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Transcript
BACTERIAL GENETICS Prof. Manal Baddour INTRODUCTION 12/12/2008 • Bacteria have four important advantages for "traditional types of genetic experiments": Bacterial Genetics 1. They are haploid (no masking). 2. New generation is produced every 20 minutes. 3. Easy to grow in ENORMOUS NUMBERS. 4. Individual members of these large populations are GENETICALLY IDENTICAL (or very nearly so). 2 DEFINITIONS 12/12/2008 The science of genetics defines and analyzes heredity, or constancy and change in the vast array of physiologic functions that form the properties of organisms. The unit of heredity is the gene, a segment of DNA that carries in its nucleotide sequence information for a specific biochemical or physiologic property. Bacterial Genetics 3 12/12/2008 The chemical basis for variation in phenotype is change in genotype, or alteration in the sequence of DNA within a gene or in the organization of genes. Restriction enzymes cleave DNA at specific sites, giving rise to DNA restriction fragments Plasmids are small genetic elements capable of independent replication Bacterial Genetics 4 12/12/2008 DNA inserted into plasmids can be placed under control of high-expression bacterial promoters that allow encoded proteins to be expressed at high levels. Bacterial genetics fostered development of genetic engineering, a technology that has been responsible for tremendous advances in the field of medicine. Bacterial Genetics 5 THE WATSON-CRICK MODEL 12/12/2008 Bacterial Genetics 6 12/12/2008 Bacterial Genetics Top: Adenine-thymine pair. Bottom: Guanine-cytosine pair. 7 THE PROKARYOTIC GENOME 12/12/2008 Prokaryotic genomes consist of a single circular DNA molecule containing from 580 kbp to more than 5220 kbp of DNA. Many bacteria contain additional genes on plasmids that range in size from several to 100 kbp. DNA circles (chromosome and plasmid), which contain genetic information necessary for their own replication, are called replicons. Bacterial Genetics 8 BACTERIAL GENES 12/12/2008 Membranes do not separate bacterial genes from cytoplasm as in eukaryotes. With few exceptions, bacterial genes are haploid. Restriction enzymes (restriction endonucleases) provide bacteria with a mechanism to distinguish between their own DNA and DNA from other biologic sources. Bacterial Genetics 9 THREE MAIN TYPES OF MUTATIONS • Bacterial Genetics a. Antibiotic resistance • StrR=streptomycin resistant; able to grow when the antibiotic streptomycin is added to the medium. • StrS=streptomycin sensitive; killed in the presence of streptomycin. 12/12/2008 FOR BACTERIA Can use upper or lower case letter for resistant vs. sensitive or a dash (Str-r). 10 b. Nutritional mutants • • Examples of nutritional mutants: Pro+ able to synthesize the amino acid proline Prounable to synthesize the amino acid proline Nutritional mutants include defects in the synthesis of: Amino acids (examples Leu, leucine; Pro, proline, Met, methionine, His, histidine) Vitamins (examples Thi, thiamine; Bio, biotin) Nucleotides Bacterial Genetics • 12/12/2008 prototrophs: able to synthesize all their components from simple molecules (from a minimal medium) auxotrophs: mutants unable to synthesize one or more of their components from simple molecules. Usually the wild type trait is the ability to synthesize the compound (they are prototrophs). Mutants usually are defective in the gene for an enzyme needed to 11 synthesize the compound and thus require that compound to be added to the medium. c. Carbon source mutants • Bacterial Genetics • 12/12/2008 Generally these cannot use particular sugars as a source of energy. Example: Mutants in genes in the lactose metabolic enzymes (lac Z or lac Y) cannot use lactose; must be supplied with glucose or some other source to grow. Lac Z+ can grow in medium containing lactose. Lac Z- can’t grow in medium containing lactose. 12 12/12/2008 Bacterial Genetics 13 12/12/2008 Bacterial Genetics Some bacterial species can invade higher organisms because they possess specific genes for pathogenic determinants. These genes are often clustered together in the DNA and are referred to as pathogenicity islands. Pathogenicity islands contain diverse genes important for pathogenesis—including adhesins, invasins, and exotoxins—as well as those that are probably involved in mobilization. 14 BACTERIAL REPLICATION 12/12/2008 Bacterial Genetics The replication of bacterial DNA begins at one point and moves in both directions (ie, bidirectional replication) from there. The two old strands of DNA are separated and used as templates to synthesize new strands (semiconservative replication). 15 PLASMIDS • • • • Bacterial Genetics • Non-essential DNA molecules in bacteria that replicate independent of the bacterial chromosome. Intracytoplasmic extrachromosomal double stranded circular DNA 12/12/2008 • Usually small circular DNAs Some are large (example, fertility plasmids) Copy number per cell can vary Encode a few genes 16 PLASMID GENES 12/12/2008 Bacterial Genetics Plasmids carry genes associated with specialized functions. Many plasmids carry genes that mediate their transfer from one organism to another as well as other genes associated with acquisition or rearrangement of DNA. Swift spread among bacterial populations of plasmid-borne resistance to antibiotics. Plasmids also carry IS elements, which are important in the formation of high-frequency recombinant (Hfr) strains. 17 Major types: Bacterial Genetics R plasmids: Resistance plasmids. Contain genes for drug or antibiotic resistance. These are the types of plasmids from which most of the recombinant DNA vectors are derived. F plasmids: Fertility plasmids. Involved in transfer of genes between bacteria by conjugation (bacterial mating). 12/12/2008 18 INSERTION SEQUENCES AND TRANSPOSONS 12/12/2008 Many DNA sequences in bacteria are mobile and can be transferred between individuals and among species. Bacterial Genetics 19 MAIN TYPES OF MOBILE ELEMENTS: • • • • 1-3 kb long can be found in plasmids or in the chromosome can mediate integration of plasmids into the chromosome; example F plasmid integration to form an Hfr contain a gene for a tranposase enzyme; regulates mobility of the element Can cause insertion mutations. Bacterial Genetics • 12/12/2008 IS elements=insertion sequences 20 12/12/2008 Bacterial Genetics Transposons are genetic elements that contain several kbp of DNA, including the information necessary for their migration from one genetic locus to another. Unlike plasmids, transposons do not contain genetic information necessary for their own replication. larger than IS elements usually contain at least one additional gene, often for antibiotic resistance often are arranged with the resistance gene in the middle, flanked by IS elements contain a gene for a transposase enzyme 21 12/12/2008 Bacterial Genetics Significance of mobile elements: Rapidly spread beneficial traits between bacteria, such as drug resistance Responsible for “horizontal transfer” of genes across species boundaries 22 BACTERIOPHAGE 12/12/2008 Bacterial Genetics Viruses associated with prokaryotes The nucleic acid molecule of bacteriophages is surrounded by a protein coat Bacteriophages exhibit a wide variety of morphologies Many phages contain specialized syringe-like structures (ie, tails) that bind to receptors on the cell surface and inject the phage nucleic acid into a host cell 23 12/12/2008 Bacterial Genetics 24 12/12/2008 Bacterial Genetics 25 12/12/2008 Bacterial Genetics 26 12/12/2008 Bacterial Genetics Lytic phages produce many copies of themselves as they kill their host cell. Temperate phages are able to enter a nonlytic prophage state in which replication of their nucleic acid is linked to replication of host cell DNA. Bacteria carrying prophages are termed lysogenic. 27 REPLICATION OF BACTERIOPHAGE 12/12/2008 Bacterial Genetics 28 12/12/2008 Bacterial Genetics 29 12/12/2008 Bacterial Genetics 30 BACTERIOPHAGE LAMBDA 12/12/2008 Bacterial Genetics 31 LYSOGENY 12/12/2008 Bacterial Genetics 32 TRANSFER OF DNA 12/12/2008 Bacterial Genetics Interstrain transfer of DNA among prokaryotes is widespread. Bacterial genetic exchange is typified by transfer of a relatively small fragment of a donor genome to a recipient cell. 33 MECHANISMS OF GENE TRANSFER 12/12/2008 Bacterial Genetics HOW DO BACTERIA EXCHANGE GENES? Three broad mechanisms mediate efficient movement of DNA between cells: conjugation, transduction, and transformation. In conjugation, only one strand of DNA is transferred. The recipient completes the structure of double-stranded DNA by synthesizing the strand that complements the strand acquired from the donor. In transduction, donor DNA is carried in a phage coat and is transferred into the recipient by the mechanism used for phage infection. Transformation, the direct uptake of donor DNA by the recipient cell, may be natural or forced. Relatively few bacterial species are naturally competent for transformation. 34 12/12/2008 BACTERIAL CONJUGATION EXCHANGE OF GENES BETWEEN BACTERIAL CELLS BY CELL TO CELL CONTACT Bacterial Genetics "Bacterial conjugation is the process in which DNA is transferred from a bacterial donor cell to a recipient cell by cell-to-cell contact. It has been observed in many bacterial species and is best understood in E.coli, in which it was discovered by Joshua Lederberg in 1951.“ 35 CONJUGATION 12/12/2008 Bacterial Genetics Plasmids are the genetic elements most frequently transferred by conjugation. Genetic functions required for transfer are encoded by the tra genes, which are carried by self-transmissible plasmids. Some self-transmissible plasmids can mobilize other plasmids or portions of the chromosome for transfer. 36 • • • Bacterial Genetics • The ability to transfer DNA by conjugation is dependent on the presence of a cytoplasmic entity termed the fertility factor, or F. Cells carrying F are termed F+ Cells without F are F-. F is a small, circular DNA element that acts like a minichromosome. It is an example of a plasmid. F contains approximately 100 genes; these give F several important properties: 12/12/2008 • 37 1. F can replicate its DNA, which allows F to be maintained in a cellular population that is dividing. Bacterial Genetics 3. F+ cells can transfer the newly synthesized copy of the circular F genome to a recipeint (F-) cell that lacks such a genome; note that a copy of F always remains behind in the donating cell. 12/12/2008 2. Cells carrying F produce pili: minute proteinaceous tubules that allow the F+ cells to attach to other cells and maintain contact with them. 4. When a donor cell transfers a copy of its cytoplasmic F to an F- cell, the recipient cell also becomes an F+ cell, because it now contains a circular F genome. 38 6. Occasionally, F leaves the cytoplasm and integrates itself into the host bacterial chromosome. When this occurs, F can also transfer the host chromosomal markers to the recipient cell along with its own DNA. Bacterial Genetics F+ cells are usually inhibited from making contact with other F+ cells and do not usually transfer the F genome to F+ cells. 12/12/2008 5. 39 12/12/2008 Bacterial Genetics 40 12/12/2008 Bacterial Genetics 41 12/12/2008 Bacterial Genetics 42 BACTERIAL TRANSFORMATION Bacterial Genetics "Transformation" is simply the process where bacteria manage to "uptake" or bring in a piece of external DNA . Usually, this process is used in the laboratory to introduce a small piece of PLASMID DNA into a bacterial cell. 12/12/2008 TRANSFER OF NAKED DNA BETWEEN BACTERIA 43 • Bacterial Genetics • some bacteria can be transformed by simply mixing DNA with bacteria for E. coli and some other bacteria, must pretreat the cells with salt solution (such as calcium chloride) to make the membrane more permeable to take up DNA typically procedure for E. coli involves pretreating cells with calcium chloride, incubation of cells with DNA on ice, brief heat shock, followed by an incubation at 37˚C to allow the cells to express the new DNA 12/12/2008 • 44 12/12/2008 Bacterial Genetics 45 DNA IS THE GENETIC MATERIAL 12/12/2008 Bacterial Genetics The First demonstration of bacterial transformation was through experiments done by Frederick Griffith (in London) in 1928. He found there were two different types of the bacterium Streptococcus pneumoniae: 46 An "S" or SMOOTH coat strain, which is lethal to mice. 12/12/2008 Bacterial Genetics An "R" or rough strain, which will not hurt the mouse. 47 12/12/2008 Griffith found that he could heat inactivate the smooth strain. Bacterial Genetics 48 12/12/2008 •However, if he were to take a mixture of the heat-inactivated S strain, mixed with the R strain, the bacteria would die. •Thus there was some Material in the heat-killed S strain that was responsible for "transforming“ the R strain into a lethal form. Bacterial Genetics 49 Bacterial Genetics Their work continued on in the U.S., and in 1944, Oswald Avery, C.M. MacLeod, and M. McCarty carefully demonstrated that the ONLY material that was responsible for the transformation was DNA - thus, DNA was the "Genetic material“ 12/12/2008 • 50 12/12/2008 Bacterial Genetics The genetic transfer of streptomycin resistance (strr) to the streptomycin sensitive (strs) cells of E.coli. The recovery of strs cells depends on the concentration of the strr DNA. 51 TRANSDUCTION CELL TO ANOTHER MEDIATED BY VIRUSES Generalized transduction: Any gene transferred. Carried out by lytic viruses. can be Specialized transduction: Only one or two genes can be transferred. Carried out by some lysogenic viruses such as bacteriophage lambda. Bacterial Genetics 12/12/2008 THE EXCHANGE OF GENES FROM ONE BACTERIAL 52 12/12/2008 Bacterial Genetics 53