Textbook assignment: Chapter 7, pages 178-215.
Major concepts
Introduction: Chapter 7 of our textbook provides a relatively detailed introduction to genetic manipulations in bacteria and also describes interactions between bacteria and their viruses (bacteriophage). This material provides a foundation for studies on the control of expression of specific genes in bacteria and their viruses, (chapter 8), and on recombinant DNA and gene cloning (Chapter 9), as well as later studies on genetic fine structure (parts of chapter 16). Because many of these topics will be taken up in much greater detail in MCDB 3500, we will move through some of them rather quickly here. You should concentrate your efforts on the materials that are covered in the notes.
Methods for culture of bacteria: Chapter 7 begins with a description of basic techniques for isolation and culture of pure strains of bacteria in the laboratory. Colony formation on a sterile semisolid medium (usually nutrient agar) allows the progeny of a single bacterium to form a visible colony containing large numbers of cells that are all derived from the original cell, and thus are all genetically identical (except for rare mutations that may have occurred during their multiplication). Such a population, consisting of many identical cells derived from a single cell, is referred to as a clone. Larger populations of genetically uniform bacteria can be obtained by transferring a single colony to a liquid culture medium (Figure 7.1).
Cloning of DNA: A few lectures from now, we will start describing the cloning of DNA sequences. The process is similar -- to isolate a single DNA sequence under conditions where many identical copies of that sequence can be obtained through appropriate experimental manipulations.
Genetic markers: In order to study genetics of bacteria, it is necessary to be able to see phenotypic properties associated with specific mutations or other inherited differences. Most of the markers used in bacterial genetics are based on ability to multiply under a particular set of conditions or ability to carry out a particular metabolic process. Culture media that support growth of some strains and not of others are referred to as selective media. Wild type bacteria are often able to grow on minimal media that consist only of an appropriate reduced carbon energy source, such as glucose, and an adequate mixture of inorganic ions. The common intestinal bacterium, Escherichia coli (E. coli), will be used as an example here. (Please note that despite extensive recent publicity about certain relatively rare strains of E. coli that are highly pathogenic, typical laboratory strains are not dangerous).
Prototrophs and auxotrophs: A wild type strain that has minimal requirements for exogenously supplied nutrients is refered to as a prototroph . A mutant strain that has lost the ability to synthesize its own supply of a particular nutrient, such as histidine or adenine or thiamine is called an auxotroph . While the prototroph can be grown on a minimal medium, a histidine auxotroph will not grow on that medium unless histidine is added. Similarly, an adenine auxotroph must have adenine added to its culture medium, etc.
Ability to utilize substrates: The ability to utilize carbon and energy sources other than glucose often requires induced expression of enzymes that are not otherwise present. Mutations either in the genes coding for those enzymes or in the regulatory systems that are involved in their activation can result in loss of ability to multiply on the alternative substrates. Such mutations (for example, loss of ability to use lactose as an alternative to glucose) are widely used as genetic markers. In the next lecture, we will examine in detail the mechanisms that control expression of the enzymes needed for utilization of lactose.
Antibiotic resistance and other markers: The ability to survive and multiply in the presence of an antibiotic is a third type of genetic marker that can be used. Such markers are often particularly useful for selecting rare transformants among a much larger population of non-transformed cells. Other markers, such as the size and shape of colonies developed from a single bacterial cell, and the ability to resist infection by certain kinds of bacterial viruses can also be used as markers.
Phenotype and Genotype: In certain cases, loss of any of several enzymes involved in a particular metabolic pathway can result in the same phenotype. Thus, for example a strain of E. coli that is isolated as an auxotroph unable to multiply in the absence of tryptophan would be designated as Trp-. Such cells might have a loss-of-function mutation in any of five different enzymes that are involved in the biosynthesis of tryptophan. These would be designated as TrpA-, TrpB-, TrpC-, TrpD- and TrpE-. Note that italics are used for genotype and regular type for phenotype. Similarly a strain unable to utilize lactose (Lac-) might be LacZ- or LacY-, depending on which gene has been mutated. In either of these examples, there can also be mutations in regulatory genes, as will be described in the next two lectures. When it is necessary to identify wild type phenotype or genotype, the minus is replaced with a plus. Antibiotic resistrance is designated with a superscript r, and susceptibility with an s.
Transformation: Transformation refers to the ability of extracellular DNA to enter a bacterial cell and recombine with the bacterial genome, thereby giving the cell new genetic properties. As described in lecture 2, transformation of bacteria by non-living material derived from other bacteria was discovered in 1928. Demonstration by Avery et al. that the transforming material was DNA, which was published in 1944, was the first direct evidence that DNA carried genetic information.
Transformation mapping: A crude form of genetic mapping can be done by determining the frequency with which two separate mutations are simultaneously reversed by transformation. Loci that are closer together are more likely to be co-transformed. The textbook analyzes in detail the mechanisms by which exogenous DNA can recombine with the DNA of the bacterial chromosome (Figures 7.10 and 7.11). You should understand in general terms how this process works, but it is not necessary to memorize the molecular details for this course. The mechanisms involved in genetic recombination are studied in detail in MCDB 3500. (For those who are curious, there is a good introductory discussion on pages 490-495 of our textbook)
Plasmids: In addition to the bacterial chromosome, which is a large circular DNA molecule (4.6 million base pairs in E. coli), bacteria often contain much smaller circular DNA molecules known as plasmids (figure 7.6). Plasmids have their own origins of replication, such that they can multiply independently within the bacterial cells. They usually contain only a few thousand base pairs of DNA and carry only a few genes, often for antibiotic resistance. Plasmids that carry antibiotic resistance genes can make their host cells resistant to antibiotics. Looking ahead, this provides the basis for selective techniques that are widely used in gene cloning.
Conjugation: Bacterial conjugation can be viewed as a primitive form of sex, in which a cell from a donor strain injects DNA into a recipient cell, where it can undergo recombination and become part of the recipient's genome (figures 7.13 and 7.14). Donor strains contain an additional genetic element, called the fertility factor (F), usually in the form of a plasmid. Cells that are receptive to conjugation lack the F factor and are sometimes designated F-. During conjugation of an F+ cell with an F-, the frequency of transformation for any genes other than the F factor is very low. (When it does happen, it is due to random chromosomal integration of the F factor, as described below).
Hfr strains: In certain F+ strains, the F factor has become integrated into the bacterial chromosome. When this happens, transfer of chromosomal DNA from the donor to the recipient F- cell begins adjacent to the integrated F factor and can progress around the entire bacterial chromosome if the process is not interrupted. Bacterial strains with integrated F factors are referred to as Hfr strains (for "high frequency of recombination").
Mechanisms of DNA transfer: The DNA transfer that occurs in conjugation begins as a single strand break in the donor chromosome (or plasmid), with only one strand transferred through the F-pilus to the recipient cell (figure 7.17). The single strand left behind in the donor and the one that is transferred to the recipient are both converted into double strands, restoring the donor chromosome and generating double stranded donor DNA in the recipient. As a result of receiving new DNA from the donor, the recipient is temporarily partially diploid. Recombination can then integrate parts of the transferred DNA into the recipient genome (figure 7.11). Any DNA that is not integrated is soon destroyed.
Chromosomal gene mapping: Genetic map distances in E. coli and other types of bacteria are based on the order and rate of transfer of genes from an Hfr donor to an F- recipient. Because the F factor can integrate into the bacterial chromosome at different sites, the transfer of genetic material can start at different genes. In addition, it can proceed around the circular E. coli genome in either direction (figures 7.19 and 7.20). However, the order of transfer of the genes is always the same (or the exact reverse). This provides relative map locations based on how rapidly the transfer of a particular gene occurs relative to that of the leading gene (table 7.4 and figures 7.19 and 7.21). The relative amount of time required for the transfer of individual genes can be determined by interrupting conjugation at various times after it was started. Map distances on the circular map of the E. coli genome are expressed as units of time, relative to transfer of the entire genome, which requires approximately 100 minutes under standard conditions (figure 7.22).
F' factors and sexduction: Sometimes an integrated F+ factor from an Hfr strain will escape from the bacterial chromosome carrying a few chromosomal genes with it in its circular plasmid DNA (figure 7.23). Such a plasmid is called an F-prime (F') factor. The bacterial genes carried on the F' plasmid are easily transmitted into F- cells by conjugation. Since the F' factor is a complete plasmid with its own origin of replication, it can be stably maintained in the recipient cells. The resultant cell contains two copies (one chromosomal and one on the F' plasmid) of all of the chromosomal genes that have been integrated into the F' factor. The process of introducing a second copy of a gene into a cell with an F' factor is sometimes called sexduction . The resultant partially diploid cell is called a merozygote
Complementation analysis: The presence of a second partial genome in a cell permits studies to see if one copy of a particular gene is dominant over the other copy in determining cellular phenotype. In addition, it becomes possible to determine whether the plasmid and the chromosomal genome can each supply a gene product that the other cannot. Figure 7.25 shows how this analysis can be done with two genes involved in lactose utilization (LacY, LacZ). If the plasmid has a good copy of Z and a bad copy of Y and the chromosome has a bad copy of Z and a good copy of Y, the two together can code for both gene products and the cell can utilize lactose. When this happens, it is called complementation because each genome complements the defect in the other genome. If both genomes have defects in the same gene, neither can make that gene product, and the cells cannot utilize lactose (there is no complementation). The complementation that occurs in merozygotes is similar in principle to the examples of complementation that we have already examined in the previous lecture for the development of Drosophila imaginal discs and for the growth of auxotrophic strains of Neurospora.
Cis and trans: Genes that are on the same chromosome (or plasmid) are called cis- and those that are on separate "chromosomes" are called trans-. (For those of you who are taking organic chemistry, please be careful not to use trans to refer to the opposite strand in double-stranded DNA. The terminology here is not the same as for trans substitutions on two carbons linked by a double bond). Complementation is an example of an interaction that can be observed when genes are in a trans orientation (on different DNA double strands in the same cell). Interaction of genes in a trans relationship usually involves synthesis of protein products that can act at a distance from the genes that coded for them. Many regulatory sequences must be in a cis relationship to the gene(s) they influence. We have already seen examples such as promoters and enhancers. In the next lecture, we will see that cis and trans interactions among genes in merozygotes have played a major role in the analysis of the regulartory mechanisms that control expression of gene products needed for lactose utilization by E. coli.
Bacteriophage : Bacteriophages are bacterial viruses. The bacteriophage particle consists of a hollow head packed with DNA, a tail structure, which is a hollow tube used to inject the DNA into the bacterial cell, and tail fibers, which are involved in attachment to the bacterial cell (Fig. 7.26). Bacteriophages inject their DNA into bacteria such as E. coli, leaving their protein coats entirely on the outside (Figure 2.4 -- this was one of the lines of evidence that DNA, rather than protein is the genetic material).
Replication of bacteriophage: Host transcriptional and translational machinery is used to produce new bacteriophage proteins (often with changes in transcriptional specificity for expression of "late" viral genes). The bacteriophage DNA is replicated to 100 fold or more. New bacteriophage particles are assembled inside the bacterial cell, and the products of late-acting genes lyse the host cell, giving rise to a "burst" of new bacteriophage particles (figure 7.27). When single bacteriophage are plated on a lawn of bacterial cells on an agar plate, the first burst initiates a second round of infection, leading to more bursts and a growing circle of bacterial lysis, which is known as a plaque (figure 7.28).
Lysogeny: The bacterophage life cycle described above results in rapid lysis of the host cells. However, some bacteriophage, such as bacteriophage lambda (normally written with the Greek letter lambda), can also enter into a stable relationship with the host cell, known as lysogeny. In this case, the bacteriophage genome becomes integrated into the bacterial genome and replicates with it indefinitely, without causing any pathology, and often protecting against other bacteriophage infections (figure 7.29). If the host cell becomes stressed or damaged, bacteriophage lambda can return to a lytic cycle, form mature phage particles, and infect other bacterial cells (figure 7.31a).
Transduction: Transduction refers to a genetic exchange in which bacteriophages carry bacterial genes from one host cell to another. There are two classes of transduction, specialized and generalized. In specialized transduction, a lysogenic phage undergoes recombination with the host genome and later when it is excised to become an independent phage genome, it carries one or more host genes with it, which can be transduced into a new host cell and recombined into the genome of that cell (Figures 7.30 and 7.31). In generalized transduction, fragments of host DNA are mistakenly psackaged into a bacteriophage in place of its own DNA (figure 7.32). When a virus particle carrying host DNA "infects" a new host cell, that DNA is injected into the host and may recombine with the host genome. Transduction can be verified through use of selective media that only support cells carrying the transduced genes. Crude mapping of closely linked genes can also be done by transduction.
Complete DNA sdequences of bacterial genomes: Modern DNA sequencing techniques have made it possible to determine the complete DNA sequences of several different bacterial species. These range in size from just under 0.6 million base pairs in Mycoplasma genitalium to over 4 6 million base pairs in E. coli (table 7.5). The relatively small genome of M. genitalium contains about 470 potential protein coding genes (Figure 7.33 and Table 7.6). The larger E. coli genome contains 4288 potential protein coding genes, including many whose functions are not yet known. (The detailed E. coli linkage map in Figure 7.22 contains 1403 genes that have been identified by standard genetic mehtods).
Genomic details: For those who may wish to pursue the details and have a powerful enough computer, web pages for the E. Coli Genome Project provide information on map position and function when known for a total of 4405 sites that appear either to be protein-coding sequences or templates for various types of cellular RNA in E. coli K12. After you have read the introductory page, you can obtain as much detail as you want (or can handle) by clicking on various options. The link entitled Browse the gene table will take you to a menu of tables listing all 4405 entries. You will need to set your browser to smallest type size and use a full screen window to be able to read entirely across the tables.
Unanswered questions: A complete genomic sequence is a major step toward the total understanding of an organism, but for large genomes, the complexity is such that many other questions related to gene expression and interactions of gene products must also be answered, even after the basic functions of all of the encoded proteins have been identified, which in itself is far from completed.