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This chapter discusses the cholera bacterium, Vibrio cholerae and its toxic viruses. The cholera bacterium causes disease and death when the infection results in a continual watery diarrhea that dehydrates the host. The diarrhea is the immediate consequence of a poisonous substance excreted by the bacterium, namely cholera toxin. However, the cholera bacterium does not carry this toxin by nature. The toxin is coded in the genes of a virus called the cholera toxin phage, or CTXphi.

This chapter discusses all aspects of the methodology used to solve isometric virus structures with separate case study examples for two demanding examples, the blue tongue virus core (BTV) and bacteriophage PRD1, the first structure of an intact virus with an internal membrane.

The discovery of bacteriophages initiated a long controversy about their nature. Were they intracellular parasites, a stage of the bacterial life cycle, or symbionts with the bacterium (which might be likened to the alga-fungus relationship in lichens)? Or were they perhaps infective entities below the level of the living, or lethal substances that triggered bacterial cells to make and release more of the same substance? These considerations reflect the uncertainty at the time, not only about the nature of bacteriophage, but also about the nature of the bacterial cell itself. This chapter discusses early studies on bacteriophages, including those by Henry J. Muller, Max Delbrück, Emory Ellis, and Salvador Luria.

This chapter begins with a discussion of studies on temperate bacteriophages. It then describes the discovery of a new and surprising form of genetic recombination called transduction, which led to Salmonella typhimurium becoming a model for bacterial genetics, and the discovery of phage lambda.

“Work efficiently, like bacteriophages with icosahedral symmetry” explains in non-technical terms how an exceptionally small virus that infects bacteria uses its geometry to efficiently encode instructions for assembling the protein shell that stores its genetic material. Readers may cut out a template and construct a model of the bacteriophage’s icosahedron-shaped head. In addition, numerous hand-drawn sketches illustrate the mathematical symmetries of the bacteriophage’s replication. Mathematics students and enthusiasts are encouraged to learn a lesson from the bacteriophage by economizing time and effort in mathematical and life pursuits. At the chapter’s end, readers may check their understanding by working on a problem. A solution is provided.

This chapter focuses on empirical tests of evolutionary theory using viruses, specifically bacteriophage or phage, beginning by reviewing phage biology and the phage life cycle, and then describing laboratory procedures for cultivating phage and assaying fitness. It also reviews the theoretical models that have been used to describe phage ecology and evolution, in order to emphasize the advantages of using phage experimental systems to test theoretical predictions. The chapter then discusses recent phage experiments that have investigated the genetic basis and molecular mechanisms underlying adaptation. Finally, it emphasizes that the insights gained from experimental evolution studies with phage have important implications not only for phage ecology and evolution, but also for predator–prey and host–parasite interactions in general.

This chapter follows radioisotopes into the laboratories of biochemists and molecular biologists, where they illuminated metabolic pathways and genetic transmission. Early isotope labelling experiments involved stable isotopes, but with the availability of radioisotopes from the AEC, biochemists began relying more on radioactive labels. The chapter features two case studies: Melvin Calvin and Andrew Benson’s elucidation of the steps of photosynthesis using carbon-14, and the role of sulfur-35 and phosphorus-32 in the 1953 Hershey-Chase experiment and other gene transfer experiments (including the so-called “suicide experiments” with phage). Both of these experiments helped establish radiolabeling as a central technique among biochemists and molecular biologists.

This essay argues that model systems in biology function as exemplars in the sense Thomas Kuhn develops in his postscript to second edition of The Structure of Scientific Revolutions, though there are distinctions. Whereas Kuhn largely emphasized the role of theory and problem-solving in terms of conceptual work, a model systems approach brings out the centrality of experimentation and analogies. Nonetheless, Kuhn’s insight that scientists solve problems by looking for similarities that are embodied in physical situations, rather than following rules or laws, pertains to much biomedical research. Examples are drawn from molecular biology, particularly on how bacteriophage and tobacco mosaic virus provided alternative models for isolating and characterizing animal viruses.

Much of the evolutionary study of species is retrospective and reconstructs the past processes leading to extant diversity. Yet the nature of species and extent of diversity has profound implications for adaptation to ongoing environmental and biotic change. This chapter considers the significance of species and species boundaries for contemporary evolution. Simple theory and evidence is presented, showing how partial gene flow between co-occurring species alters the dynamics of evolution in changing environments. The chapter then focuses on gene transfer in microbial populations, showing how plasmids and phage target transfer to particular subsets of genes, and thereby optimize adaptation in fluctuating environments. The costs and benefits depend on the ecological interactions among the donor and recipient species. Different mechanisms have a different range of transfer, with phage being mainly restricted to species, but plasmids often transferring traits across greater taxonomic distances.

In addition to grazing, another form of top-down control of microbes is lysis by viruses. There is probably a virus or several for every one organism in the biosphere, but the most common viruses are thought to be those that infect bacteria. Viruses come in many varieties, but the simplest is a form of nucleic acid wrapped in a protein coat. The form of nucleic acid can be virtually any type of RNA or DNA, single or double stranded. Few viruses in nature can be identified by traditional methods, because their hosts cannot be grown in the lab. Direct count methods have found that viruses are very abundant, being about 10-fold more abundant than bacteria, but the ratio of viruses to bacteria varies greatly, especially so in soils. Viruses are thought to account for about 50 per cent of bacterial mortality, but the percentage varies from zero to 100 per cent, depending on the environment and time. In addition to viruses of bacteria and cyanobacteria, studies by microbial ecologists conducted to date have examined viruses of phytoplankton and the possibility that when viral lysis ends, phytoplankton blooms. While viral lysis and grazing are both top-down controls on microbial growth, they differ in several crucial respects. Unlike grazers, which often completely oxidize prey organic material to carbon dioxide and inorganic nutrients, viral lysis releases the organic material from hosts more or less without modification. Perhaps even more important, viruses may facilitate the exchange of genetic material from one host to another.

This chapter shows Ole Maaloe heading full-speed into a study of bacteriophages, that is, viruses that infect and replicate in bacterial cells, while Niels Jerne was beginning to identify himself as an immunologist. Max Delbruck and other geneticists used the tiny phage as a model organism to understand the molecular mechanism of heredity, and during his stay at Cal Tech in the spring of 1949, Maaloe had been seriously bitten by the “phage bug.” He returned to Copenhagen full of energy and ideas and, with Orskov's indulgence, established his one-man branch of the internationally dispersed phage group in the Standardization Department. The following spring he started a series of experiments on how changes in temperature affect the reproduction of phages in the bacterial cell.

Pseudomonas aeruginosa and Pseudomonas syringae are opportunistic pathogens, the former of animals and the latter of plants, that usually enter their hosts through wounds. They share many features relating to virulence. The two bacteria have a core genome that is similar in size to the postulated ‘minimal bacterial genome’. The non-core genome is very large and reflects the metabolic and ecological diversity of the genus. Both species produce a wide range of virulence factors that are controlled by quorum sensing and many are injected into cells by a type III secretion system. Two virulence factors that are shared are the production of exoenzymes to destroy host tissue and the polysaccharide alginate that promotes biofilm formation. The latter plays a key role in Pseudomonas aeruginosa infections of patients with cystic fibrosis. The production of Pf bacteriophages is a key virulence determinant of Pseudomonas aeruginosa. There is no counterpart in Pseudomonas syringae.

This chapter reviews the research that set the stage for Joshua Lederberg’s surprising discovery of bacterial conjugation. While the foundational research of Gregor Mendel and his principles of inheritance had been effectively combined with Darwinian evolution, producing the Modern Synthesis in the mid-forties, bacteria did not fit into this grand synthesis. Most biologists believed that bacteria were too primitive to have real genes. But Delbruck, Hershey and Luria organized the Phage School, leading a novel approach to discovering the molecular biology of the gene by studying bacteriophages. Microbiologists like Tracy Sonneborn and Carl Lindegren turned to alternative microorganisms—protists, fungi, and yeast—to develop new model systems that offered advantages over the classical genetics organisms of animals and plants. The research of Edward Tatum and Jacques Monod indicated that mutations seemed to explain variation in bacteria. For many years, however, bacteriologists had known that bacteria reproduced by fission. The lack of any genetic hybridization seemed to argue against using bacteria to study basic genetic processes.

This chapter relates how, in the 1950s, Esther and Joshua Lederberg and their colleagues uncovered a whole new kind of genetic transfer involving plasmids and viruses. In plants and animals, genetic recombination is integrated within the processes of sexual reproduction. Imagine if you could trade genes with strangers at will! That’s what bacteria can do. Esther Lederberg’s discoveries of the F-plasmid and the λ‎ bacteriophage were happy accidents that occurred while she working to complete her dissertation research. Serendipity happens to those who are very attentive, broadly experienced, and open to surprises. Esther Lederberg discovered a transferable factor, the F-factor, that could transform recipients into donors. Then she discovered a lysogenic virus, hiding harmlessly inside the chromosome of its bacterial host. These two surprising discoveries showed that bacteria could transfer genes and pieces of chromosomes horizontally, as opposed to the classical inheritance of plants and animals which pass on genetic traits vertically, down through generations.

Microorganisms are ubiquitous but easy to ignore. They live in a hostile world. Bacteria are subject to attack by viruses called bacteriophages, and environmental conditions can easily change to be better or worse for their existence. One might wonder how a single-celled organism might leverage death to its advantage. In fact, these organisms communicate with each other in a process called quorum sensing, leading some cells to secrete an extracellular death factor. They also use this process when they develop biofilms. Some fungi are single-celled and others are multicellular. Forms of programmed cell death are used by the multicellular fungi too. Other examples of quorum sensing are seen in the protista.

This chapter considers two of the most important legacies of the Lederbergs’ pioneering work: the discoveries of the model organisms that would dominate molecular biology, E. coli and λ‎ bacteriophage. The Lederbergs’ introduction of E. coli as a convenient model organism shifted the direction of molecular genetics. Barbara McClintock’s discovery of jumping genes remained unappreciated for decades, until the field of molecular biology caught up to validate her transposable elements in bacteria. The discovery of restriction enzymes—the molecular scissors for precisely cutting DNA at specific sites, a prerequisite for genetic recombination techniques—emphasized the versatility of bacteriophage λ‎ as a powerful experimental tool. The discovery of specialized transduction by Larry Morse and Esther Lederberg hinted at the mechanisms of “host restriction.” Werner Arber and Daisy Dussoix discovered restriction endonucleases by building upon Esther Lederberg’s research with λ‎ phage and the differences between E. coli B and K-12.

In this chapter, we discuss the origin and early evolution of genetic replication. The argument is complex, so we start with a brief outline. Section 4.2 discusses the nature of replication. We draw a distinction between simple replicators, limited hereditary replicators and indefinite hereditary replicators. Continued evolution requires indefinite hereditary replicators: it seems that such replicators depend on some form of template reproduction. In section 4.3, we point out that there is an error threshold for the accuracy of replication: for a given total quantity of genetic information–for example, for a fixed number of bases—there is an upper limit on the error rate of replication. If the error rate rises above this limit, natural selection cannot maintain the information. This leads to what we have called Eigen’s paradox. In the absence of specific enzymes, replication accuracy is low. Hence the total genome size must be small–almost certainly, less than 100 nucleotides. The genome is therefore too small to code for accurate replication machinery. There is a catch-22 situation: no enzymes without a large genome, and no large genome without enzymes. The next three sections discuss possible solutions to the paradox. Section 4.4 considers populations of replicating RNA molecules. We point out that the dynamics of replication are such as to lead to the stable coexistence of a diverse population, but we do not think that this constitutes a solution to the paradox. Section 4.5 discusses the hypercycle, a particular relationship between replicators that makes it possible for a greater total quantity of information to be maintained with a given accuracy of replication. We argue that the further evolution of hypercycles requires that they be enclosed within compartments, because otherwise they are sensitive to parasitic replicators. We also discuss, rather inconclusively, the possibility that, even in the absence of compartments, cooperation might evolve, by a processes analogous to kin selection, if the components of the hypercycle were confined to a surface. Finally, we discuss an alternative model, the stochastic corrector model. This also depends on the existence of compartments, but emphasizes the importance of stochastic effects arising if there are small numbers of each kind of molecule in a compartment. Essentially, small numbers serve to generate variation upon which selection can act.

The most fundamental distinction in biology is between nucleic acids, with their role as carriers of information, and proteins, which generate the phenotype. In existing organisms, nucleic acids and proteins mutually presume one another. The former, owing to their template activity, store the heritable information: the latter, by enzymatic activity, read and express this information. It seems that neither can function without the other. Which came first, nucleic acids or proteins? There are three possible answers: (1) nucleic acids; (2) proteins; (3) neither: they coevolved. In this chapter, we discuss various possible answers to this ‘chicken or egg?’ problem. In section 5.2, we discuss what seems to us the most likely answer, that at first RNA performed both functions, as replicator and enzyme. In section 5.3, we consider an alternative view, in which protein enzymes existed either before, or alongside, the first nucleic acids. In section 5.4, we ask whether, perhaps, the first replicators were not nucleic acids. Finally, in section 5.5, we ask why, given that the genetic message is carried by nucleic acids, there are only four nucleotides and two base pairs. So far, we have tacitly assumed nucleic acids preceeded proteins, without stating the main reason. Nucleic acids came first because they can perform both functions: they are replicable, and they can have enzymatic activity. For many years, a common opinion was that to be replicable almost amounted to self-replicative ability, but that it was far-fetched to assume enzymatic activity. Today, there is increasing evidence that RNA can act as an enzyme, but we are more aware of the difficulty of self-replication. It should have been expected on theoretical grounds that RNA could act as an enzyme: the possibility was discussed by Woese (1967), Crick (1968) and Orgel (1968). Consider first why proteins can act as enzymes. An enzyme has a well-determined three-dimensional structure of chemical groups that, in most cases, arises automatically from the primary structure. Substrates of the enzyme are bound by the chemical groups on the surface. This means that the reactants will be kept in close proximity, and hence experience a much higher local concentration of each other than in solution. This by itself increases the rate of the reaction.