Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin
Transformation
As discussed at the beginning of the Introduction, most organisms exhibit some mechanism for exchanging genes. The first demonstration of genetic exchange in bacteria was made by Fred Griffith in 1928. He was studying two variants of pneumococci, now called Streptococcus pneumoniae. One variant formed smooth-appearing colonies on plates and was pathogenic in mice. The other variant formed rough-appearing colonies on plates and did not kill mice. Only live, and not dead, smooth-colony-forming bacteria could cause disease, since the disease requires that the bacteria multiply in the infected mice. However, when Griffith mixed dead smooth-colony formers with live rough-colony formers and injected the mixture into mice, the mice became sick and died. Moreover, he isolated live smooth-colony formers from the dead mice. Apparently, the dead smooth-colony formers were “transforming” some of the live rough-colony formers into the pathogenic, smooth-colony-forming type. The “transforming principle” given off by the dead smooth-colony formers was later shown to be DNA, since addition of purified DNA from the dead smooth-colony formers to the live rough-colony formers in a test tube transformed some members of the rough type to the smooth type (see Avery et al., Suggested Reading). This method of exchange is called transformation (see chapter 6), and this experiment provided the first direct evidence that genes are made of DNA. Later experiments by Alfred Hershey and Martha Chase in 1952 (see Suggested Reading) showed that phage DNA alone is sufficient to direct the synthesis of more phages.
Conjugation
In 1946, Joshua Lederberg and Edward Tatum (see Suggested Reading) discovered a different type of gene exchange in bacteria. When they mixed some strains of E. coli with other strains, they observed the appearance of recombinant types that were unlike either parent. Unlike transformation, which requires only that DNA from one bacterium be added to the other bacterium, this means of gene exchange requires direct contact between two bacteria. It was later shown to be mediated by a genetic element that replicated separately from the chromosome, called a plasmid, in a process that was subsequently called conjugation (see chapter 5).
Transduction
In 1953, Norton Zinder and Joshua Lederberg (see Suggested Reading) discovered yet a third mechanism of gene transfer between bacteria. They showed that a phage of Salmonella enterica serovar Typhimurium could carry DNA from one bacterium to another. This means of gene exchange is called transduction and is now known to be quite widespread (see chapter 7).
Recombination within Genes
At the same time, experiments with bacteria and phages were also contributing to the view that genes were linear arrays of nucleotides in the DNA. By the early 1950s, recombination had been well demonstrated in higher organisms, including fruit flies. However, recombination was thought to occur only between mutations in different genes and not between mutations in the same gene. This led to the idea that genes were like “beads on a string” and that recombination is possible between the “beads,” or genes, but not within a gene. In 1955, Seymour Benzer disproved this hypothesis by using the power of phage genetics to show that recombination is possible within the rII genes of phage T4. He mapped numerous mutations in the rII genes, thereby demonstrating that genes are linear arrays of mutable sites in the DNA. Later experiments with other phage and bacterial genes showed that the sequence of nucleotides in the DNA directly determines the sequence of amino acids in the protein product of the gene.
Semiconservative DNA Replication
In 1953, James Watson and Francis Crick published their structure of DNA. One of the predictions of this model is that DNA replicates by a semiconservative mechanism, in which specific pairing occurs between the bases in the old and the new DNA strands, thus essentially explaining heredity. In 1958, Matthew Meselson and Frank Stahl used bacteria to confirm that DNA replicates by this semiconservative mechanism.
mRNA
The existence of mRNA was also first indicated by experiments with bacteria and phages. In 1961, Sydney Brenner, François Jacob, and Matthew Meselson used phage-infected bacteria to show that ribosomes are the site of protein synthesis and confirmed the existence of a “messenger” RNA that carries information from the DNA to the ribosome.
The Genetic Code
Also in 1961, Francis Crick and his collaborators used phages and bacteria to show that the genetic code is unpunctuated, three lettered, and redundant. These researchers also showed that not all possible codons designate an amino acid and that some are “nonsense.” These experiments laid the groundwork for Marshall Nirenberg and his collaborators to decipher the genetic code, in which a specific three-nucleotide set encodes one of 20 amino acids. The code was later verified by the examination of specific amino acid changes due to mutations in the lysozyme gene of phage T4.
The Operon Model
François Jacob and Jacques Monod published their operon model for the regulation of the lactose utilization genes of E. coli in 1961 as well. They proposed that a repressor blocks RNA synthesis on the lac genes unless the inducer, lactose, is bound to the repressor (see chapter 11). Their model has served to explain gene regulation in other systems, and the lac genes and regulatory system continue to be used in molecular genetic experiments, even in systems as far removed from bacteria as animal cells and viruses.
Enzymes for Molecular Biology
The early 1960s saw the start of the discovery of many interesting and useful bacterial and phage enzymes involved in DNA and RNA metabolism. In 1960, Arthur Kornberg demonstrated the synthesis of DNA in the test tube by an enzyme from E. coli. The next year, a number of groups independently demonstrated the synthesis of RNA in the test tube by RNA polymerases from bacteria. From that time on, other useful enzymes for molecular biology were isolated from bacteria and their phages, including additional RNA and DNA polymerases, polynucleotide kinase, DNA ligases, topoisomerases, and many phosphatases.
From these early observations, the knowledge and techniques of molecular genetics exploded. For example, in the early 1960s, techniques were developed for detecting the hybridization of RNA to DNA and DNA to DNA on nitrocellulose filters. These techniques were used to show that RNA is made on only one strand in specific regions of DNA, which later led to the discovery of promoters and other regulatory sequences. By the late 1960s, restriction endonucleases had been discovered in bacteria and shown to cut DNA in specific places (see Linn and Arber, Suggested Reading). By the early 1970s, these restriction endonucleases were being exploited to introduce foreign genes into E. coli (see Cohen et al., Suggested Reading), and by the late 1970s, the first human gene had been expressed in a bacterium. Also in the late 1970s, methods to sequence DNA by using enzymes from phages and bacteria were developed.
In 1988, a thermally stable DNA polymerase from a thermophilic bacterium was used to invent the technique called the polymerase chain reaction (PCR). This extremely sensitive technique allows the amplification of genes and other regions of DNA, facilitating their cloning and study. Thermally stable DNA polymerases are now an essential tool for genome sequencing.
Synthetic Genomics
More recently, advances in DNA synthesis and DNA recombination have been ushering in a new age of bacterial molecular genetics under the name of synthetic genomics, where massive strands of DNA large enough to comprise entire genomes can be made from the building blocks of DNA. In 2010,