Download Presented By: Chantille Haynes, Hilary Price, and Richard Dalton

Presented By: Chantille Haynes, Hilary Price, and Richard Dalton
Working with Microorganisms
-Individual bacteria = invisible to the naked eye .
-Colonies of bacteria= visible to the naked eye.
-Cells in colonies are genetically identical.
-Colonies form as bacteria divide by binary fission.
-Do colonies grow forever? NO!
- How do you grow a bacterial colony?
Bacterial Phenotypes
- Prototrophic- Self-feeding on a minimal media. Synthesize all the molecules of which they are composed.
-Auxotrophic- Will not grow unless specific nutrients are added to the minimal media. (ie. if a bacterium cannot
synthesize adenine (ad¯) then it must be added to the media).
- Resistance- Bacterial wild types are affected by various inhibitors. Mutants can form colonies in the presence of
inhibitors (ie. streptomycin).
-Utilizing Energy Sources- Bacterial wild types can utilize many different sugars as energy sources. Mutations can
arise that inactivate the ability to use a particular energy source.
-Note- Testing the phenotype of a culture can be done by inoculating a few cells from the culture onto the appropriate
medium and determining whether or not a colony appears.
Bacterial Selective Systems
- Used to collect a mass of bacterial mutagens.
-Are experimental protocol designed to allow the desired mutant types to survive and propagate.
- Allows researcher to single out a particular mutant in an abundance of wild type cells.
-Example: antibiotic resistance
-Examples: revertants
-Example: Mutations that change from protrophy to auxotrophy.
-Add penicillin to a suspension of bacterial.
-The auxotrophic mutants survive and the prototrophic cells die.
-Mutants are isolated by washing the filter.
-The washed cells must be plated on a range of different media,
each supplemented with one specific chemical.
-This will aid in determining the specific requirements
of any given auxotroph. What results might we expect?
Bacterial Conjugation
- The union of two compatible bacterial cells, first by direct contact, and then by a bridge like connection, allowing the
transfer of genetic material from the donor cell to the recipient cell.
-Discovered by Joshua Lederberg and Edward Tatum in 1946.
- Conjugation and gene transfer in Escherichia coli are driven by the fertility factor or sex factor.
-This genetic element is not found in all E. coli. F+ = a cell carrying the F plasmid. F- = a cell lacking the F plasmid.
Recombination Between Donor and Recipient DNA
Bacterial genetic crosses:
Hfr (donor) X F- (recipient)
Steps leading up to recombination:
- Conjugation (cell fusion).
- Hfr chromosome replication (beginning at the origin).
- Linear transfer of genetic information into the F- cell.
- Linear ssDNA is polymerized into linear dsDNA.
Genes are transmitted in the order they appear on the chromosome!
Exogenote » Linear dsDNA donor fragment.
Endogenote » Circular F- chromosome.
Two crossover events result in the integration of part of the exogenote (donor genome) into the endogenote (recipient
genome).
This is bacterial recombination.
The ability to cross bacteria and detect recombination events allows for chromosome mapping.
Two methods of chromosome mapping based on bacterial conjugation:
(1) Mapping by Interrupted Conjugation.
- Low-resolution map.
- Analyzes large regions of the genome.
(2) High-Resolution Mapping by Recombinant Frequency.
- High-resolution map.
- Analyzes small regions of the genome.
F Factors Carrying Bacterial Genes
An integrated F factor can also exit the recipient bacterial chromosome.
Usually, this involves a precise excision...
BUT!
If an alternate insertion sequence (IS) element is involved, part of the bacterial chromosome can be liberated into
the plasmid!
F' » Plasmids carrying bacterial genes.
Sexduction » Creation of a merozygote by an F' element.
Allow haploid cells to be studied as diploid cells.
ex. Determination of dominanace.
Bacterial Transformation
Transformation » The directed modification of a genome by the external application of DNA from a cell of different
genotype.
Recipient bacteria must be in a physiological state of competence for transformation to occur.
Bacterial transformation can be demonstrated by genes for drug resistance.
- resistant culure + sensitive culture = resistant culture
- Frequency of resistant cultures depends on the amount of transforming (resistant) DNA added.
- DNA is inserted via double crosover.
Transfomation can also be used to asses the tightness of gene linkages.
- DNA extraction for transformation causes breakage of the DNA.
- The closer two genes are, the less likely a break will occur between them.
- Frequency of cotransformants indicates genes carried on the same piece of DNA.
ex.
The diagnostic linkage for DNA from an a+/b+ and a-/b- cross is:
a+b+/T > a+b-/T X a-b+/T
There is a greater frequency of cotransformants than individual transforamations. Therefore,
linked!
a and b are closely
The frequency of cotransforamtion of two genes can be used to map the distance between them.
Bacteriophage Genetics
What is a phage and why is it of interest? "Eater of Bacteria"
Phage Infection
Attaches to Bacterium
Injects genetic material through cell wall into cytoplasm
Phage essentially takes over the machinery of the host cell.
Uses the machinery to produce phage components.
The Lytic Cycle
Phages are very small
- 25 to 200 nm in length (0.025um - 0.2 um)
- However we can produce visible phenotypes
Phages can be genetically analyzed
-Phage Cross
-Have specific parental strains
-Infection of bacteria with strains
-Analysis of Lysate (Progeny Phase)
phagein = "to eat"
Bacterial Genomes and Phages
Phages can be divided into two types depending on if integration occurs.
1) Virulent Phages
-always lyse the infected cell
2) Temperate Phages
-integration of phage chromosome into bacterial genome
- if the phage genetic material is replicated along with the
bacterial chromosome, termed prophage.
Bacterial Lysogenic State
-phage DNA is incorporated into bacterium
-the bacterium becomes resistant to any other
infection
-this 'immunity' can be transferred to other generations
-However not totally safe, prophage can excise from
the bacterial chromosome and enter the lytic cycle
Zygotic Induction and lysogeny
Integration of Phage Lambda
- attachment site
-homologous site between the genes gal and bio in the bacterial chromosome
-crossover occurs
-integration is promoted by proteins which promote recombination
between the homologous pieces of bacterial DNA
Sometimes phages accidentally take up the genetic information from bacteria and
transfer it to other bacteria. This is termed Transduction.
Two types of tranduction:
1. Generalized Transduction
2. Specialized Transduction
Cotransduction and Linkage
-the closer two genes are on a chromosome the more likely they
will be cotransduced
-experiments can be done to give percentages in which both
genes are cotransduced
-the higher the percentage of cotransduction, the closer the two genetic markers,
the shorter the map distance will be between them
-used to create a genetic map