprise. Anti biotics, after all, are used to suppress and kill bacteria, and bacteria, like every other living thing, have no greater evo lutionary imperative than to stay alive. Our overuse of antibiotics methodi cally rewards any bacterium fortunate enough to carry a mutation that confers even slight resistance to the substance in its environment. The less-fortunate