Microbial Genetics Lectures

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Microbial Genetics Lectures

• John Buchanan
• Lecture 1
• Mutation (239-248)
• Types of mutations
• Detection of mutations
• Recombination and Plasmids (285-306)
• Plasmids
• Transposable Elements
• Transformation
• Conjugation
• Recombination

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• Genetics branch of biology that deals with
  heredity, especially the mechanisms of hereditary
  transmission and the variation of inherited
  characteristics among similar or related
  organisms.
• At the most basic level is the study of genes

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• Genes are the fundamental unit of heredity
• DNA sequence in the chromosome
• Transcribed into mRNA
• Translated into proteins which make cells work

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• Genes are copied (DNA replication) almost exactly
  from parent cell to daughter and from parent to
  offspring
• DNA replication is EXTREMELY high fidelity
• (1 wrong nucleotide every 10 Billion)
• The copying of genes from one generation to the
  next is crucially important
• Too many mistakes (MUTATIONS) and gene integrity
  is lost and the system falls apart

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• Anyone who has never made a mistake has never
  tried anything new. - Albert Einstein
• A mistake may turn out to be the one thing
  necessary to a worthwhile achievement
• - Henry Ford
• Maybe mistakes are not such a bad thing?

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• Although multiple redundant systems are in place
  to prevent alteration of genetic material,
  mistakes still occur
• Mutations
• The wonder of all of these systems is not just
  their high fidelity, but also that mistakes are
  the source of amazing diversity and adaptation to
  changing environments
• Mistakes are the driving forces that lead to the
  tremendous variation in life we see today
• Reoccurring theme of these two lectures
  Appreciate the system AND its errors, as both are
  important

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Mutation A stable, heritable change in the
genomic nucleotide sequence
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Mutations affect bacterial cell phenotype

• Morphological mutations-result in changes in
  colony or cell morphology
• Lethal mutations-result in death of the organism
  (this is a bit of a dead end)
• Conditional mutations-are expressed only under
  certain environmental conditions
• Biochemical mutations-result in changes in the
  metabolic capabilities of a cell
• 1) Auxotrophs-cannot grow on minimal media
  because they have lost a biosynthetic capability
  require supplements
• 2) Prototrophs-wild type growth characteristics
• Resistance mutations-result in acquired
  resistance to some pathogen, chemical, or
  antibiotic

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How do mutations occur?Spontaneous mutations

• Spontaneous mutations - Arise occasionally in all
  cells are often the result of errors in DNA
  replication (random changes)
• Errors in replication which cause point
  mutations other errors can lead to frameshifts
• Point mutation - mismatch substitution of one
  nucleotide base pair for another
• Frameshift mutation - arise from accidental
  insertion or deletion within coding region of
  gene, results in the synthesis of nonfunctional
  protein

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Types of Mutations

• Point mutation affects only 1 bp at a single
  location
• Silent mutation a point mutation that has no
  visible effect because of code degeneracy
• Missense mutation a single base substitution
  in the DNA that changes a codon from one amino
  acid to another
• Nonsense mutation converts a sense codon to a
  nonsense or stop codon, results in shortened
  polypeptide

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Base-pair substitution point mutation
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Silent mutation
Missense mutation
Nonsense mutation
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Types of Mutations

• Point mutation affects only 1 bp at a single
  location
• Silent mutation a point mutation that has no
  visible effect because of code degeneracy
• Missense mutation a single base substitution
  in the DNA that changes a codon from one amino
  acid to another
• Nonsense mutation converts a sense codon to a
  nonsense or stop codon, results in shortened
  polypeptide
• Frameshift mutation arise from accidental
  insertion or deletion within coding region of
  gene, results in the synthesis of nonfunctional
  protein

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Frame-shift mutation - Insertion
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Frameshift mutation - Deletion
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How do mutations occur?Induced mutations

• Induced mutations-caused by mutagens
• Mutagens Molecules or chemicals that damage DNA
  or alter its chemistry
• Base analogs are incorporated into DNA during
  replication and exhibit base-pairing properties
  different from the bases they replace
• Specific mispairing occurs when a mutagen changes
  a bases structure and thereby alters its pairing
  characteristics (e.g., alkylating agents)
• Intercalating agents insert into and distort the
  DNA, and thus induce single nucleotide pair
  insertions or deletions that can lead to
  frameshifts
• Many mutagens (e.g., UV radiation, ionizing
  radiation, some carcinogens) can severely damage
  DNA so that it cannot act as a replication
  template cell repair mechanisms can restore the
  DNA, however they are very error prone and lead
  to mutations

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Other Types of Mutations

• Point mutation affects only 1 bp at a single
  location
• Frameshift mutation arise from insertion or
  deletion within coding region of gene, results in
  the synthesis of nonfunctional protein
• Insertion/deletion mutation Larger stretch of
  DNA added or deleted from a gene that alters gene
  expression
• Forward mutation a mutation that alters
  phenotype from wild type
• Reverse mutation a second mutation which may
  make the mutant appear wt (in same gene)

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Mutant Detection

• In order to study microbial mutants, one must be
  able to detect them and isolate them from the wt
  organisms
• Visual observation of changes in colony
  characteristics
• Mutant selection-achieved by finding the
  environmental condition in which the mutant will
  grow but the wild type will not (useful for
  isolating rare mutations)

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Application of Microbial Genetics
Ames Test for carcinogenicity
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Ames Test for carcinogenicity
Application of Microbial Genetics
Auxotroph (tryp- mutant)
Selective media to look for mutants
Looking for reversion mutants
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Recombination and Plasmids285-306
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Plasmids

• Plasmids are small ds DNA molecules, usually
  circular that can exisit independently of the
  host chromosome. They have their own replication
  origin so can replicate automonously (episomes)
  and have relatively few genes (lt30) that are not
  essential to the host.

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Types of Plasmids

• Conjugative plasmids have genes for pili and can
  transfer copies of themselves to other bacteria
  during conjugation
• Fertility factor or F factor - These plasmids can
  also intergrate into the host chromosome or be
  maintained as an episome (independent of
  chromosome)
• R factor - Also conjugative plasmids which have
  genes that code for antibiotic resistence for the
  bacteria harboring them. These do not integrate
  into the host chromosome.
• Col Plasmids - harbor Bacteriocins which are
  proteins that destroy other bacteria (eg cloacins
  kill Enterobacter species)
• Virulent plasmids - have genes which make
  bacteria more pathogenic because the bacteria is
  better able to resist host defenses or produce
  toxins/invasins

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Bacterial Conjugation

• The transfer of genetic information via direct
  cell-cell contact
• This process is mediated by fertility factors (F
  factor) on F plasmids
• Basic Conjugation
• F / F- mating
• An F plasmid moves from the donor (F) to a
  recipient (F-)
• The F plasmid is transferred via a sex pilus and
  then copied thus the recipient becomes F and
  the donor remains F
• In gram-positive bacteria, the sex pilus is not
  necessarily required for transmission generally
  fewer genes are transferred
• The F factor codes for pilus formation which
  joins the donor and recipient and for genes which
  direct the replication and transfer of a copy of
  the F factor to the recipient
• The F factor can remain on a plasmid or it can
  integrate into the bacterial chromosome via IS
  sequences. This type of donor is called and Hfr
  strain (High frequency recombination)
• F- When the F factor in an Hfr strain leaves the
  chromosome, sometimes is makes an error in
  excision and picks up some bacterial genes

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HFR Strain
F Strain
F Factor
F plasmid
Bacterial chromosome
Bacterial chromosome
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Hfr X F Mating

• Similar to the F X F cross

conjugal bridge
Sex pilus
F - cell
Hfr
F - cell
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Transposable Elements

• Transposons - DNA segments that carry genes that
  allow them to move about the chromosome
  (transposition)
• Unlike plasmids or phages, they are unable to
  reproduce or exist apart from the host chromosome

Cut and Paste
Transposon
Bacterial chromosome
Bacterial chromosome
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Transposable Elements

• Transposons - DNA segments that carry genes that
  allow them to move about the chromosome
  (transposition)
• Unlike plasmids or phages, they are unable to
  reproduce or exist apart from the host chromosome
• Insertion sequences - IS elements- short sequence
  of DNA containing only genes required for
  transposition Flanked by inverted repeats (IR) -
  identical or similar sequences 15-25 bp in
  reversed orientation
• Transposase - enzyme that recognizes the IR and
  promotes transposition

IS10 1329 bp
Inverted repeat (IR)
IR
Transposase (402 amino acids)
Bacterial chromosome
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TRANSPOSITION MECHANISM OF INSERTION SEQUENCES
Insertion of IS into chromosomal DNA target
sequence catalysed by transposase
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TRANSPOSITION MECHANISM OF INSERTION SEQUENCES
TCGAT
AGCTA
Gap filled by DNA polymerase and DNA ligase
TCGAT AGCTA
TCGAT AGCTA
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Transposable Elements

• Transposons - DNA segments that carry genes that
  allow them to move about the chromosome
  (transposition)
• Unlike plasmids or phages, they are unable to
  reproduce or exist apart from the host chromosome
• Insertion sequences - IS elements- short sequence
  of DNA containing only genes required for
  transposition Flanked by inverted repeats (IR) -
  identical or similar sequences 15-25 bp in
  reversed orientation
• Transposase - enzyme that recognizes the IR and
  promotes transposition
• Composite transposon (Tn)- contains other genes
  in addition to transposase like antibiotic
  resistance genes or toxins

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STRUCTURE OF COMPOSITE TRANSPOSONS
Tn10 9,300 bp
Tetracycline resistance gene
IS10R
Bacterial chromosome
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Transposable Elements

• Transposons - DNA segments that carry genes that
  allow them to move about the chromosome
  (transposition)
• Unlike plasmids or phages, they are unable to
  reproduce or exist apart from the host chromosome
• Insertion sequences - IS elements- short sequence
  of DNA containing only genes required for
  transposition Flanked by inverted repeats (IR) -
  identical or similar sequences 15-25 bp in
  reversed orientation
• Transposase - enzyme that recognizes the IR and
  promotes transposition
• Composite transposon (Tn)- contains other genes
  in addition to transposase like antibiotic
  resistance genes or toxins
• Importance
• Can insert within a gene to cause a mutation or
  stimulate DNA rearrangement leading to deletions
  of genetic material
• Can have termination sequences to block
  translation or transcription
• Can have promoters which activate genes near pt
  of insertion
• Can move antibiotic resistance genes around
• Can be on plasmids to aid in insertion of F
  plasmids into host chromosome
• Some bear transfer genes (Tn916) and can move
  between bacteria through conjugation
  (conjugative transposon)

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TRANSPOSABLE GENETIC ELEMENTS CAUSE INSERTION
MUTATIONS
Lactose operon E. coli lac
AATTC TTAAG
Chromosomal DNA
AATTC TTAAG
AATTC TTAAG
Tn10
Mutated lactose operon E. coli lac-
Tetracycline resistance gene
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DNA Transformation

• Transformation-a naked DNA molecule from the
  environment is taken up by the cell and
  incorporated in some heritable form. This process
  is random and any portion of the genome may be
  transferred
• A competent cell is one that is capable of taking
  up DNA
• Competent bacteria must be in a certain stage of
  growth (usually exponential) and secrete a small
  protein (competency factor) that stimulates
  production of new protein required for DNA uptake
• Gene transfer by this process occurs in soils and
  marine environments so it is an important route
  of genetic exchange in nature
• Artificial transformation - carried out in
  laboratory to transfer plasmid DNA, a common
  method for introducing recombinant DNA into
  bacterial cells. eg CaCl2 or electroporation

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Oswald T. Avery
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R strain
S strain
S strain
Competent cell
Plasmid
Treatment
Competent cell
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Bacterial Recombination-process by which one or
more nucleic acid molecules are rearranged or
combined to produce a new nucleotide sequence

• Types of recombination
• General recombination involves exchange between
  homologous DNA sequences
• Site-specific recombination is the nonhomologous
  insertion of DNA into a chromosome often occurs
  during viral genome or transposon integration
  into the host, a process catalyzed by enzymes
  specific for the host sequence
• Replicative recombination accompanies replication
  and is used by some genetic elements that move
  about the genome
• All can lead to Horizontal Gene Transfer

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General recombination
HFR Strain
F Strain
F Factor
Homologous recombination
F plasmid
Bacterial chromosome
Bacterial chromosome
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DNA RecombinationHorizontal Gene Transfer

• Horizontal gene transfer-transfer of genes from
  one independent organism to another (compared to
  vertical gene transfer-transmission of genes from
  parents to offspring)
• Intracellular fates of transferred DNA
• Integration into the host chromosome
• Independent functioning and replication without
  integration
• Survival without replication
• Degradation by host nucleases (host restriction)
• Mechanisms of horizontal gene transfer
• Conjugation is direct transfer from donor
  bacterium to recipient while the two are
  temporarily in physical contact
• Transformation is transfer of a naked DNA
  molecule
• Transduction is transfer mediated by a
  bacteriophages (viruses that infect bacteria)

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How do mutations occur?Directed mutations

• Hypothesis only
• Organism induces hypermutation in DNA in response
  to environmental stimuli
• For example
• Bacteria baseline mutation rate 1 in 10 billion
• Bacteria in nutritionally poor medium 1 in 1
  million
• Some mutations allow survival in poor medium
• Fundamental question Can organisms upregulate
  their mutation rate in response to environmental
  stress?