1-16 Work on microbes paves the way to taking control of the genome

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• E. coli continues to help scientists answer many basic questions about biology and is a indispensable tool in genetics and genomics.
• Joshua Lederberg discovered plasmids, small, circular, self-replicating pieces of DNA.
• The discovery of restriction enzymes allowed scientists to build chimeric DNA molecules, and gave birth to the technique of cloning. It was now possible to isolate any DNA molecule, mix it with a plasmid and created recombinant molecules. These could be grown in E.coli, and other microbes, to make as many copies as the scientist needed.
• The polymerase chain reaction allows the amplification of any DNA sequence over a million fold. °CR has found hundreds of applications in science and medicine.
• DNA sequencing, independently developed by Walter Gilbert and Fred Sanger, has allowed the determination of the sequence of millions of base pairs of DNA. Genomic sequences of organisms, including humans and many important pathogens, are generating new insights into us, as well as the diseases caused by certain microbes. This large amount of data has spawned the fields of genomics and bioinformatics.

One final thread we will trace is the rise of molecular microbiology and the technique of genomics. This pathway begins in 1885 with the seemingly unimportant isolation of a common intestinal microorganism by Theodor Escherich. The microbe is eventually renamed Escherichia coli in his honor. It could not have been understood at the time, but this bacterium would play a central role in our understanding of life. E. coli achieved this position because it has several properties that made it invaluable as an experimental system:

1. E. coli is easy to take care of. It can grow on a wide variety of media, at temperatures ranging from 20-45 °C, in the presence or absence of air.
2. E. coli grows quickly. Under normal laboratory conditions it goes through a complete life cycle and double its population about every 30 minutes. With this rapid growth rate it is possible to generate large populations in just a few hours. Most experiments can be set up one day and the results observed the next.
3. It can survive storage for long periods of time without losing viability using relatively simple preservation techniques.
4. It was available. There is an element of arbitrariness that this microbe came to serve such a vital role in biology. Many other bacteria probably could have filled this position, but E. coli was the one chosen.

Several key discoveries laid the foundation for future developments in molecular biology. The first of these was the discovery of autonomously replicated pieces of DNA separate from the bacterial chromosome. plasmids, as Joshua Lederberg called them, were capable of carrying genes that could be regulated and moved from one microbe to another independently of the bacterial chromosome. The ability of some plasmids to move between different cells meant that different bacteria could rapidly inherit genes carried on these plasmids. It was found that one class of plasmids encoded resistance genes that would enable the bacteria to grow in the presence of an antibiotic. It later became clear it was possible to select for the presence of these plasmids by demanding growth of the bacterial strain on medium containing the antibiotic to which the plasmid encoded resistance.

A second discovery that greatly impacted modern genetic engineering was that bacteria have their own parasites. As mentioned earlier, there are viruses that infect and kill bacteria, just as viruses infect animals and plants. The analysis of bacterial viruses was a major avenue to understanding the bacteria themselves.

For example, investigation of the ability of some viruses to grow well on certain strains of bacteria and not others lead to the discovery of restriction-modification systems in bacteria. These systems allow bacteria to recognize and destroy foreign DNA and consist of two parts. A modification enzyme that labels DNA as belonging to the bacterium and a restriction enzyme that recognizes a certain 4-8 base pair sequence in the DNA and cuts it in two if it has not been modified. The landmark paper of Hamilton Smith and Kent Wilcox showed that restriction enzymes were capable of cleaving double-stranded DNA into discrete pieces. The authors correctly hypothesized that the enzyme was recognizing a sequence of DNA and cutting it.

Further studies on restriction enzymes led to the birth of genetic engineering. Janet Mertz and Ronald Davis made restriction enzymes into tools for the manipulation of DNA when they showed that many of the breaks these enzymes made in the DNA produced single-stranded complementary ends. Stanley Cohen, Annie Chang, Robert Helling and Herbert Boyer then demonstrated that any DNA can be broken into fragments with restriction enzymes and by mixing that with a plasmid digested in a similar manner, it was possible to create recombinant molecules. Moving the plasmid into a microbe and growing it on selective medium could produce any desired amount of these recombinant molecules.

Another discovery was "brewing" in the western United States. In the mid 1960s, Thomas Brock was on a trip to Yellowstone and became intrigued by the mats of green, brown and pink material that were found in many of the hot springs. Brock was sure that these were living communities of microorganisms, yet the springs were at temperatures near boiling. Subsequent research proved his hypothesis correct and lead to the isolation of a microbe, Thermus aquaticus, capable of growth at 85 °C.

Organisms capable of growing at this high a temperature were unheard of at the time and the discovery spawned two major developments. First, work with T. aquaticus and other unusual microbes by Woese and many others lead to the discovery of the Archaea. Second, the enzyme responsible for replicating DNA in T. aquaticus was found to be extremely heat stable, surviving temperatures of 95 °C and copying DNA at 72 °C. This enzyme allowed the following important breakthrough. In the early '70s, H. Gobind Khorana and colleagues had suggested a method to amplify DNA in a test tube. As they envisioned it, the method used short oligonucleotides, DNA polymerase and repeated cycles of heating and cooling. However, the polymerase was killed during each heating step, so massive amounts of prohibitively expensive enzyme were required. When Kary Mullis reinvented the method in 1985, the thermostable polymerase of T. aquaticus was readily available, which made the procedure wildly easier and cheaper to apply. He termed it polymerase chain reaction (always referred to as °CR) and it has become a pivotal technique in many areas of science, from detecting bacteria in the environment to the analysis of evidence at crime scenes.

In 1977 Walter Gilbert and Fred Sanger independently developed methods for determining the exact sequence of bases in DNA. These techniques became immediately useful in determining the sequence of numerous important genes being investigated in countless laboratories.

Further refinement has made DNA sequencing more efficient. It became efficient enough that in 1985 Robert Sinsheimer convened a meeting of a number of biologists active in genetics and gene mapping. Out of this meeting came a proposal to sequence the entire three billion base pairs of the human genome. This was equivalent at the time of proposing a mission to the moon, technically possible, but with unpredictable value. The initial cost of the project was estimated to be $10 a base pair or a staggering 30 billion dollars. There was resistance from a significant portion of the scientific community, fearing that the project would siphon funds away from other worthy scientific projects. However, the clarity of the goal ignited the imagination of Congress and the public, creating unstoppable momentum for the project. After about a decade of effort, a preliminary draft of the entire genome was released in the spring of 2001, earlier than projected and dramatically under budget. The project itself drove the development of sequencing technology, allowing the determination of millions of base pairs of sequence per day at a cost of less than 3 cents per base at large sequencing facilities.

Along with the human genome, hundreds of other organisms have already been sequenced and there will be thousands of sequenced genomes before the decade is out. These include the genomes of many pathogenic bacteria, the mouse, the fruit fly and the nematode Caenorhabditis elegans, the last being an experimental model for development in eukaryotes. The analysis and application of this sequence data to investigate biological problems has resulted in the development of the field of of genomics and bioinformatics. In their infancy, these fields are already producing astonishing findings that are accelerating the progress of fields such as evolutionary biology, immunology, bacterial pathology, bacterial physiology and cancer treatment. Figure 1-26 lists important events in understanding the genome.

Let's end this chapter with a defense of fundamental scientific research. That is, research without an obvious applied goal or application. If you look at the initial results generated in many of these important analyses, the research would have seemed esoteric and inconsequential. Is the analysis of microbes in the colon important? Who cares why a virus can grow in one strain of bacteria and not another? Why should the government support Tom Brock's isolation of microbes from the hot springs of Yellowstone National Park? What use is knowing the DNA sequence of a virus or a bacterial operon? Yet, each of these insights began a thread of inquiry that changed the world. The take-home message is that basic research creates insights and applications beyond the imagination of even the scientists performing the work and should be supported.

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