Microbial Genetics Lectures - PowerPoint by cGYCwrJ

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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
•   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
•   Genes are the fundamental unit of heredity
     – DNA sequence in the
       chromosome
     – Transcribed into mRNA
     – Translated into proteins which
       make cells work
• 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
• 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?
–     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
Mutation = A stable, heritable change in the
      genomic nucleotide sequence
         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
                  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
                            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
Base-pair substitution – point mutation
                    Silent mutation




                                      Missense mutation




Nonsense mutation
                            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
Frame-shift mutation -
Insertion
Frameshift mutation -
Deletion
                        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
                           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)
                 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)
                     Ames Test for
  Application of
Microbial Genetics   carcinogenicity
                                                 Ames Test for
  Application of
Microbial Genetics                               carcinogenicity
                     Auxotroph (tryp- mutant)



                                       Selective media to
                                       look for mutants




                                                            Looking for
                                                            reversion
                                                            mutants
Recombination and Plasmids
         285-306
                        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
  (<30) that are not essential to the host.

                     Bacterial cell

                                          Plasmid DNA
   Chromosomal
      DNA
                            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
                           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
      F+ Strain           HFR Strain


                             F Factor




              F plasmid
  Bacterial                  Bacterial
chromosome                 chromosome
                   Hfr X F– Mating

• Similar to the F+ X F– cross



                    conjugal
 Sex pilus           bridge




             Hfr               F - cell   F - cell
                       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                                         Bacterial
chromosome                                        chromosome
                       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


         Inverted                 IS10
        repeat (IR)              1329 bp                        IR

                    Transposase (402 amino acids)




                                 Bacterial
                               chromosome
    TRANSPOSITION MECHANISM OF INSERTION SEQUENCES


        IR                              IR

     ACAGTTCAG                      CTGAACTGT
                    Transposase
     TGTCAAGTC                      GACTTGACA

   Insertion of IS
 into chromosomal
DNA target sequence
                            Cut
    catalysed by
    transposase
                       TCGAT
                       AGCTA
Chromosomal
    DNA
                     Cut
 TRANSPOSITION MECHANISM OF INSERTION SEQUENCES

          IR                         IR

TCGAT   ACAGTTCAG                 CTGAACTGT
                    Transposase               AGCTA
        TGTCAAGTC                 GACTTGACA


    Gap filled by
   DNA polymerase
   and DNA ligase

          IR                        IR

TCGAT   ACAGTTCAG                 CTGAACTGT TCGAT
                    Transposase
AGCTA   TGTCAAGTC                 GACTTGACA AGCTA
                       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
STRUCTURE OF COMPOSITE TRANSPOSONS



             Tn10
             9,300 bp    Tetracycline
                          resistance
                             gene

 IS10L                         IS10R




             Bacterial
           chromosome
                       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)
 TRANSPOSABLE GENETIC ELEMENTS CAUSE INSERTION MUTATIONS



                   Lactose operon
                     E. coli lac+
                        AATTC
                        TTAAG
Chromosomal
    DNA




       AATTC              Tn10              AATTC
       TTAAG                                TTAAG
          Mutated lactose operon               Tetracycline
                E. coli lac-                    resistance
                                                   gene
                          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
Oswald T. Avery
                   R strain       S strain




S strain

                 Competent cell




                                         Plasmid


           Treatment
                                    Competent cell
    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
                     General recombination


      F+ Strain                              HFR Strain


                                                F Factor
          Homologous
         recombination




              F plasmid
  Bacterial                                     Bacterial
chromosome                                    chromosome
                 DNA Recombination:
               Horizontal 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)
                 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?

								
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