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Hypothesis: The nucleation of biofilms is known to be affected by both the chemistry and topography of the underlying substrate, particularly when topography includes nanoscale (

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 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.

The specification of floral organ identity in the higher dicots depends on the function of a limited set of homeotic genes, many of them members of the MADS-box gene family. Two such genes, APETALA3 (AP3) and PISTILLATA (PI), are required for petal and stamen identity in Arabidopsis; their orthologs in Antirrhinum exhibit similar functions. To understand how changes in these genes may have influenced the morphological evolution of petals and stamens, we have cloned twenty-six homologs of the AP3 and PI genes from two higher eudicot and eleven lower eudicot and magnolid dicot species. The sequences of these genes reveal the presence of characteristic PI- and AP3-specific motifs. While the PI-specific motif is found in all of the PI genes characterized to date, the lower eudicot and magnolid dicot AP3 homologs contain distinctly different motifs from those seen in the higher eudicots. An analysis of all the available AP3 and PI sequences uncovers multiple duplication events within each of the two gene lineages. A major duplication event in the AP3 lineage coincides with the base of the higher eudicot radiation and may reflect the evolution of a petal-specific AP3 function in the higher eudicot lineage.

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.

The evolution of any given protein reflects the interplay between proximal selective forces involving the conservation of protein structure and function and more general populational factors that shape the action and efficiency of natural selection. In an attempt to address that interplay, we have analyzed patterns of amino acid replacement within a well-conserved molecule, alcohol dehydrogenase (ADH), in the Drosophilidae. A sliding window, moved along the protein sequence in order to quantify the extent of change at each amino acid position, reveals heterogeneous amounts of replacement across the molecule when all ADH sequences are analyzed simultaneously. Surprisingly, the replacement profile for ADH differs significantly in the melanogaster, mulleri, and Hawaiian subgroups, reflecting the imprint of the differing evolutionary histories of each of these assemblages on the evolution of this conservative molecule.

If genes have been assembled from exon subunits, the frequency with which exons are reused leads to an estimate of the size of the underlying exon universe. An exon database was constructed from available protein sequences, and homologous exons were identified on the basis of amino acid identity; statistically significant matches were determined by Monte Carlo methods. It is estimated that only 1000 to 7000 exons were needed to construct all proteins.

In order to explore the variety of possible responses available to a ribozyme population evolving a novel phenotype, five Tetrahymena thermophila group I intron ribozyme pools were evolved in parallel for cleavage of a DNA oligonucleotide. These ribozyme populations were propagated under identical conditions and characterized when they reached apparent phenotypic plateaus; the populations that reached the highest plateau showed a near 100-fold improvement in DNA cleavage activity. A detailed characterization of the evolved response in these populations reveals at least two distinct phenotypic trajectories emerging as a result of the imposed selection. Not only do these distinct solutions exhibit differential DNA cleavage activity, but they also exhibit a very different correlation with a related, but unselected, phenotype: RNA cleavage activity. In turn, each of these trajectories is underwritten by differing genotypic profiles. This study underscores the complex network of possible trajectories through sequence space available to an evolving population and uncovers the diversity of solutions that result when the process of experimental evolution is repeated multiple times in a simple, engineered system.

The Drosophila olfactory genes OS-E and OS-F are members of a family of genes that encode insect odorant-binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants through the aqueous fluid within olfactory sensilla to the underlying receptor proteins. The recent discovery of a large family of olfactory receptor genes in Drosophila raises new questions about the function, diversity, regulation, and evolution of the OBP family. We have investigated the OS-E and OS-F genes in a variety of Drosophila species. These studies highlight potential regions of functional significance in the OS-E and OS-F proteins, which may include a region required for interaction with receptor proteins. Our results suggest that the two genes arose by an ancient gene duplication, and that in some lineages, one or the other gene has been lost. In D. virilis, the OS-F gene shows a different spatial pattern of expression than in D. melanogaster. One of the OS-F introns shows a striking degree of conservation between the two species, and we identify a putative regulatory sequence within this intron. Finally, a phylogenetic analysis places both OS-E and OS-F within a large family of insect OBPs and OBP-like proteins.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.

DNA polymorphism in the Y chromosome, examined at a 729-base pair intron located immediately upstream of the ZFY zinc-finger exon, revealed no sequence variation in a worldwide sample of 38 human males. This finding cannot be explained by global constraint on the intron sequence, because interspecific comparisons with other nonhuman primates revealed phylogenetically informative sequence changes. The invariance likely results from either a recent selective sweep, a recent origin for modern Homo sapiens, recurrent male population bottlenecks, or historically small effective male population sizes. A coalescence model predicts an expected time to a most recent common ancestral male lineage of 270,000 years (95 percent confidence limits: 0 to 800,000 years).

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

Lederman then said, "I don't 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, Fermilab's 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 Fermilab's discovery in 1995 of the top quark, the last of the six quarks. The book draws to a close at that point. But Fermilab's 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. Fermilab's 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

The catalogue of mosaic proteins showing evidence of exon-shuffling continues to expand. The repeated use of exon modules suggests that current protein diversity could have been generated from a finite set of such exon modules, and that the size and character of this underlying exon universe can still be glimpsed in extant proteins.

We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.