1. Mechanisms of Transfer and Features in Common.

• survival- for bacteria to survive they must change, evolve, and this can be accomplished by the accumulation of mutations and/or the acquisition of "new" genetic information.
• acquisition of "new" genetic information- The 3 mechanisms that allow DNA to be transferred from one cell, called the donor cell, to another cell, called the recipient cell, differ in how the DNA is transferred, how much genetic information is transferred, and the types of genes transferred, i.e., chromosomal versus plasmid genes.
• mechanisms of transfer-
  • DNA-mediated transformation- free or naked DNA (isolated from or released from a donor cell) is taken up by a recipient cell.
  • transduction- donor DNA is transferred from donor to recipient cell by a bacteriophage.
  • conjugation- cell to cell contact mediated by the sex-pilus is required for DNA transfer.
• features in common between mechanisms of transfer-
  • there is always a unidirectional transfer of DNA, not a reciprocal exchange.
  • only a portion of donor chromosomal DNA is transferred or in the case of a plasmid the entire plasmid is transferred.
  • for donor chromosomal DNA to become an inheritable property of recipient, DNA must integrate, insert itself, into the recipient genome. Insertion occurs by the breakage and reunion model (homologous recombination). This is not required of plasmids. Why?
  • all have potential to change genotype/phenotype of recipient cell. To demonstrate that transfer has occurred there must be a difference(s) between the donor, recipient, and the resulting recombinant cell(s). In most cases looking for phenotypic difference, or a selection procedure that will select for a particular type of recombinant.

2. Mechanism of Transfer:

• DNA-mediated transformation- many bacteria, both Gram-positive and negatives, are naturally transformable, while others including yeast and mammalian cells can be artificially transformed. Will discuss transformation that occurs in the Gram-positive bacterium Streptococcus pneumoniae.
  • source of DNA- lysed cell or extracted DNA from a donor cell.
    
  • competence state- in order for recipient cells to take up the DNA, recipient must be in a state of competence. In S. pneumoniae competence is established late in exponential phase of growth, and is the result of having a few cells synthesize and release a small protein called competence factor, which induces cells in the population to synthesize 8-14 new proteins that establish the competence state (two of these proteins are a DNA binding protein and a nuclease).
    
  • binding/entry of DNA- recipient cell binds fragments of DNA (~400 genes), which are cut into small fragments of ds-DNA (~40 genes). Entry of DNA is accompanied by the degradation of one of the two strands.
    
  • integration of DNA into recipient cell- if homology exists between transferred DNA and recipients genome, have homologous recombination that leads to the formation of a heterogenote (two strands are not completely complimentary).
    
  • DNA replication- resolves heteroduplex, and gives a recombinant cell.
    
• Transduction:
  • Generalized transduction- Can involve a lytic or temperate bacteriophage. In generalized transduction can have transfer of any group of genes of the infected host, but the maximum amount of host DNA that can be transferred is equivalent to the size of the phage genome (~100 genes).
    • during phage gene expression, host chromosome is fragmented.
      
    • during maturation, by mistake, host DNA is packaged into phage heads (no phage DNA in these heads). These particles are called generalized transducing viruses.
      
    • upon infection of new host, previous host DNA is injected into cell, and if this DNA is homologous with recipients DNA, will recombine.
      
  • Specialized transduction- occurs with temperate bacteriophage that are in the prophage state, and transfer only a few chromosomal genes, and only those bacterial genes to one side or the other of where prophage existed.
    • during the excision of the prophage from host chromosome, an error occurs in that DNA to one side or the other of where prophage existed is cut out with phage genes (a complete genomes worth of DNA is cut out). Excised DNA contains chromosomal genes plus most of phage genome (some of phage genome left in chromosome!).
      
    • This excised DNA is replicated and packaged into phage heads. These viruses are call specialized transducing particles.
      
    • upon infection of new host, DNA is injected, and if homology exists with recipient DNA recombination occurs. This type of infection never leads to a productive phage infection. Why?
      
• Conjugation:
  • F-plasmid- donor cell, designated the male cell, synthesizes a sex-pilus which is encoded by genes on the F (fertility) plasmid. The F-plasmid can replicate independent of chromosome (cell called an F⁺ cell) or can integrate into host chromosome (called an Hfr cell). Unlike a prophage, F-plasmid genes are expressed. F-plasmid can intergrate into several, but specific sites on chromosome. Cell lacking F-plasmid is recipient cell, also designated the F^- cell or female cell.
    
  • F'- occasionally when F-plasmid excises itself from chromosome can take chromosomal genes to either side of plasmid, but unlike prophage does not leave any plasmid genes behind.
    
  • role of sex-pilus- sex-pilus allows contact with an F- cell. Sex-pilus retracts, bring mating cell together and allowing DNA transfer to occur.
    
  • types of mating-
    • F⁺ X F^-- in this type of mating the entire F-plasmid is transferred to F^- cell, and no chromosomal genes are transferred. Donor retains copy of F-plasmid. No integration of F-plasmid required.
    • Hfr X F^-- in mating of this type, chromosomal genes to one side or the other of where F-plasmid is integrated are transferred in a sequential manner. Since conjugation bridge fragile, rarely, if ever, is the entire donor chromosome transferred (F-plasmid genes would be last to be transferred, and time required would be 100 minutes). By interrupting mating, can map location of genes on chromosome (~30-40 genes transferred per minutes, and 8 to 10 minute conjugations experiments are very realistic). Integration of transferred DNA required. Why?
    • F' X F^-- mating is similar to that of an F⁺ cell, but recipient cell may know have two copies for certain genes (one copy on plasmid and another on chromosome of recipient).

3. R-plasmids and their importance: Contain gene(s) for resistance to antibiotics and/or heavy (toxic) metals. Some R-plasmids behave life F-plasmid, and thus allow transfer, say, of R-plasmid from a nonpathogenic bacteria to a pathogenic bacterium.

Learning Objectives:

• What is meant by homologous recombination?
• What are the basic features of DNA-mediated transformation?
• How are bacterial genes transferred in transduction, and what is the deference between generalized and specialized transduction.
• How is gene transfer accomplished in conjugation, and how do the different matings that can occur influence the number and types of genes that are transferred.
• You should be familiar with the following words: competence, plasmid, R-factors.

7/22/09