Download Genetic Analysis and Mapping in Bacteria and Bacteriophages

Genetic Analysis and Mapping in Bacteria and Bacteriophages
Why study bacteria and viruses?
 Initially, bacterial and viral genetic systems were studied with the hope that they were
simplified versions of the genetic systems found in higher organisms
 They have served as excellent model systems for certain aspects of genetics
 We have many interactions with bacteria and viruses - some beneficial; some detrimental
 A better understanding of their genetics will allow us to better control this aspect of our
lives
 Their genetic systems can be easily manipulated
 Bacteria and viruses have served as incredible tools for many of our greatest discoveries
and biotechnological inventions and innovations
 Because of their unique relationship with us, bacteria and viruses have also opened a
window into our understanding of human origins and migrations
Basic terminology
 Prototrophic (wild-type) bacteria can synthesize all of the compounds they need
o
We can grow prototrophs on minimal media
 Auxotrophic (mutant) bacteria cannot synthesize all of the compounds so they require
minimal media supplemented with the necessary growth ingredient(s)
 Complete media contains all of the substances necessary for growth of the bacteria
Bacterial cultures
 Bacteria are typically cultured in tubes (liquid) or on plates (solid) containing sterile media
 We use different media for different bacteria and for different purposes
 If we plate an appropriate number of bacteria, each colony will represent the progeny of a
single bacterium
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This allows us to estimate the number of bacteria in our original sample
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It also allows us to analyze the phenotype of a colony and assume that it is the
phenotype of the original bacterium
The bacterial genome
 Chromosomes
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Most bacteria contain a single, circular chromosome
 Plasmids
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Small, circular DNA molecules
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Autonomous, extrachromosomal genetic elements
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Usually not essential to bacterial function but can be
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Many of the plasmids first isolated and characterized carried genes for antibiotic
resistance
Joshua Lederberg and Edward Tatum used two strains of auxotrophic bacteria to demonstrate
that genetic exchange was occurring
Bernard Davis showed that cell-to-cell contact was required
Genetic exchange in bacteria
1. Conjugation - bacterial mating
 Genetic material is passed from the donor to the recipient through cell-to-cell contact
 We can focus on F plasmid conjugation
o
The end result is that both cells now contain the F plasmid
o
They can now both act as donors in subsequent matings
 Episomes, such as the F factor, can either exist as freely-replicating plasmids or by
integrating into the chromosome
 At a low frequency, a F factor can integrate into the chromosome, giving a Hfr strain
 But once a Hfr strain does form, it can transfer chromosomal genes at a high-frequency
 The recipient cell will usually still be F- because the entire F factor is only rarely transferred
 Mapping the bacterial chromosome
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Initial mapping efforts for the bacterial chromosome involved the isolation and mating
of numerous Hfr strains
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The location and orientation of oriT determines the order of genes transferred
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Analysis of several Hfr strains indicated that the bacterial chromosome is circular
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At a low frequency, the F factor present in the chromosome of a Hfr strain will excise
precisely to produce a F+ strain
If, however, the excision is imprecise, the result will be a F’ strain
This strain would transfer the A and E genes with every mating
This process is called sexduction and leads to partial diploids - merozygotes
2. Transformation
 Uptake of naked DNA fragments from the surrounding medium
 Requires both the uptake of DNA and its incorporation into the bacterial chromosome or
plasmid
 Cells that can take up DNA are competent
 DNA binding proteins are required
 Even competent bacteria are only capable of taking up DNA during certain stages of growth
 Transformation has also been used to map genes
o
It can tell us if two genes lie close to each other
o
If they do, they are likely to be cotransformed
Viral genetics
 Viruses are simple replicating structures containing nucleic acid surrounded by a protein
coat
Viral life cycle
 Lytic phages reproduce strictly through the lytic cycle
 Since bacteriophages kill their host bacteria, we can count the number of phage in a sample
by doing a plaque assay
 Each plaque represents an individual phage particle
 Phage genes can be mapped by recombination frequencies in a manner similar to eukaryotic
gene mapping
Viral life cycle
 Temperature phages can move through either the lytic or the lysogenic cycle
3. Transduction
 Movement of DNA is facilitated by a bacterial virus (bacteriophage)
Transduction
 Two types
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Generalized transduction
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Specialized transduction
Generalized transduction
 1952 - Joshua Lederberg and Norton Zinder were working with Salmonella typhimurium
 They were looking to perform the same type of experiment that Davis had done previously
 The filter prevented bacteria from passing through but not bacteriophage
 Generalized transduction is so-named because genes located anywhere on the chromosome
can be transferred
Specialized transduction
 Only temperate bacteriophage can be involved because the phage has to be able to go
through both the lysogenic and lytic cycle
 Imprecise excision of the prophage brings along some chromosomal genes, often at the
expense of some phage genes
When genes are located near each other, they are more likely to be cotransduced
Section 8.8 is not required