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Microbiology, Fifth Edition
Microbiology, 5/e
Lansing M Prescott, Augustana College
Donald A Klein, Colorado State University
John P Harley, Eastern Kentucky University
Microbial Recombination and Plasmids

Study Outline

  1. Introduction
    1. Recombination-process by which one or more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence
    2. In eucaryotes, recombination occurs during meiosis and results from crossing-over between homologous chromosomes (chromosomes containing identical sequences of genes)
  2. Bacterial Recombination: General Principles
    1. Types of recombination
      1. General recombination usually involves a reciprocal exchange in which a pair of homologous sequences breaks and rejoins in a crossover; nonreciprocal general recombination involves the incorporation of a single strand into the chromosome to form a stretch of heteroduplex DNA
      2. Site-specific recombination is the nonhomologous insertion of DNA into a chromosome; often occurs during viral genome integration into the host, a process catalyzed by enzymes specific for the virus and its host
      3. Replicative recombination accompanies replication and is used by some genetic elements that move about the genome
    2. Horizontal gene transfer-transfer of genes from one mature, independent organism to another (compare this to vertical gene transfer-transmission of genes from parents to offspring)
      1. Exogenote-donor DNA that enters the bacterium by one of several mechanisms
      2. Endogenote-the genome of the recipient
      3. Merozygote-a recipient cell that is temporarily diploid for a portion of the genome during the gene transfer process
      4. Most linear DNA fragments are not stably maintained unless integrated into the endogenote
    3. Types of horizontal exogenote transfer
      1. Conjugation is direct transfer from donor bacterium to recipient while the two are temporarily in physical contact
      2. Transformation is transfer of a naked DNA molecule
      3. Transduction is transfer mediated by a bacteriophage
    4. Intracellular fates of exogenote
      1. Integration into the host chromosome
      2. Independent functioning and replication of the exogenote without integration (a partial diploid clone develops)
      3. Survival without replication (only the one cell is a partial diploid)
      4. Degradation by host nucleases (host restriction)
  3. Bacterial Plasmids
    1. Plasmids-small, circular DNA molecules that are not part of the bacteriumís chromosome
      1. Have their own replication origins, replicate autonomously, and are stably inherited
      2. Can be eliminated from a cell by a process called curing, which can occur either spontaneously or as a result of treatments that inhibit plasmid replication but do not affect host cell reproduction
      3. Episomes are plasmids that can exist either with or without being integrated into the host chromosome
      4. Conjugative plasmids usually have genes for sex pili and can transfer copies of themselves to other bacteria during conjugation
    2. Fertility factors-episomes that can direct the formation of sex pili and transfer copies of themselves during conjugation
    3. Resistance factors-(R plasmids); have genes for resistance to various antibiotics; some are conjugative; however, they usually do not integrate into the host chromosome
    4. Col plasmids-provide a competitive advantage to the bacteria having them; carry genes for the synthesis of bacteriocins (e.g., colicins) that are directed against other bacterial species; some col plasmids are conjugative and may also carry resistance genes
    5. Other types of plasmids include virulence plasmids, which make the bacterium more pathogenic by conferring resistance to host defense mechanisms or by carrying a gene for the production of a toxin, and metabolic plasmids, which carry genes for enzymes that utilize certain substances as nutrients (aromatic compounds, pesticides, etc.)
  4. Transposable Elements
    1. Transposition-the movement of pieces of DNA around in the genome
      1. Transposons-segments of DNA that can move about chromosomes
      2. Transposons differ from bacteriophages in that they lack an infectious viral life cycle, and they differ from plasmids in that they are unable to replicate independently
    2. Insertion sequences (IS elements) contain genes only for those enzymes required for transposition (e.g., transposase); they are bound on both ends by inverted terminal repeat sequences
    3. Composite transposons carry other genes in addition to those needed for transposition (e.g., for antibiotic resistance, toxin production, etc.)
    4. Movement of the transposon occurs typically by replicative transposition, during which a replicated copy of the transposon inserts at the target site on the DNA, while the original copy remains at the parental site
    5. Effects of transposable elements
      1. Insertional mutagenesis can cause deletion of genetic material at or near the target site, arrest of translation or transcription due to stop codons or termination sequences located on the inserted material, and activation of genes near the point of insertion due to promoters located on the inserted material
      2. Fusion of plasmids and insertion of F plasmids into chromsosome
      3. Generation of plasmids with resistance genes
      4. Conjugative transposons can move between bacteria through the process of conjugation
    6. Transposable elements are present in eucaryotes, bacteria, and archaea
  5. Bacterial Conjugation
    1. The transfer of genetic information via direct cell-cell contact; this process is mediated by fertility factors (F plasmids)
    2. F+ ¥ F- mating
      1. In E. coli and other gram-negative bacteria, an F plasmid moves from the donor (F+) to a recipient (F-) while being replicated by the rolling circle mechanism
        1. The displaced strand is transferred via a sex pilus and then copied to produce double-stranded DNA; the donor retains the other parental DNA strand and its complement; thus the recipient becomes F+ and the donor remains F+
        2. Chromosomal genes are not transferred
      2. In gram-positive bacteria, the sex pilus is not necessarily required for transmission; generally fewer genes are transferred
    3. Hfr conjugation
      1. F plasmid integration into the host chromosome results in an Hfr strain of bacteria
      2. The mechanics of conjugation of Hfr strains are similar to those of F+ strains
      3. The initial break for rolling-circle replication is at the integrated plasmidís origin of transfer site
        1. Part of the plasmid is transferred first
        2. Chromosomal genes are transferred next
        3. The rest of the plasmid is transferred last
      4. Complete transfer of the chromosome takes approximately 100 minutes, but the conjugation bridge does not usually last that long; therefore, the entire F factor is not usually transferred, and the recipient remains F-
    4. F¢ conjugation (sexduction)
      1. When an integrated F plasmid leaves the chromosome incorrectly, it may take with it some chromosomal genes from one side of the integration site; this results in the formation of an abnormal plasmid called an F¢ plasmid
      2. The F¢ cell (cell harboring an F¢ plasmid) retains its genes, although some of them are in the chromosome and some are on the plasmid; in conjugation, an F¢ cell behaves as an F+ cell, mating only with F- cells
      3. The chromosomal genes included in the plasmid are transferred with the rest of the plasmid, but other chromosomal genes usually are not
      4. The recipient becomes an F¢ cell, and a partially diploid merozygote
  6. DNA Transformation
    1. Transformation-a naked DNA molecule from the environment is taken up by the cell and incorporated into its chromosome in some heritable form
    2. A competent cell is one that is capable of taking up DNA and therefore acting as a recipient; only a limited number of species are naturally competent; the mechanics of the natural transformation process differ from species to species
    3. Species that are not normally competent (such as E. coli) can be made competent by calcium chloride treatment or other methods, which makes the cells more permeable to DNA
  7. Transduction
    1. Transduction-transfer of bacterial genes by viruses (bacteriophages); occurs as the result of the reproductive cycle of the virus
      1. Lytic cycle-a viral reproductive cycle that ends in lysis of the host cell; viruses that use this cycle are called virulent bacteriophages
      2. Lysogeny-a reproductive cycle that involves maintenance of the viral genome (prophage) within the host cell (usually integrated into the host cellís chromosome), without immediate lysis of the host; with each round of cell division, the prophage is replicated and inherited by daughter cells; bacteriophages reproducing by this mechanism are called temperate phages; certain stimuli (e.g., UV radiation) can trigger the switch form lysogeny to the lytic cycle
    2. Generalized transduction-any part of the bacterial genome can be transferred; occurs during the lytic cycle of virulent and temperate bacteriophages
      1. The phage degrades host chromosome into randomly sized fragments
      2. During assembly, fragments of host DNA of the appropriate size can be mistakenly packaged into a phage head (generalized transducing particle)
      3. When the next host is infected, the bacterial genes are injected and a merozygote is formed
        1. Preservation of the transferred genes requires their integration into the host chromosome
        2. Much of the transferred DNA does not integrate into the host chromosome, but is often able to survive and be expressed; the host is called an abortive transductant
    3. Specialized (restricted) transduction
      1. Transfer of only specific portions of the bacterial genome; carried out only by temperate phages that have integrated their DNA into the host chromosome at a specific site in the chromosome
        1. The integrated prophage is sometimes excised incorrectly and contains portions of the bacterial DNA that was adjacent to the phageís integration site on the chromosome
        2. The excised phage genome is defective because some of its own genes have been replaced by bacterial genes; therefore, the bacteriophage cannot reproduce
        3. When the next host is infected, the donor bacterial genes are injected, leading to the formation of a merozygote
      2. Low frequency transduction lysates-lysates containing mostly normal phages and just a few specialized transducing phages
      3. High frequency transduction lysates-lysates containing a relatively large number of specialized transducing phages; created by coinfecting a host cell with a helper phage (normal phage) and a transducing phage; the helper phage allows the transducing phage to replicate, thus increasing the number of transducing phages in the lysate
  8. Mapping the Genome
    1. Hfr mapping involves the use of an interrupted mating experiment to locate the relative position of genes
      1. The conjugative bridge is broken and the Hfr ¥ F- mating is stopped at various intervals
      2. While the bridge is intact, chromosome transfer occurs at a constant rate
      3. The order and timing of gene tranfser directly reflects the order of genes on the chromosome
      4. Interrupted mating is good for mapping genes that are 3 minutes or more apart; however the instability of the conjugation bridge makes it nearly impossible to map genes that are very distant; several Hfr strains with different integration sites are used to generate overlapping maps, which can then be pieced together to form the entire genome map
    2. Transformation mapping-the frequency with which two genes simultaneously transform a recipient cell (cotransformation) indicates the distance between the genes; the higher the frequency of cotransformation, the closer the two genes are; overlapping maps can be pieced together to complete the genome map
    3. Generalized transduction maps-as with transformation mapping, the frequency of cotransduction indicates the distance of two genes from each other
    4. Specialized transduction maps provide distances from integration sites, which themselves must be mapped by conjugation mapping techniques
  9. Recombination and Genome Mapping in Viruses
    1. Recombination maps are generated from crossover frequency data obtained when cells are infected with two or more phage particles simultaneously
    2. Heteroduplex mapping-wild type and mutant chromosomes are denatured and allowed to reanneal together; homologous regions pair normally, but mutant regions form bubbles that can be seen in electron micrographs; generates a physical map of the viral genome
    3. Restriction endonuclease mapping-locates deletions and other mutations by examining the electrophoretic mobility (size) of the fragments generated
    4. Sequence mapping-small phage genomes can be directly sequenced to map genes








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