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Chapter 14 The Prokaryotic Chromosome: Genetic Analysis in Bacteria

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Title: Chapter 14 The Prokaryotic Chromosome: Genetic Analysis in Bacteria


1
Chapter 14The Prokaryotic Chromosome Genetic
Analysis in Bacteria
2
Outline of Chapter 14
  • General overview of bacteria
  • Range of sizes
  • Metabolic activity
  • How to grow them for study
  • The bacterial genome
  • Structure
  • Organization
  • Transcription
  • Replication
  • Evolution of large, circular chromosomes
  • Structure and function of small circular plasmids
  • Gene transfer in bacteria
  • Transformation
  • Conjugation
  • Transduction
  • A comprehensive example
  • Genetic tools to dissect bacterial chemotaxis

3
General overview of bacteria
  • One of the three major lineages of life
  • Eukaryotes organisms whose cells have encased
    nuclei
  • Prokaryotes lack a nuclear membrane
  • Archea
  • 1996 complete genome of Methanococcus jannaschii
    sequenced
  • More than 50 of genes completely different than
    bacteria and eukaryotes
  • Of those that are similar, genes for replication,
    transcription, and translation are same as
    eukaryotes
  • Genes for survival in unusual habitats similar to
    some bacteria
  • Bacteria
  • Similar genome structure, morphology, and
    mechanisms of gene transfer to archea
  • Evolutionary biologist believe earliest single
    celled organism, probably prokaryote existed 3.5
    billion years ago

4
A family tree of living organisms
Fig. 14.1
5
Diversity of bacteria
  • Outnumber all other organisms on Earth
  • 10,000 species identified
  • Smallest 200 nanometers in diameter
  • Largest 500 micrometers in length (10 billion
    times larger than the smallest bacteria)
  • Habitats range from land, aquatic, to parasitic
  • Remarkable metabolic diversity allows them to
    live almost anywhere

6
Common features of bacteria
  • Lack defined nuclear membrane
  • Lack membrane bound organelles
  • Chromosomes fold to form a nucleoid body
  • Membrane encloses cells with mesosome which
    serves as a source of new membranes during cell
    division
  • Most have a cell wall
  • Mucus like coating called a capsule
  • Many move by flagella

7
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8
Power of bacterial genetics is the potential to
study rare events
  • Bacteria multiply rapidly
  • Liquid media E. coli grow to concentration of
    109 cells per milliliter within a day
  • Agar media single bacteria will multiply to 107
    108 cells in less than a day
  • Most studies focus on E. coli
  • Inhabitant of intestines in warm blooded animals
  • Grows without oxygen
  • Strains in laboratory are not pathogenic
  • Prototrphic makes all the enzymes it needs for
    amino acid and nucleotide synthesis
  • Grows on minimal media containing glucose as the
    only carbon source
  • Divides about once every hour in minimal media
    and every 20 minutes in enriched media
  • Rapid multiplication make it possible to observe
    very rare genetic events

9
The bacterial genome is composed of one circular
chromosome
  • 4-5 Mb long
  • Condenses by supercoiling and looping into a
    densely packed nucleoid body
  • Chromosomes replicate inside cell and cell
    divides by binary fission

Fig. 14.4 b
10
E. coli lysed to release chromosome
Fig. 14.4 a
11
How to find mutations in bacterial genes
  • Mutations affecting colony morphology
  • Mutations conferring resistance to antibiotics or
    bacteriophages
  • Mutations that create auxotrophs
  • Mutations affecting the ability of cells to break
    down and use complicated chemicals in the
    environment
  • Mutations in essential genes whose protein
    products are required under all conditions of
    growth

12
How to identify mutations by a genetic screen
  • Genetic screens provide a way to observe
    mutations that occur very rarely such as
    spontaneous mutations (1 in 106 to 1 in 108
    cells)
  • Replica plating simultaneous transfer of
    thousands of colonies from one plate to another
  • Treatments with mutagens increase frequency of
    mutations
  • Enrichment procedures increase the proportion
    of mutant cells by killing wild-type cells
  • Testing for visible mutants on a petri plate

13
Bacteria nomenclature
  • wild-type
  • mutant gene -
  • three lower case, italicized letters a gene
    (e.g., leu is wild type leucine gene)
  • The phenotype for a bacteria at a specific gene
    is written with a capital letter and no italics
    (e.g., Leu is a bacteria with that does not need
    leucine to grow, and Leu- is a bacteria that does
    need leucine to grow.)

14
Structure and organization of E. coli chromosome
  • 4.6 million base pairs
  • open reading frames (ORFs)
  • 90 of genome encodes protein (compare that to
    humans!)
  • 4288 genes, 40 of which we do not know what they
    do.
  • almost no repeated DNA
  • 427 genes have a transport function, other
    classes also identified
  • bacteriophage sequences found in 8 places (must
    have been invaded by viruses at least 8 times
    during history.

15
Insertion sequences dot the E. coli chromosome
  • Transposable elements place DNA sequences at
    various locations in the genome.
  • Geneticists use transposable elements to insert
    DNA at various locations in bacterial genomes.
  • If you were to insert a piece of DNA into a
    bacterial genome using a transposable element,
    can you think of a molecular method that you
    could use to find out which gene you inserted the
    DNA into?

16
  • Transposable elements in bacteria

Fig. 14.6
17
Transcription in bacteria
  • Transcription machinery moves clockwise
  • Different strands code for different genes
  • Several genes may be transcribed in one segment
  • RNA polymerase may transcribe adjacent genes at
    the same time in a counterclockwise direction
  • Highly transcribed genes generally oriented in
    direction of replication fork movement

18
DNA replication in E. coli
Fig. 14.7
19
Plasmids smaller circles of DNA that do not
carry essential genes
  • Plasmids vary in size ranging from 1kb 3 Mb.
  • Plasmids can carry genes that confer resistance
    to antibiotics and toxic substances.
  • Plasmids are not needed for reproduction or
    normal growth, but they can be beneficial.
  • Plasmids can carry genes from one bacteria to
    another. Bacteria can thus become resistant to a
    drug, put the resistance gene in the plasmid, and
    transfer it to other bacteria. This transfer of
    plasmid DNA can even occur across species.

20
Some plasmids contain multiple antibiotic
resistance genes
21
Gene Transfer in Bacteria
Fig. 14.9
22
Transformation
  • Fragments of donor DNA enter the recipient and
    alter its genotype
  • Natural transformation recipient cell has
    enzymatic machinery for DNA import
  • Artificial transformation damage to recipient
    cell walls allows donor DNA to enter cells
  • Treat cells by suspending in calcium at cold
    temperatures
  • Electroporation mix donor DNA with recipient
    bacteria and subject to very brief high-voltage
    shock

23
Mechanism of natural transformation
Fig. 14.10
24
Conjugation A type of gene transfer requiring
cell-to-cell contact
Fig. 14.11
25
The F plasmid and conjugation
Fig. 14.12 a
26
The process of conjugation
27
The F plasmid occasionally integrates into the E.
coli chromosome
  • Hfr cells have integrated part of chromosome
  • Episomes plasmids that can integrate into host
    chromosome
  • Exconjugate recipient cell with integrated DNA
  • Integrated plasmid can initiate DNA transfer by
    conjugation, but may take some of bacterial
    chromosome as well

Fig. 14.13
28
Gene transfer in a mating between Hfr donor and
F- recipient
Fig. 14.14
29
Mapping genes in Hfr and F- crosses by
interrupted mating experiments
30
Interrupted mating studies confirm bacterial
chromosome is a circle
  • Cross between Hfr and F-
  • The F plasmid integrates into different locations
    in different orientations into the circular donor
    chromosome

Fig. 14.16 a, b
31
Partial genetic map of the E. coli chromosome
Fig. 14.16 c
32
Recombination analysis improves accuracy of map
  • Interupted mating experiments accurate to only 2
    minutes
  • Frequency of recombination between genes is more
    accurate
  • Start by considering only exconjugates that have
    all of the genes to be mapped (select for the
    last gene transferred)
  • Living cells must have even number of crossovers
  • Consider as a three-point cross

33
Mapping genes using a three-point cross
Fig. 14.17
34
Different classes of crossovers quadruple
crossover is least frequent
Fig. 14.17 c
35
F plasmids can be used for complementation
studies
  • F plasmids replicate as discrete circles of DNA
    inside host cells.
  • Transferred in same manner as F plasmids
  • A few chromosomal genes will always be
    transferred as part of the F plasmid
  • Can create partial diploids
  • Merozygotes partial diploids in which two gene
    copies are identical
  • Heterogenotes partial dipoids carrying
    different alleles of the same gene

36
F plasmid formation and transfer
Fig. 14.18 a, b
37
Complementation testing using F plasmids
  • Creation of a heterogenote
  • Phenotype of partial diploid establishes whether
    mutations complement each other or not

Fig. 14.18 c
38
Transduction Gene transfer via bactgeriophages
  • Bacteriophages
  • Widely distributed in nature
  • Infect, multiply, and kill bacterial host cells
  • Transduction - may incorporate some of bacterial
    chromosome into its own chromosome and transfer
    it to other cells
  • Bacteriophage particles are produced by the lytic
    cycle
  • Phage inject DNA into cell
  • Phage DNA expresses its genes in host cell and
    replicate
  • Reassemble into 100-200 new phage particles
  • Cells lyse and phage infect other cells
  • Lysate is population of phage after lytic cyle is
    complete

39
Generalized transduction
Fig. 14.19
40
Mapping genes by generalized transduction
  • Frequency of recombination between genes
  • P1 bacteriophage often used for mapping
  • 90kb can be constransduced corresponding to about
    2 recombination or 2 minutes
  • First find approximate location of gene by mating
    mutant strain to different Hfr strains
  • P1 transduction then used to map to specific
    location

41
Fig. 14.20
42
Temperate phage can integrate into bacterial
genome through lysogenic cycle creating a prophage
Fig. 14.21
43
  • Recombination between att sites on the phage and
    bacterial chromosomes allows integration of the
    prophage

Fig. 14.22 b
44
  • Errors in prophage excision produce specialized
    transducing phage
  • Adjacent genes are included in circular phage DNA
    that forms after excision

Fig. 14.22 c
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