Bacterial Genetics

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• Bacterial Genetics
• Bacteria are essential organisms in demonstrating
  structure and function of DNA
• Their haploid genetic state allows expression of
  a genetic trait in presence of a single mutant
  gene
• Rapid growth allows observations of transmission
  of trait through generations
• Large populations allow for isolation spontaneous
  mutants
• Low cost of maintenance and propagation allows
  for many experimental procedures

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• Bacteria are used to demonstrate genetic
  phenomena of mutations
• Point mutations - permanent, sudden qualitative
  alterations, that arise as a result of addition,
  deletion, or substitution of one or more bases in
  region of gene
• Spontaneous mutations - result from chemical and
  physical components in organisms environment
• Induced mutations genetic changes resulting
  from organisms exposure to artificial physical or
  chemical mutagen

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• Bacteria are used to demonstrate genetic
  phenomena of genetic transfer
• Conjugation mating process between sexually
  differentiated bacterial strains that allow
  unidirectional transfer of genetic material
• Transduction a bacteriophage-mediated transfer
  of genetic material from one cell to another
• Transformation a genetic alteration in a cell
  resulting from the introduction of free DNA from
  the environment across the cell membrane

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• Conjugation
• Differentiation or existence of different mating
  strains in bacteria is determined by presence of
  fertility factor, or F Factor.
• Cells that lack F Factor are recipients of
  genetic material during conjugation F-
• If cells possess the F Factor and it is
  extrachromosomal (plasmid) F
• If F Factor is incorporated into bacterial
  chromosome, resulting cells are designated Hfr
  (high frequency recombinants)

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Conjugation
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• Transformation
• Process mixing DNA from one strain of lysed
  cells with another strain of living cells. The
  DNA enters the viable cells and is incorporated
  into the bacterial chromosome. The new DNA is
  expressed and genetic capability of cell may
  change.
• The DNA donors and DNA recipients must differ in
  some way. One strain has a marker such as
  resistance to an antibiotic.
• In this exercise, a gene responsible for
  conferring resistance to the antibiotic
  Streptomycin is transferred to cells that are
  sensitive to it.

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Transformation
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Transduction
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AMES TEST A bacterial test system for chemical
carcinogenicity Various chemical that may cause
malignant transformations in humans Genetic
Aspect Carcinogenicity is linked to
mutagenicity - 90 of chemicals proved to be
carcinogens are mutagens-based on base
substitution and frame shift mutations
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Since chemical nature of DNA is common to all
organisms, allows for the use of bacterial test
system for rapid detection of possible
mutagens--called the Ames Test. Organism used
HIS- and BIO- Auxotrophic strain of Salmonella
typhimurium Mechanism Role of S-9 Review
of Procedure
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AMES TEST
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Cultivation and Enumeration of Bacteriophages 191
7 Bacteriophage term coined----means bacteria
eater Virus that attacks bacteria Structure
Protein coat w/ polyhedral head (contains
nucleic acid) and helical tail.
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Attachment adsorbs onto bacterial cell surface
by means of tail fibers and base plates Injects
nucleic acid into the bacterium during
penetration (tail sheath contracts, driving the
tail core through cell wall) Plaque formation
New phages are released from original infected
cell to infect other cells which are lysed
produces a clearing or plaque
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DNA Isolation From Saccharomyces cerevisiae
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• Why Isolate DNA?
• A. First step in genetic analysis
• 1. Plasmid DNA/Genomic DNA
• 2. PCR, REA, Cloning, Southern
• Blotting
• B. Yeast DNA-more in Saccharomyces
• than bacteria easier to see

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• IV. Purposes of each step
• Salt solutionneutralizes the negative charge of
  the DNAs phosphate units
• Detergent (Dawn)helps to remove cellular
  membrane and allows access to DNA (emulsifies or
  breaks up the fats in the cell membrane)
• Meat tenderizerdestroys enzymes which could
  digest the DNA heat enhances this action
• EthanolSeparation of layersDNA (white stringy
  matter) rises into the alcohol layer while
  protein and cellular debris is in the bottom

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DNA FINGERPRINTING -DNA Extraction and
purification -Restriction enzymes (restriction
digest) -Gel electrophoresis -RFLP
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What banding pattern will the pBIO209 plasmid
have after restriction digest? Lane 2 EcoRI
only Lane 3 HindIII only Lane 4 ApaI
HindIII Lane 5 EcoRI HindIII ApaI
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Biofilms

• Biofilms composed of populations or communities
  of microorganisms adhering to environmental
  surfaces.
• Microbial Mats - specialized microbial
  communities composed mainly of photosynthetic
  prokaryotes.

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Biofilms
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Microbial Mats
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• Forms on
• On solid substrates in contact with moisture
• On soft tissue surfaces in living organisms
• At liquid air interfaces
• On rock and other substrate surfaces in marine or
  freshwater environments
• Living organisms- e.g. tissue surfaces, mucosal
  surfaces, and teeth

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• Methods Adherence
• Glycocalyx (capsule)
• Sticky surface for other bacteria to bind to.
• Benefits of Biofilms
• Water Treatment
• Septic systems

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• Disadvantages of Biofilms
• Dental caries
• Contamination of artificial surfaces used to
  treat patients
• Resistant to antibiotics

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• Formation Steps
• Reversible Adsorption of bacteria
• Irreversible attachment of bacteria
• Growth and Division of Bacteria
• Exopolymer production and biofilm formation
• Attachment of other organisms to biofilm

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Regulation of Gene Expression

• Operon Model
• Promoter
• Operator
• Structural genes
• Inducible operon
• Repressible operon

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• Nar operon-encodes E. coli genes for nitrate
  reductase
• Ara operon-encodes E. coli genes for catabolism
  of arabinose
• Regulatory genes for these operons
• Produces a protein to inhibit transcription
• Promote binding of RNA polymerase
• In this exp. ara structural genes replaced with
• a gene pGLO that encodes green fluorescent
  protein

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Bioinformatics

• What
• branch of science devoted to computational
  information management systems used to collect,
  store, analyze, and disseminate biological
  information
• Why
• Identification and classification of bacteria
• Classical vs. New methods

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DNA Sequencing

• Correlation between similarity of DNA sequences
  and relatedness
• More similar sequences more closely related
• Most useful 16S rRNA DNA segment
• Easily compared because found in all organisms
  with the same function

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rRNA Gene Sequencing

• Key advantages
• All cells contain ribosomes and rRNA
• Greater relation equates to fewer differences in
  sequences
• RNA genes are highly conserved
• Does not require cell to be cultured

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Signature Sequences

• 5-10 base pair sequence always found in a
  particular group of organisms
• Specific location in sequence

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The Point (and todays lab)

• Use database to compare gene sequences
• Ribosomal Database Project
• Enables (most likely) identification of an
  unknown

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What To Do

1. Get a laptop and open an internet browser
2. Go to rdp.cme.msu.edu
3. Under the page heading, click on SEQMATCH
4. Cut and paste a 16S rRNA sequence, from the
   document provided, into the appropriate box
5. Click on Submit
6. Identify the organism based upon the results of
   the analysis
7. Repeat until all organisms have been identified

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Biosafety Levels
Level 1 Organisms not known to cause human
disease minimal hazard Level 2 Agents of
moderate potential hazard Level 3 Clinical,
diagnostic, teaching, research with indigenous
agents serious hazard Level 4 Exotic
organisms life threatening disease
potential LAST SLIDE
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