Robert L. Dorit

Resistance is futile: the bacteriocin model for addressing the antibiotic resistance challenge

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.

Rethinking the composition of a rational antibiotic arsenal for the 21st century

The importance of the human microbiome in health may be the single most valuable development in our conception of the microbial world since Pasteurs 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.

Routes of Resistance

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

By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

Bacterial RNase P is an essential ribonucleoprotein composed of a catalytic RNA component (encoded by the rnpB gene) and an associated protein moiety (encoded by rnpA). We construct a system that allows for the deletion of the essential endogenous rnpA copy and for its simultaneous replacement by a heterologous version of the gene. Using growth rate as a proxy, we explore the effects on fitness of heterologous replacement by increasingly divergent versions of the RNase P protein. All of the heterologs tested complement the loss of the endogenous rnpA gene, suggesting that all existing bacterial versions of the rnpA sequence retain the elements required for functional interaction with the RNase P RNA. All replacements, however, exact a cost on organismal fitness, and particularly on the rate of growth acceleration, defined as the time required to reach maximal growth rate. Our data suggest that the similarity of the heterolog to the endogenous version — whether defined at the sequence, structure or codon usage level — does not predict the fitness costs of the replacement. The common assumption that sequence similarity predicts functional similarity requires experimental confirmation and may prove to be an oversimplification.

Deconvoluting the effects of surface chemistry and nanoscale topography: Pseudomonas aeruginosa biofilm nucleation on Si-based substrates

HYPOTHESISnThe nucleation of biofilms is known to be affected by both the chemistry and topography of the underlying substrate, particularly when topography includes nanoscale (<100u202fnm) features. However, determining the role of topography vs. chemistry is complicated by concomitant variation in both as a result of typical surface modification techniques. Analyzing the behavior of biofilm-forming bacteria exposed to surfaces with systematic, independent variation of both topography and surface chemistry should allow differentiation of the two effects.nnnEXPERIMENTSnSilicon surfaces with reproducible nanotopography were created by anisotropic etching in deoxygenated water. Surface chemistry was varied independently to create hydrophilic (OH-terminated) and hydrophobic (alkyl-terminated) surfaces. The attachment and proliferation of Psuedomonas aeruginosa to these surfaces was characterized over a period of 12u202fh using fluorescence and confocal microscopy.nnnFINDINGSnThe number of attached bacteria as well as the structural characteristics of the nucleating biofilm were influenced by both surface nanotopography and surface chemistry. In general terms, the presence of both nanoscale features and hydrophobic surface chemistry enhance bacterial attachment and colonization. However, the structural details of the resulting biofilms suggest that surface chemistry and topography interact differently on each of the four surface types we studied.

When comparative information leads us astray: the receptor-binding region of colicin E9.

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.n

Essential is Not Irreplaceable: Fitness Dynamics of Experimental E. coli RNase P RNA Heterologous Replacement

While critical cellular components—such as the RNA moiety of bacterial ribonuclease P—can sometimes be replaced with a highly divergent homolog, the cellular response to such perturbations is often unexpectedly complex. RNase P is a ubiquitous and essential ribonucleoprotein involved in the processing of multiple RNA substrates, including tRNAs, small non-coding RNAs and intergenic operons. In Bacteria, RNase P RNAs have been subdivided—based on their secondary and tertiary structures—into two major groups (A and B), each with a distinct phylogenetic distribution. Despite the vast phylogenetic and structural gap that separates the two RNase P RNA classes, previous work suggested their interchangeability. Here, we explore in detail the functional and fitness consequences of replacing the endogenous Type-A Escherichia coli RNase P RNA with a Type-B homolog derived from Bacillus subtilis, and show that E. coli cells forced to survive with a chimeric RNase P as their sole source of RNase P activity exhibit extremely variable responses. The chimeric RNase P alters growth rates—used here as an indirect measure of fitness—in unpredictable ways, ranging from 3- to 20-fold reductions in maximal growth rate. The transcriptional behavior of cells harboring the chimeric RNAse P is also perturbed, affecting the levels of at least 79 different transcripts. Such transcriptional plasticity represents an important mechanism of transient adaptation which, when coupled with the emergence and eventual fixation of compensatory mutations, enables the cells to overcome the disruption of this tightly coevolving ribonucleoprotein.

The Evolutionary Histories of Clinical and Environmental SHV β-Lactamases are Intertwined

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 blaSHV genes isolated both from clinical and non-clinical sources and show that clinically important resistance determinants arise repeatedly from within a diverse pool of blaSHV genes present in the environment. While our results argue against the notion of a single common origin for all clinically derived blaSHV 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.

Truth and Consequences

This paper reviews the extent and consequences of grade retention in elementary and secondary school. The first part of the paper reviews recent proposals for test-based grade promotion and retention. These are based on politically, attractive, but scientifically unsupported claims about the benefits of retention, and minority students are more likely to be subject to these claims. The second part of the paper outlines what is known about rates, trends, and differentials in grade retention in the United States. Sound data are scarce, but current retention rates are much higher than is generally believed. At least 15% of students are retained between ages 6 to 8 and 15 to 17, and a substantial amount of retention occurs before or after these ages. Retention rates are much higher for boys and members of minority groups than for girls and the white majority. Retention rates have also grown substantially over the past two decades. A review of the scientific evidence about retention shows that the academic benefits of retention are both temporary and costly. When previous academic performance and relevant social characteristics are controlled, past grade retention accelerates current school dropout rates. There is no evidence for claims that new retention polices will be coupled with effective remediation of learning deficits that would be worth their cost or would offset the well-established long-term negative effects of retention. (Contains 10 figures and 61 references.) (SLD) Reproductions supplied by EDRS are the best that can be made from the original document. Should We End Social Promotion? Truth and Consequences

In the Land of Plenty

Lederman then said, I dont know if we have such a thing as a prairie hat, provoking more laughter. His subse quent speech encouraged his people to keep physics going at Fermilab for another 10 to 15 years. And indeed, Fermilabs program continued to flour ish after Lederman stepped down the following year, and also beyond the 1993 decision of Congress to terminate the Superconducting Super Collider. The Main Injector, an upgrade of the Main Ring accelerator in the early 1990s, led to Fermilabs discovery in 1995 of the top quark, the last of the six quarks. The book draws to a close at that point. But Fermilabs story was not over then; it continues today. Since the discovery of the top quark, the laboratory has continued to pursue a rich program of research in physics. However, the Tevatron will shut down within the next few years. Internation al attention has shifted to the Large Hadron Collider in Europe. Fermilabs long-term future is cur rently unclear, although research con tinues there on questions of neutrino mass, the Higgs boson, supersymmetry and new properties of matter. Ambi tious plans are in the works for new high-intensity beams, for astrophysical initiatives, and for perhaps being the eventual site of the International Linear