Molecular Genetics of Bacteria, 4th Edition

Book Preface

WITH THE ADDITION OF TWO NEW COAUTHORS, the fourth edition of the textbook Molecular Genetics of Bacteria has been substantially revised and some new sections have been added. We tried to do this without increasing the length of the book, which, at more than 700 pages, was already quite long. While the book retains the same number and order of chapters, many topics have been moved or integrated more completely into the text to reflect a more modern perspective. The purpose was to convey more accurately how one approaches questions in modern bacterial genetics, using the full repertoire of methods now available. Also, to make room for the new material, we made the philosophical decision to condense or eliminate descriptions of methods where they seemed unnecessarily detailed for a textbook.

Chapter 1, on DNA structure, DNA replication, and chromosome segregation, was expanded to include updates in our understanding, including how replication proceeds through obstacles typically found during normal DNA replication in bacteria, while some aspects of repair-associated replication were moved to later chapters. The chapter was also significantly expanded with new information about how numerous cell processes coordinate for the efficient processing and organizing of chromosomes after DNA replication. Scientists now more fully appreciate how sequences “hidden” in the structure guide a variety of systems that aid in repairing, segregating, packaging, and pumping the chromosome for exquisite genome stability in bacteria. In chapter 2, which covers bacterial gene expression, the translation section has been reorganized to follow the same order as the transcription section. It begins with initiation of translation and then discusses elongation followed by termination, rather than following the more historical order with the genetic code coming first. We reasoned that this order makes more sense since most students already have had some exposure to translation and the genetic code. More information on RNA degradation is now included, and the sections on gene regulation have been moved to chapter 12. The protein transport section has been moved from chapter 2 to chapter 14 (see below), where it can be better integrated with other topics of protein export. Chapter 3, on bacterial genetic analysis, also now takes a less historical approach. Rather than beginning with a review of classical genetic analysis and then contrasting it with bacterial genetic analysis as in previous editions, the chapter now begins with bacterial genetic analysis, again assuming that students have already had some general genetics. Furthermore, rather than putting more recently developed methods such as site-specific mutagenesis, recombineering, etc., into a separate section, we have integrated throughout the text all the methods available nowadays to use in a genetic analysis. The discussion of mapping by Hfr crosses has been sharply condensed, since it is likely that no one will ever again perform the laborious task of constructing the genetic map of a bacterium. The relative ease of DNA sequencing now allows the placing of mutations on the sequenced genomes of bacteria by direct comparison of sequences rather than by Hfr mapping. Transduction and transformation (including electroporation) are used extensively for genetic manipulations, so their use is still covered in some detail. Chapter 4 has been updated with more information about how plasmids are typically used in the laboratory setting in work with model organisms and beyond as well, including updates on our understanding of partitioning systems. Chapter 5 was extensively updated to illustrate the hodgepodge organization of conjugal elements and advances in our understanding of conjugation and to more fully integrate the important role of integrating conjugative elements in bacterial genomes (including a focus on one of these elements from B. subtilis on the front cover). Chapter 6 is updated throughout and focuses on similarities and differences between different transformation systems. The bacteriophage chapters (chapters 7 and 8) have been updated, and new material has been added. Some highlights from phage genomics are now covered, as are phage defense mechanisms, including CRISPR. The section on phage lysis is expanded, as is the text box on phage display, whose power is now demonstrated with some current uses. The interaction between lysogenic phages and genetic islands has been updated and moved into the text, as have some more recently developed techniques using lysogenic phages, for example, in detecting protein-protein interactions. Chapter 9, which covers transposable elements and site-specific recombination, has been updated to clarify the basic molecular biology of these elements, and it includes updated sections describing how they are used in the laboratory today. Chapter 10 was significantly reorganized to stress the role of homologous recombination in the repair of DNA double-strand breaks that occur at interruptions in the template DNA during replication. The role of homologous recombination in repair explains the underpinning of the evolution of the process and also clarifies how the process works in concert with DNA replication.
A more comprehensive treatment for how DNA double-strand breaks are repaired across different types of bacteria, using systems found in all domains of life, is also included. Chapter 11 was updated to discuss many advances in the field of repair, including an expanded understanding of the regulation of multiple DNA polymerases found in bacteria with the SOS response. Chapters 12 and 13 have been reorganized so that chapter 12 is now focused on mechanisms of regulation of individual genes and operons and chapter 13 is mostly concerned with examples of global regulatory systems that utilize these mechanisms. There is also more emphasis on posttranscriptional regulation in both chapters, and global regulatory mechanisms in Escherichia coli are contrasted with those in Bacillus subtilis. Chapter 14 is probably the most changed chapter. It now contains our entire discussion of protein export, including the Sec and Tat systems as well as the secretion systems of gramnegative (i.e., Proteobacteria) and gram-positive (i.e., Firmicutes) bacteria. Most notably, it now contains a new section on bacterial cell biology, including cell wall synthesis and cell division and their regulation, as well as a new box on the evolution of cytoskeletal filaments, and it introduces the use of Caulobacter crescentus as a model system for these studies. Chapter 14 finishes with sporulation in B. subtilis, probably the best understood bacterial developmental system.

As in earlier editions, we do not mention the names of most investigators who have made major contributions to bacterial molecular genetics. We include only those names that have become icons in the field because they are associated with certain seminal experiments (e.g., Meselson and Stahl or Luria and Delbrück), models (e.g., Jacob and Monod), or structures (e.g., Watson and Crick). Many other names are available in the suggested reading lists, where we give some of the original references to the developments under discussion, and in the credit lines for sources of figures and tables, which are given at the end of the book.

Again we are indebted to a number of people who helped us in various ways. Some read sections of the book at our request and made valuable suggestions. Some, who have used the book for teaching, have pointed out ways to make it more useful for them and their students. Others have noticed factual errors or errors of omission and have pointed out references that helped us check our facts. In addition to those who commented on earlier editions, many of whose contributions have carried over, this list includes Dennis Arvidson, Dominique Belin, Melanie Berkmen, Helmut Bertrand, Lindsay Black, Rob Britton, Yves Brun, Rich Calendar, George Chaconas, Dhruba Chattoraj, Carton Chen, Todd Ciche, Laszlo Csonka, Gary Dunny, Marie Elliot, Laura Frost, Barbara Funnell, Peter Geiduschek,

Graham Hatfull, John Helmann, Ann Hochschild, Susan Lovett, Ken Marinas, Norman Pace, Steven Sandler, Joel Schildbach, Linda Sherwood, Chris Waters, Robert Weiss, Joanne Willey, Steve Winans, Ry Young, and Steve Zinder. Special thanks go to Lee Kroos, who agreed to update an entire section. Yet others furnished original figures that we could incorporate into the text; some of them are mentioned in the figure credits. However, in the end, any mistakes and omissions were all ours.

As with the first three editions, it was a great pleasure to work with the professionals at ASM Press. The former director of ASM Press, Jeff Holtmeier, helped us prepare for the fourth edition. We have been fortunate to continue to work with Kenneth April, the production manager, who coordinated the entire project. We have also had the good fortune to work again with two of the same professionals who did a masterful job with the first three editions: Susan Brown Schmidler, who created the book and cover design; Terese Winslow, who created the cover illustration; and Elizabeth McGillicuddy, who copy edited the manuscript. We also thank Patrick Lane of ScEYEnce Studios for bringing an attractive aestheticism to the rendering of our hand-drawn illustrations into the final figures.

Larry Snyder Joe Peters Tina Henkin Wendy Champness

About the Authors

Larry (Loren R.) Snyder, PhD, is a professor emeritus of microbiology and molecular genetics at Michigan State University, where he taught microbial genetics and microbiology to undergraduate and graduate students for about 40 years. He received his BS in mathematics and zoology at the University of Minnesota in Duluth and his PhD in biophysics at the University of Chicago before doing postdoctoral work at the International Laboratory of Genetics and Biophysics in Naples, Italy, and at the Curie Institute and Faculty of Sciences at the University of Paris as a Jane Coffin Childs postdoctoral fellow. He was a visiting professor at Harvard University and the University of Tel Aviv. Most of his research was on the interaction between bacteriophage T4 and its host, Escherichia coli, and was supported by the National Science Foundation (NSF) and the National Institutes of Health (NIH). At Michigan State, he served as acting chair of the Department of Microbiology for one year and was a founding co-principal investigator of the NSF Center for Microbial Ecology and director of the Howard Hughes Medical Institute Undergraduate Research Program. He was awarded the College of Natural Science Alumni Association Meritorious Faculty Award in 2002.

Joseph E. Peters, PhD, is an associate professor of microbiology at Cornell University, where he has been teaching bacterial genetics and microbiology since 2002. He received his BS from Stony Brook University and his PhD from the University of Maryland at College Park. He did postdoctoral work at the Johns Hopkins University School of Medicine, in part as an NSF-Alfred P. Sloan Foundation postdoctoral research fellow in molecular evolution. His research has focused on the intersection of DNA replication, recombination, and repair and how it relates to evolution, especially in the area of transposition. Research in his laboratory is funded by the NSF and NIH. He is the chair of the advisory board for the NSF-funded E. coli

Genetic Stock Center, the chair of the American Society for Microbiology’s Division of Genetics and Molecular Biology, and the director of graduate studies for the field of microbiology at Cornell.

Tina M. Henkin, PhD, is a professor of microbiology, chair of the Department of Microbiology, and Robert W. and Estelle S. Bingham Professor of Biological Sciences at Ohio State University, where she has been teaching bacterial genetics and microbiology since 1995. She received her BA in biology at Swarthmore College and her PhD in genetics at the University of Wisconsin—Madison, and she did postdoctoral work at the Tufts University School of Medicine. Her research focuses on gene regulation and regulatory RNAs in bacteria. Research in her laboratory is funded by the NIH. She is a fellow of the American Academy of Microbiology, the American Association for the Advancement of Science, and the American Academy of Arts and Sciences; a member of the National Academy of Sciences; and a cowinner of the National Academy of Sciences Pfizer Prize in Molecular Biology for her work on riboswitch RNAs.

Wendy Champness, PhD, is a professor emerita of microbiology and molecular genetics at Michigan State University, where she taught microbial genetics and microbiology to undergraduate and graduate students for more than 25 years. She received her BS and PhD degrees at Michigan State, where she was an NSF predoctoral fellow. She did postdoctoral work at the Massachusetts Institute of Technology as a Jane Coffin Childs postdoctoral fellow and was a visiting scientist at the John Innes Research Centre in Norwich, United Kingdom, and at the University of Tel Aviv. Most of her research was on the regulation of antibiotic synthesis genes in Streptomyces, and research in her laboratory was supported by grants from the NSF and NIH. She was a charter member of the NSF Center for Microbial Ecology at Michigan State and was a member of the editorial board of the Journal of Bacteriology for 12 years as well as an associate editor of the journal Microbiology.

Introduction

THE GOAL OF THIS TEXTBOOK is to introduce the student to the field of bacterial molecular genetics. From the point of view of genetics and genetic manipulation, bacteria are relatively simple organisms. There also exist model bacterial organisms that are easy to grow and easy to manipulate in the laboratory. For these reasons, most methods in molecular biology and recombinant DNA technology that are essential for the study of all forms of life have been developed around bacteria. Bacteria also frequently serve as model systems for understanding cellular functions and developmental processes in more complex organisms. Much of what we know about the basic molecular mechanisms in cells, such as translation and replication, has originated with studies of bacteria. This is because such central cellular functions have remained largely unchanged throughout evolution. Ribosomes have similar structures in all organisms, and many of the translation factors are highly conserved. The DNA replication apparatuses of all organisms contain features in common, such as sliding clamps and editing functions, which were first described in bacteria and their viruses, called bacteriophages. Chaperones that help other proteins fold and topoisomerases that change the topology of DNA were first discovered in bacteria and their bacteriophages. Studies of repair of DNA damage and mutagenesis in bacteria have also led the way to an understanding of such pathways in eukaryotes. Excision repair systems, mutagenic polymerases, and mismatch repair systems are remarkably similar in all organisms, and defects in these systems are responsible for multiple types of human cancers.

In addition, as our understanding of the molecular biology of bacteria advances, we are finding a level of complexity that was not appreciated previously. Because of the small size of the vast majority of bacteria, early on, it was impossible to recognize the high level of organization that exists in bacteria, leading to the misconception that bacteria were merely “bags of enzymes,” where small size allowed passive diffusion to move cellular constituents around. However, it is now clear that positioning of enzymes within the bacterial cell is highly controlled. For example, despite the lack of a specialized membrane structure called the nucleus (the early defining feature of the “prokaryote” [see below]), the genome of bacteria is exquisitely organized to facilitate its repair and expression during DNA replication. In addition, advances facilitated by molecular genetics and microscopy have made it clear that many cellular processes occur in highly organized subregions within the cell. Once it was appreciated that bacteria evolved in the same basic way as all other living organisms, the relative simplicity of bacteria paved the way for some of the most important scientific advances in any field, ever. It is safe to say that a bright future awaits the fledgling bacterial geneticist, where studies of relatively simple bacteria, with their malleable genetic systems, promise to uncover basic principles of cell biology that are common to all organisms and that we can now only imagine. However, bacteria are not just important as laboratory tools to understand other organisms; they are important and interesting in their own right. For instance, they play an essential role in the ecology of Earth. They are the only organisms that can “fix” atmospheric nitrogen, that is, convert N2 to ammonia, which can be used to make nitrogen-containing cellular constituents, such as proteins and nucleic acids. Without bacteria, the natural nitrogen cycle would be broken. Bacteria are also central to the carbon cycle because of their ability to degrade recalcitrant natural polymers, such as cellulose and lignin. Bacteria and some types of fungi thus prevent Earth from being buried in plant debris and other carbon-containing material. Toxic compounds, including petroleum, many of the chlorinated hydrocarbons, and other products of the chemical industry, can also be degraded by bacteria. For this reason, these organisms are essential in water purification and toxic waste cleanup. Moreover, bacteria and archaea (see below) produce most of the naturally occurring so-called greenhouse gases, such as methane and carbon dioxide, which are in turn used by other types of bacteria. This cycle helps maintain climate equilibrium. Bacteria have even had a profound effect on the geology of Earth, being responsible for some of the major iron ore and other mineral deposits in Earth’s crust. Another unusual feature of bacteria and archaea is their ability to live in extremely inhospitable environments, many of which are devoid of life except for microbes. These organisms are the only ones living in the Dead Sea, where the salt concentration in the water is very high. Some types of bacteria and archaea live in hot springs at
temperatures close to the boiling point of water (or above in the case of archaea), and others survive in atmospheres devoid of oxygen, such as eutrophic lakes and swamps. Bacteria that live in inhospitable environments sometimes enable other organisms to survive in those environments through symbiotic relationships. For example, symbiotic bacteria make life possible for Riftia tubeworms next to hydrothermal vents on the ocean floor, where living systems must use hydrogen sulfide in place of organic carbon and energy sources. In this symbiosis, the bacteria obtain energy and fix carbon dioxide by using the reducing power of the hydrogen sulfide given off by the hydrothermal vents, thereby furnishing food in the form of high-energy carbon compounds for the worms, which lack a digestive tract. Symbiotic cyanobacteria allow fungi to live in the Arctic tundra in the form of lichens. The bacterial partners in the lichens fix atmospheric nitrogen and make carbon-containing molecules through photosynthesis to allow their fungal partners to grow on the tundra in the absence of nutrient-containing soil. Symbiotic nitrogen- fixing Rhizobium and Azorhizobium spp. in the nodules on the roots of legumes and some other types of higher plants allow the plants to grow in nitrogen- deficient soils. Other types of symbiotic bacteria digest cellulose to allow cows and other ruminant animals to live on a diet of grass. Bioluminescent bacteria even generate light for squid and other marine animals, allowing illumination and signaling in the darkness of the deep ocean.

Bacteria are also worth studying because of their role in disease. They cause many human, plant, and animal diseases, and new diseases are continuously appearing. Knowledge gained from the molecular genetics of bacteria helps in the development of new ways to treat or otherwise control old diseases that can be resistant to older forms of treatment, as well as emerging diseases. Some bacteria that live in and on our bodies also benefit us directly. The role of our commensal bacteria in human health is only beginning to be appreciated. It has been estimated that of the 1014 cells in a human body, only 10% are human! Of course, bacterial cells are much smaller than our cells, but this shows how our bodies are adapted to live with an extensive bacterial flora, which helps us digest food and avoid disease, among other roles, many of which are yet to be uncovered. Bacteria have also long been used to make many useful compounds, such as antibiotics, and chemicals, such as benzene and citric acid.

Bacteria and their bacteriophages are also the source of many of the useful enzymes used in molecular biology.

In spite of substantial progress, we have only begun to understand the bacterial world around us. Bacteria are the most physiologically diverse organisms on Earth, and the importance of bacteria to life on Earth and the potential uses to which bacteria can be put can only be guessed at. Thousands of different types of bacteria are known, and new insights into their cellular mechanisms and their applications constantly emerge from research with bacteria. Moreover, it is estimated that less than 1% of the types of bacteria living in the soil and other environments have ever been isolated, including entire phyla that have been identified using culture-independent mechanisms (see Pace, Suggested Reading). Undiscovered bacteria may have all manner of interesting and useful functions. Clearly, studies of bacteria will continue to be essential to our future efforts to understand, control, and benefit from the biological world around us, and bacterial molecular genetics will be an essential tool in these efforts. However, before discussing this field, we must first briefly discuss the evolutionary relationship of the bacteria to other organisms.