The Genetics of Bacteria and Their Viruses

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• Chapter 7
• The Genetics of Bacteria and Their Viruses

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Plasmids

• Many DNA sequences in bacteria are mobile and can
  be transferred between individuals and among
  species.
• Plasmids are circular DNA molecules that
  replicate independently of the bacterial
  chromosome.
• Plasmids often carry antibiotic resistance genes
• Plasmids are used in genetic engineering as gene
  transfer vectors

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F factor and Conjugation

• F (fertility) factor is a conjugative plasmid
  transferred from cell to cell by conjugation
• F factor is an episomea genetic element that can
  insert into chromosome or replicate as circular
  plasmid
• The F plasmid is a low-copy-number plasmid 100
  kb in length and is present in 12 copies per
  cell
• It replicates once per cell cycle and segregates
  to both daughter cells in cell division

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F factor and Conjugation

• Conjugation is a process in which DNA is
  transferred from bacterial donor cell to a
  recipient cell by cell-to-cell contact
• Cells that contain the F plasmid are donors and
  are designated the F
• Cells lacking F are recipients and are designated
  the F
• The transfer is mediated by a tube-like structure
  called a pilus, formed between the cells, through
  which the plasmid DNA passes

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Figure 07.03 Transfer of F from an F to an F-
cell.
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Transposable Elements

• Transposable elements are DNA sequences that can
  jump from one position to another or from one DNA
  molecule to another
• Bacteria contain a wide variety of transposable
  elements
• The smallest and simplest are insertion
  sequences, or IS elements, which are 13 kb in
  length and encode the transposase protein
  required for transposition and one or more
  additional proteins that regulate the rate of
  transposition

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Transposable Elements

• Other transposable elements in bacteria contain
  one or more genes unrelated to transposition that
  can be mobilized along with the transposable
  element this type of element is called a
  transposon
• Transposons can insert into plasmids that can be
  transferred to recipient cells by conjugation
• Transposable elements are flanked by inverted
  repeats and often contain multiple antibiotic
  resistance genes

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Figure 07.04 Transposable elements in bacteria.
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Figure 7.5 Cointergrate formed between two
plasmids by recombination between homologous
sequences present in both plasmids
Figure 05 Cointegrate
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Transposable Elements

• Integron is a DNA element that encodes a
  site-specific recombinase and a recognition
  region that allows other sequences with similar
  recognition regions to be incorporated into the
  integron by recombination.
• The elements that integrons acquire are known as
  cassettes
• Integrons may acquire multiple-antibiotic-resistan
  ce cassettes
• Bacteria with resistance to multiple antibiotics
  are an increasing problem in public health

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Figure 06 Site-specific recombinase
Figure 7.6 Site-specific recombinase
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Figure 07 Mechanism by which an integron
sequentially captures cassettes by site-specific
recombination
Figure 7.7 Mechanism by which an integron
sequentially captures cassettes by site-specific
recombination
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Figure 7.8 Mechanism of cassette excision
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Bacterial Genetics

• Three principal types of bacterial mutants use in
  bacterial genetics
• Antibiotic-resistant mutants are able to grow in
  the presence of an antibiotic.
• Nutritional mutants are unable to synthesize an
  essential nutrient and thus cannot grow unless
  the required nutrient is supplied in the medium.
  Such a mutant bacterium is said to be an
  auxotroph.
• Carbon-source mutants cannot utilize particular
  substances as sources of energy or carbon atoms.

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Figure 09 Bacterial colonies on petri dish
Figure 7.9 Bacterial colonies on petri dish
Courtesy of Dr. Jim Feeley/CDC
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Bacterial Transformation

• The process of genetic alteration by pure DNA is
  transformation.
• Recipient cells acquire genes from DNA outside
  the cell.
• DNA is taken up by the cell and often recombines
  with genes on bacterial chromosome.
• Bacterial transformation showed that DNA is the
  genetic material.

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Cotransformation of Linked Genes

• Cotransformation genes located close together
  are often transferred as a unit to recipient
  cell.
• Cotransformation of two genes at a frequency
  substantially greater than the product of the
  single-gene transformation frequencies implies
  that the two genes are close together in the
  bacterial chromosome.
• Genes that are far apart are less likely to be
  transferred together
• Cotransformation is used to map gene order

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Figure 07.10 Cotransformation of linked markers.
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Conjugation

• In bacterial mating, conjugation, DNA transfer is
  unidirectional
• F factor can integrate into chromosome via
  genetic exchange between IS elements present in F
  and homologous copy located anywhere in bacterial
  chromosome
• Cells with the F plasmid integrated into the
  bacterial chromosome are known as Hfr cells
• Hfr High Frequency of Recombination

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Hfr

• In an Hfr cell the bacterial chromosome remains
  circular, though enlarged 2 percent by the
  integrated F-factor DNA
• When an Hfr cell undergoes conjugation, the
  process of transfer of the F factor is initiated
  in the same manner as in an F cell
• However, because the F factor is part of the
  bacterial chromosome, transfer from an Hfr cell
  also includes DNA from the chromosome

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Figure 11 Integration of F
Figure 7.11 Integration of F
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Hfr and Conjugation

• Transfer begins within an integrated F factor and
  proceeds in one direction
• A part of F is the first DNA transferred,
  chromosomal genes are transferred next, and the
  remaining part of F is the last
• The conjugating cells usually break apart long
  before the entire bacterial chromosome is
  transferred, and the final segment of F is almost
  never transferred
• The recipient cell remains F

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Figure 07.12 Stages in the transfer and
production of recombinants.
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Chromosome Mapping

• It takes 100 minutes for an entire bacterial
  chromosome to be transferred and about 2 minutes
  for the transfer of F
• The difference reflects the relative sizes of F
  and the chromosome (100 kb versus 4600 kb)
• Regions in the transferred DNA may incorporate
  into the recipient chromosome and replace
  homologous regions
• This results in recombinant F cells containing
  one or more genes from the Hfr donor cell

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Table T01 Data showing the production of
recombinants when mating is interrupted at
various times
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Chromosome Mapping

• Genes in the bacterial chromosome can be mapped
  by Hfr x F mating

Figure 07.13AE Time-of-entry mapping.
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Chromosome Mapping

• Circular genetic map of E. coli shows map
  distances of genes in minutes

Figure 07.13F Time-of-entry mapping.
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Figure 07.14 Circular genetic map of E. coli.
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Figure 15 Formation of an F lac plasmid
Figure 15 Formation of an F lac plasmid by
aberrant excision of F from an Hfr chromosome
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Transduction

• In the process of transduction, bacterial DNA is
  transferred from one bacterial cell to another by
  a phage
• A generalized transducing phage transfers DNA
  derived from any part of the bacterial chromosome
• A specialized transducing phage transfers genes
  from a particular region of the bacterial
  chromosome.

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Transduction

• A generalized transducing phage P1 cuts bacterial
  chromosome into pieces and can package bacterial
  DNA into phage particles transducing particle
• Transducing particle will insert transduced
  bacterial genes into recipient cell by infection
• Transduced genes may be inserted into recipient
• chromosome by homologous recombination

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Figure 07.16 Transduction.
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Transduction

• A typical P1 transducing particle contains from
  100 to 115 kb of bacterial DNA or about 50 genes
• The probability of simultaneous transduction of
  both markers (cotransduction) depends on how
  close to each other the genes are. The closer
  they are, the greater the frequency of
  cotransduction
• Cotransduction provides a valuable tool for
  genetic linkage studies of short regions of the
  bacterial genome

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Figure 07.17 Demonstration of linkage of the gal
and bio genes.
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Transduction

• Specialized transducing phages transduce
  bacterial genes at the site of prophage insertion
  into the bacterial chromosome
• Transduction of bacterial genes occurs by
  aberrant excision of viral DNA, which results in
  the incorporation of bacterial genes into phage
  chromosome

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Temperate Bacteriophages

• Temperate bacteriophages have two life cycles
• lytic cycle infection that results in
  production of progeny phage and bacterial cell
  lysis
• lysogeny nonproductive viral infection results
  in insertion of viral DNA into bacterial
  chromosome
• Viral DNA integration site-specific insertion
  into bacterial chromosome

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Lytic Cycle

• The reproductive cycle of a phage is called the
  lytic cycle
• In lytic cycle
• Phage DNA enters the cell and replicates
  repeatedly Cell ribosomes produce phage proteins
• Phage DNA and proteins assemble into new phage
  particles
• Bacterium is split open (lysis), releasing phage
  progeny with parental genotypes

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Figure 07.18A The absence of a phage.
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Figure 07.18B Large plaques in lawn of E.coli.
Courtesy of CDC
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Lytic Cycle

• When two phage particles that have different
  genotypes infect a single bacterial cell, new
  genotypes can arise by genetic recombination
• This process differs from genetic recombination
  in eukaryotes
• the number of participating DNA molecules varies
  from one cell to the next
• reciprocal recombinants are not always recovered
  in equal frequencies from a single cell

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Figure 7.19 Progeny of a phage cross
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Fine Structure of the Gene

• The mutation and mapping studies of rII locus of
  phage T4 performed by S. Benzer provided an
  experimental proof to important conclusions
• Genetic exchange can take place within a gene and
  probably between any pair of adjacent nucleotides
• The unit of mutation is an individual pair of
  nucleotides
• Mutations are not produced at equal frequencies
  at all sites within a gene

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Figure 07.20 Array of deletion mutations used to
divide the rII locus of phage T4.
Adapted from S. Benzer, Proc. Natl. Acad. Sci.
USA 47(1961) 403-426.
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Figure 07.21 Genetic map of part of the rII
locus of phage T4.
Adapted from S. Benzer, Proc. Natl. Acad. Sci.
USA 47(1961) 403-426
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Lysogenic Cycle

• All phage species can undergo a lytic cycle
• Phages capable of only the lytic cycle are called
  virulent
• The alternative to the lytic cycle is the
  lysogenic cycle no progeny particles are
  produced, the infected bacterium survives, and a
  phage DNA is transmitted to each bacterial
  progeny cell when the cell divides
• Those phages that are also capable of the
  lysogenic cycle are called temperate

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Lysogenic Cycle

• In the lysogenic cycle, a replica of the
  infecting phage DNA becomes integrated into the
  bacterial chromosome
• The inserted DNA is called a prophage, and the
  surviving bacterial cell is called a lysogen
• Many bacterial generations, after a strain has
  become lysogenic, the prophage can be activated,
  excised from the chromosome, and the lytic cycle
  can begin

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Figure 07.22 The general mode of lysogenization.
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Bacteriophage ?

• E. coli phage ? is a temperate phage capable of
  both lytic and lysogenic, cycles
• The DNA of ? is a linear molecule with cohesive
  ends (cos) that pairing yields a circular
  molecule
• In lysogen prophage ? is linearly inserted
  between the gal and bio genes in the bacterial
  DNA
• The sites of ? integration in the bacterial and
  phage DNA are called the bacterial attachment
  site and the phage attachment site

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Figure 7.23 Linear DNA molecule showing the
cohesive ends
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Figure 26 Geometry of integration and excision
of phage
Figure 7.24 Geometry of integration and excision
of phage ?
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Bacteriophage ?

• Prophage genetic map is a permutation of the
  genetic map of the phage progeny obtained from
  standard phage crosses.
• Upon induction, the prophage ? is usually excised
  from the chromosome precisely. However, once in
  every 106 or 107 the excision error leads to
  formation of aberrant phage particles that can
  carry either the bio genes (cut at the right) or
  the gal genes (cut at the left)

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Figure 07.25 Aberrant excision leading to the
production of specialized l transducing phages.