Chapter 14 The Prokaryotic Chromosome: Genetic Analysis in Bacteria

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Chapter 14The Prokaryotic Chromosome Genetic
Analysis in Bacteria
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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

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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

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A family tree of living organisms
Fig. 14.1
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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

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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

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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

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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
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E. coli lysed to release chromosome
Fig. 14.4 a
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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

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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

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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.)

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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.

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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?

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• Transposable elements in bacteria

Fig. 14.6
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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

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DNA replication in E. coli
Fig. 14.7
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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.

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Some plasmids contain multiple antibiotic
resistance genes
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Gene Transfer in Bacteria
Fig. 14.9
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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

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Mechanism of natural transformation
Fig. 14.10
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Conjugation A type of gene transfer requiring
cell-to-cell contact
Fig. 14.11
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The F plasmid and conjugation
Fig. 14.12 a
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The process of conjugation
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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
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Gene transfer in a mating between Hfr donor and
F- recipient
Fig. 14.14
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Mapping genes in Hfr and F- crosses by
interrupted mating experiments
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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
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Partial genetic map of the E. coli chromosome
Fig. 14.16 c
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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

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Mapping genes using a three-point cross
Fig. 14.17
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Different classes of crossovers quadruple
crossover is least frequent
Fig. 14.17 c
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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

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F plasmid formation and transfer
Fig. 14.18 a, b
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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
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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

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Generalized transduction
Fig. 14.19
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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

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

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

Fig. 14.22 c