Anecdotal , Historical and Critical Commentaries on Genetics Letting Escherichia coli Teach Me About Genome Engineering

A career of following unplanned observations has serendipitously led to a deep appreciation of the capacity that bacterial cells have for restructuring their genomes in a biologically responsive manner. Routine characterization of spontaneous mutations in the gal operon guided the discovery that bacteria transpose DNA segments into new genome sites. A failed project to fuse l sequences to a lacZ reporter ultimately made it possible to demonstrate how readily Escherichia coli generated rearrangements necessary for in vivo cloning of chromosomal fragments into phage genomes. Thinking about the molecular mechanism of IS1 and phage Mu-transposition unexpectedly clarified how transposable elements mediate large-scale rearrangements of the bacterial genome. Following up on lab lore about long delays needed to obtain Mu-mediated lacZ protein fusions revealed a striking connection between physiological stress and activation of DNA rearrangement functions. Examining the fate of Mudlac DNA in sectored colonies showed that these same functions are subject to developmental control, like controlling elements in maize. All these experiences confirmed Barbara McClintock’s view that cells frequently respond to stimuli by restructuring their genomes and provided novel insights into the natural genetic engineering processes involved in evolution. THIS article is the reminiscence of a bacterial geneticist studying the processes of mutation and DNA rearrangements. I want to emphasize how my experience was full of surprises and unplanned discoveries that took me ever deeper into the mechanisms and regulation of natural genetic engineering by Escherichia coli cells. For the benefit of younger molecular geneticists, there are at least three points to be made. First, you can find something truly novel only when you do not know exactly what you are looking for. If the experiment comes out just as you planned, you have not really learned anything you did not already know or suspect. Second, routine characterization of your experimental material is critical because it will tell you where your understanding is incomplete—but only when the characterizations do not come out as you expect. In other words, it can be a good thing if an experimental result does not fit your expectations. Third, science will inevitably lead us in the future to think about the subjects that we are studying in ways that we cannot currently predict. When I began my research, we thought we understood the basics of genome expression and mutation because we knew about DNA, RNA polymerase, and the triplet code for amino acids. The worlds of transcriptional regulation beyond simple repressor–operator models, signal transduction, chromatin formatting, transcript processing, protein modifications, and regulatory RNAs were all in the future. In my particular field, the molecular basis of genetic change, discoveries about mobile genetic elements, reverse transcription, programmed genome rearrangements, and other aspects of what I call ‘‘natural genetic engineering’’ were yet to be made. The following account relates my own experimental journey into a new way of thinking about the molecular and cellular basis of genetic change. After detailing the journey, I will explain why and how I believe that this new mode of thought is likely to influence our ideas about evolution, the most basic of biological subjects. I would be remiss if I did not acknowledge the powerful influence of Barbara McClintock on my thinking. After meeting her in 1976, I realized that she possessed an unmatched depth of experience about all aspects of biology, from natural history to the current status of molecular genetics. We engaged in a 16-year dialogue up to her death in 1992 (Shapiro 1992c). Only after many years did I finally come to appreciate the wisdom of her insistence that the ability of cells to sense and respond to ‘‘genome shock’’ was just as important in determining what happens to their genomes as are the biochemical Address for correspondence: Department of Biochemistry and Molecular Biology, University of Chicago, Gordon Center for Integrative Science, 979 E. 57th St., Chicago, IL 60637. E-mail: jsha@uchicago.edu Genetics 183: 1205–1214 (December 2009) mechanisms that they use in repairing and restructuring DNA molecules (McClintock 1984). DISCOVERING DNA INSERTIONS When I was a student at the University of Cambridge in the mid-1960s, I planned to study the effect of transcription on mutagenesis. Sydney Brenner at the MRC Laboratory for Molecular Biology suggested that a positive selection system for loss-of-function mutations would be the most sensitive experimental arrangement. The E. coli gal operon offered such a system in which the resulting mutations could easily be analyzed. A defect in the galU locus (Figure 1) made the bacteria sensitive to galactose (Gal phenotype). These galU mutants accumulated Gal-1-P, and the phosphorylated sugar inhibited growth. However, when the bacteria lost the galK-encoded galactokinase activity, they no longer accumulated Gal-1-P and shifted from a Gal to a Gal phenotype. Therefore, GalK mutants could be selected as colonies on medium containing galactose and glycerol. Since galU was not linked to the galETK operon (Shapiro 1966), it was easy to separate the GalK-inactivating mutations from the original galU mutation for reversion studies and mapping. Following this plan, I isolated a series of spontaneous Gal mutants and proceeded to characterize the underlying gal operon mutations (Adhya and Shapiro 1969). Of almost 200 spontaneous GalK-inactivating mutations, 14 proved to be pleiotropic or polar and inactivated at least two of the gal operon’s three cistrons. Of these, 10 failed to revert, and most of those were easy to interpret as deletions, which was later confirmed by mapping studies (Shapiro and Adhya 1969). But the pleiotropic gal mutations that reverted proved to be hard to understand using existing ideas about the mechanisms of genetic change. They reverted spontaneously and could be mapped to upstream regions of the galETK operon (Figure 1), but they were not traditional point mutations because reversion was not increased by either base substitution or frameshift mutagens. Other pleiotropic gal mutations isolated by the Lederberg and Starlinger groups behaved similarly (Morse 1967; Saedler and Starlinger 1967). As I wrote my thesis in 1967, it occurred to me that the mysterious pleiotropic gal mutations might result from insertion of extra DNA into the operon (Shapiro 1967). For my postdoc, I went to the Institut Pasteur, where I learned to do CsCl density-gradient centrifugation so that I could confirm the insertion hypothesis using ldgal transducing phages: the ldgal particles carrying the mutations had extra DNA and were denser than the parental phage (Figure 2; Shapiro 1969; Cohen and Shapiro 1980). The Starlinger group quickly repeated these results ( Jordan et al. 1968), and electron microscope heteroduplex studies subsequently showed that the same DNA segments inserted into various positions in the gal and lac operons (Fiandt et al. 1972; Hirsch et al. 1972). Thus, we learned that bacteria have the capacity to move specific segments of DNA through the genome, and these segments came to be called ‘‘insertion sequences’’ or IS elements (Starlinger and Saedler Figure 1.—The gal and trp regions of the E. coli chromosome. The dashed line indicates .400 kb between the two operons. This distance makes galU mutations easy to separate from gal operon mutations that inactivate galK expression. The positions of four strongly polar insertion mutations are indicated by the vertical arrows. The figure is based on the deletion mapping of Shapiro and Adhya (1969). Figure 2. —The results of CsCl equilibrium density-gradient centrifugation of a mixture of l-phages (l 857, plaqueforming phage; l dg, phage carrying the wild-type gal operon; l dgS114 , phage carrying an insertion mutation). The fractions on the left contain the densest particles. From Shapiro (1969) with permission. 1206 J. A. Shapiro