Lecture 6: Genetic Analysis and Mapping in Bacteria and Bacteriophages Flashcards
What are the benefits of using microorganisms in research?
- Bacteria and bacteriophages essential in genetic study
- Bacteria and virus research is valuable due to:
- Exhibit extremely short reproductive cycles
- Can be studied in pure culture
Define Adaptation hypothesis
- Interaction of bacteriophage and bacterium is essential to acquisition of immunity to phage
- Exposure to the phage “induces” resistance in the bacteria
Define Spontaneous mutation
- Considered primary source of genetic variation in bacteria
- Occurs in the presence or absence of bacteriophage T1
- Explains origin of resistance in E. coli
Explain Prototroph
- Wild Type
- Can grow on minimal medium
- Can synthesize all essential organic compounds (human body can not)
Explain Auxotroph
- Mutant
- Needs complete medium
- Has lost ability, via mutation, to synthesize essential compounds
At what stage would you expect to see growth?
- LOG PHASE (EXPONENTIAL GROWTH) shown here!
- Lag: not as active
- Stationary phase: Equally dying
Explain Genetic recombination
- Provides basis for development of chromosome mapping methodology
- Genetic information is transferred
- Results in altered genotype
Explain Vertical Gene transfer
- Transfer of genetic information between members of SAME species
- Parent —> Offspring
Explain Horizontal Gene transfer
- Transfer of genetic information between related but distinct species
- Plays significant role in evolution of bacteria
- SAME GENERATION
T/F: A bacterial cell WIHTOUT a fertility factor can NOT donate DNA during conjugation
TRUE! Fertility factor (plasmid) must be present for conjugation to occur
Explain F Factor (Fertility factor)
- Unidirectional transfer of genetic material
- F+ cells serve as D N A donors
- F− cells are the recipients
- F+ cells contain fertility factor
- Confers ability to donate D N A during conjugation (has pili)
Explain Conjugation in bacteria
- Bacterial sex
- Genetic information from one bacterium is transferred
to another - Recombines at independent locations to become wild-
type cells - Prototrophs result from two auxotrophs
Is cell-to-cell contact essential for chromosome transfer?
YES! Especially for the transfer of the F-factor!
Understand how Conjugation with Pilus occurs
- Note: E. coli may or may NOT contain the F factor
- If F factor is present:
- Cell forms sex pilus - serves as a donor of genetic information
- Copy of F factor is transferred from F+ cell to the F- recipient, converting the recipient to the F+ state
What is Hfr (high-frequency recombination)
- Hfr (high-frequency recombination)
* Special class of F + cells - Hfr strain donates genetic information to F− cell
* Recipient does not become F +
* F + × F− → recipient becomes F + (low rate of recombination)
* H f r × F− → recipient remains F− (high rate of recombination)
What is Time Mapping?
- Chromosome of Hfr strain transferred linearly
- Gene ORDER and DISTANCE between genes can be PREDICTED served as basis for first genetic map of E. coli
Gene transfer by Hfr strains led to the understanding that E. coli chromosome is _______
CIRCULAR
The first part of the donor chromosome to enter the
recipient is known as _____
The Origin
aka The point of integration of the F factor into the chromosome
Will Hfr always give you an F plasmid?
It depends.
Explain Plasmids
- Double-stranded closed CIRCULAR extrachromosomal function of DNA
- Exist in multiple copies in cytoplasm
- Contain one or more genes
- F factor can integrate into chromosome
- Replicate independently of the bacterial chromosome
What two components do R plasmids have?
Note: high copy # meaning they are extremely present!
- RTF - Resistance transfer factor
- r-determinants
Define RTF - Resistance Transfer Factor
Encodes genetic information for transfer between plasmids
Define r-determinants
Confer antibiotic resistance
Explain Col plasmids
- ColE1 derived from E. coli
- Encode colicins: proteins toxic to bacterial strains that do NOT harbor same plasmid
- Kill neighboring bacteria
- Not transmissible to other cells (R plasmids are)
Define colicins
Proteins toxic to bacterial strains that do NOT harbor same plasmid
Compare Endo and Exo-nuclease
- Endonucleases: Cut nucleotides found within the DNA strand.
- Exonuclease: Cuts nucleotides found on DNA at the 3’ or the 5’ ends.
Define Transformation
- Small pieces of extracellular DNA are taken up by bacterial cell
- Integrated stably into the chromosome
What are the 2 steps of transformation
- Entry of foreign DNA into recipient cell
- Recombination between foreign DNA and homologous region in recipient chromosome
What are the 2 outcomes of transformation
- Completion of both steps required for genetic recombination
- First step alone results in additional foreign DNA to cytoplasm but NOT chromosome (Known as EPISOMAL EXPRESSION)
Explain Heteroduplex
- Recombinant region that holds one host strand and one mutant strand
- Strands are from different sources
- Contains mismatch of base pairs
Define Cotransformation
- Simultaneous transfer of genes
- Genes are close enough to be linked
- Are carried on single segment of 10,000-20,000 nucleotide pairs
- Frequency of two unlinked genes transforming simultaneously lower than that of linked genes
Define Bacteriophages
- Viruses that have bacteria as their host
- Reproduce via bacterial genetic recombination mechanism:
transduction
Explain the structure of Bacteriophage T4
- Head of virus: DNA contained in icosahedral protein coat
- Tail: Contractile sheath surrounding central core
- Tail fibers contain binding sites that recognize bacterial outer surface
Understand the Phage Life Cycle
- Phage absorbed to bacterial host cell
- Phage DNA injected; host DNA degraded
- Phage DNA replicated and phage protein components made
- Mature phages assembled
- Host cell lysed; phage released (Lytic viruses due to immediate reaction!)
T/F: A lot of viruses are host specific?
TRUE!
An e-coli virus only infects E. coli
Phages exist in two forms. Name them!
- Lytic (Virulent)
- Lysogenic (Temperate)
Define Plaque Assay
- Determines number of phages produced after
infecting bacteria - Entails serial dilutions of virally infected bacteria
- Counting plaques (areas clear of bacteria) on
plates determines number of phages in original
culture
Define Lysogeny
- Phage D N A integrates into bacterial chromosome—coexists
- Replicated along with the chromosome
- Passed to daughter cells
- No lysis of host cell
Define Temperate Phages
Can either lyse cell or
behave as prophage
Define Virulent Phage
Can only lyse cell
Define Prophage
- Viral D N A integrated into bacterial chromosome
- In lysogenic stage: Capable of being lysed as a result of
induced viral reproduction - A bacterium harboring a prophage has been lysogenized
The viral DNA is classified as an ______
- Episome
- A genetic molecule that can replicate either in the
cytoplasm of a cell or as part of its chromosome
Define Transduction
Bacterial recombination
mediated by bacteriophages
Explain the Lederberg-Zinder experiment
- Used Salmonella (auxotrophic
strains) - Led to discovery of phage
transduction in bacteria
Define Generalized Transduction
Process of transduction where bacterial recombination is mediated by bacteriophage
Define Cotransduction
- Two genes are close enough to be transduced simultaneously
- Two independent transduction events may occur if genes are not close enough
T/F: All Lysogenic phages will eventually become a Lytic phage
TRUE!!!
If only a lytic phage, will not become a lysogenic virus
Bacteriophage Mutations affect what?
Plaque morphology
Define Mixed Infection Experiments
- Two distinct mutant strains simultaneously infected bacteria
- Viral particles used exceeded bacterial cells to ensure simultaneous infection
- Demonstrated intergenic (two loci) recombination occurs in bacteriophages
Define Complementation
- During simultaneous infection, mutant strains give each
other lacking genes - Restores wild type
Define Complementation groups (cistrons)
- Failed to complement each other
- Successfully complemented each other
Define Cistrons
- Smallest functional
genetic unit - Describes
complementation group - Represents a gene
Define Recombinational Analysis
- The percentage of recombinants can be determined by counting the plaques
at the appropriate dilution - The frequency of recombination is an estimate of the distance between the
two mutations within the cistron.
