8: The Genetics of Bacteria and Their Viruses
F+ Cell
a cell in the F factor that has the F factor, have the genes for the synthesis of the F Pillai
F- Cell
a cell in the F factor that is the bacterial cell with just the main chromosome
What structures would be found in an F+ cell?
a chromosome, along with an autonomous F factor
What structures would be found in an Hfr cell?
a chromosome, along with an integrated F factor from the F+ cell
What is the outcome of a successful bacterial transformation?
a heteroduplex that includes the donor DNA
Transduction
a parasexual process in bactera that includes transfer of bacterial DNA by bacteriophage (bacteriophage = virus) --> DNA material being exchanged is made of a virus
Conjugation
a parasexual process in bacteria that includes direct transfer of DNA from one bacterium to another
Transformation
a parasexual process in bacteria that includes uptake of free DNA
Bacterial Transformation
a parasexual process in which donor DNAs enter the cell and become single-stranded to be incorporated into the host chromosome
What is the difference between prototrophs and auxotrophs?
ability to synthesize essential amino acids and metabolites on their own (prototrophs can, auxotrophs cannot)
F+ --> Hfr Conjugation Technique
an "interruption" when the chromosome or genetic material is being exchanged --> a natural separation between the F- cell and the Hfr cell before the chromosome is complete (very rarely will a complete Hfr cell take place)
F Factor
associated with conjugation, where the synthesis of the genes of the F pillai occur
Plasmid
associated with conjugation; they are mini chromosomes, an extra DNA source located inside the bacterium --> can be as many as 100 different plasmids in a bacteria
Bacteriophage
associated with transduction, viruses that infect bacteria
If you were looking at an image, how can you tell if conjugation has occurred? (3)
(1) you can see the F- cell, and (2) now you have an Hfr cell because the F factor has been integrated into the chromosome, (3) and you can see the conjugation channel
What cells are associated with bacterial conjugation? (4)
1) F+ 2) F- 3) F' 4) Hfr
What are the different cells within the F factor in E. coli? (conjugation) (3)
1) F- cell 2) F+ cell 3) Hfr cell
Explain the process involved in the formation of an F' plasmid... (3)
1) Hfr cell --> contains bacterial chromosome with integrated F factor 2) F factor is excised 3) F' cell --> contains bacterial chromosome
How is the basic genetics of bacteria different from eukaryotes? (2)
1) bacteria have one main chromosome --> eukaryotes have several chromosomes 2) single main chromosome on bacteria is circular --> eukaryotes have linear chromosomes
How were the phenotypes of bacteria studied? (3)
1) colony color and morphology (ex: round or smooth) --> could distinguish different bacteria mutants based on colony color 2) nutritional mutants for energy sources --> bacteria can be nutritional sources (ex: if lactose, requires lactose or don't require lactose) 3) prototrophs (wild types) and auxotrophs (mutants)
What is an overview of bacterial transformation? (explain the general process) (4)
1) donor DNA fragments bind to proteins on the cell surface 2) donor DNA fragment is transported into the host cell and becomes single-stranded 3) single-stranded donor DNA fragment is incorporated into the host chromosome 4) degradation of donor DNA fragment and formation of a heteroduplex
Explain the process of recombination in bacteria for two crossovers... (2)
1) gene transfer in bacteria produces a partially diploid recipient cell containing a fragment of the donor cell's chromosome 2) two crossovers insert a segment of the donor cell's chromosome into the intact circular chromosome of the recipient cell --> the replaced recipient fragment is degraded
Explain the process of recombination in bacteria for one crossover... (2)
1) gene transfer produces a partially diploid bacterium --> single crossover 2) a single crossover between the linear donor fragment and the intact circular chromosome of the recipient produces an unstable linear chromosome
What are the important components of bacteriophage lambda? (3)
1) head 2) DNA (within the head) 3) sheath
What are the important components of bacteriophage T4? (6)
1) head --> contains the DNA 2) neck and collar 3) core 4) sheath 5) endplate 6) fibers
Explain how you could distinguish between the three parasexual processes in bacteria... (5)
1) if cell contact required --> conjugation 2) if no cell contact required --> either transformation or transduction 3) if DNA is sensitive to DNase --> transformation 4) if not sensitive to DNase --> either conjugation or transduction 5) if no cell contact and not sensitive --> transduction
Explain the results of Davis' experiment #3 on transformation... (3)
1) in experiment 2 --> strain A and strain B can't grow on minimal medium, but when they are mixed, they could 2) now when you have strain A and B in this U-tube and keep them separated by a filter, strain A and B cannot grow on a minimal medium 3) this could mean that there is something required, some type of cellular contact required for the genetic exchange of materials
Explain the F+ --> Hfr conjugation technique... (6)
1) integration of the F factor (F+ cell) 2) formation of the Hfr cell 3) the origin of transfer determined, conjugation 4) chromosome transfer begins 5) chromosome transfer and replication continue 6) conjugation interrupted --> F- cell, exoconjugates --> Hfr cell
Explain the mechanism of bacterial transformation... (6)
1) large donor DNA binds to the cell surface 2) nuclease nicks and degrades one strand of donor DNA as the other strand is transported into the cell 3) competence-induced proteins bind to single-stranded donor fragment 4) single-strand of donor DNA enters the host cell and is integrated into the chromosome 5) a heteroduplex chromosome is produced by recombination 6) replication of the heteroduplex recombinant chromosome produces one transformed cell (a+) and one non-transformed cell (a-)
Explain Griffith's experiment #1 on transformation... (9)
1) morphology --> S = smooth, R = resistance (rough) METHOD A 2) type IIIS (virulent) bacteria are injected into mouse 3) RESULTS --> mouse dies, type IIIS (virulent) bacteria recovered METHOD B 4) type IIR (nonvirulent) bacteria injected into mouse 5) RESULTS --> mouse lives, no bacteria recovered METHOD C 6) heat-killed type IIIS bacteria injected into mouse 7) RESULTS --> mouse lives, no bacteria recovered METHOD D 8) a mixture of type IIR bacteria and heat-killed type IIIS bacteria injected into mouse 9) RESULTS --> mouse dies, type IIIS (virulent) bacteria recovered
What were the conclusions of experiment #2 on transformation? (3)
1) neither strain A nor strain B can grow on unsupplemented minimal medium --> by themselves they are auxotrophic 2) when the two strains are mixed, some type of genetic exchange occurred that allowed them to grow and form colonies on a minimal medium --> new strain is prototrophic 3) genetic exchange occurs between strain A and strain B, making the new strain capable of synthesizing all of the requirements for growth on a minimal medium
Explain the basic genetics of bacteria... (4)
1) one main chromosome with a few thousand genes --> most bacteria is contained on the few thousand genes 2) variable number of plasmids --> plasmids aka minichromosomes 3) asexual reproduction by simple fission --> simple fission aka binary fission 4) parasexual processes occur
How do we generate Hfr cells? (4)
1) recombination between insertion sequences 2) bacterial chromosome and F plasmid with insertion sequence on both sides, so able to integrate into the chromosome 3) the integration becomes an Hfr string 4) F-plasmid x bacterial chromosome with IS-elements --RECOMBINATION--> integrated F-plasmid yielding an Hfr strain
Why were bacteria and their viruses chosen to be a model organism? (5)
1) small size 2) rapid reproduction 3) selective media (ex: by using antibiotics) 4) genetic variability --> mutants in bacteria are easily able to be detected 5) complete genome sequences
Explain what was happening in Griffith's experiment #1... (5)
1) the S smooth was encapsulated bacteria 2) when the S bacteria were heat-killed, it resulted in the protein shell being killed, which was a protective covering on the bacteria --> why mouse lived in method c 3) in method d there was a mixture of S and R --> when the S was heat-killed, it avoided the immune system which allows the bacteria to survive and kill the mouse 4) so, in a mixture of S and R, when S was heat-killed, the protective covering was degraded by the heat, however, the bacterial still lived 5) there was some type of exchange going on between the type IIR and the type IIIS that allowed the heat-killed IIIS and type IIR to kill the mouse
How does conjugation take place? (2)
1) the donor cell has surface appendages called F Pillai 2) the synthesis of F Pillai (those genes) are on a structure called the F factor --> that structure is the plasmid (the extra chromosome structure)
What are the 3 different mechanisms of genetic exchange for bacteria?
1) transformation 2) conjugation 3) transduction
Explain the process of chromosome mapping using conjugation... (6)
1) using Hfr and F- strains or donor and recipient cells 2) at different times (you can see the different genes on the chromosomes), you can see the different marker genes 3) in the lab, they allow these two different cells to mix, and then they will break this contact by throwing cells in a blender or shaking them vigorously 4) at different time points, they will place these cells on different selective media and see what the recombinants are 5) based on the recombinants, they can tell you which genes are present --> markers are present 6) gene order can be determined by comparing transfer time with origin of transfer
In E. coli, how long does it take to map all of the genes?
100 minuets
What organism would exhibit a plasmid inside a bacterial cell?
E. coli --> plasmids are used for conjugation
How is recombination in bacteria different from eukaryotes?
BACTERIA --> gene transfer is unidirectional, only from donor cell to recipient cell EUKARYOTES --> reciprocal exchange of genetic information
What was the third experiment to prove transformation?
Bernard Davis' U-Tube in 1950
What is the only way viruses can reproduce?
By infecting living host cells
What is a hallmark of transduction?
DNA material being exchanged is made of a virus (bacteriophage)
Integration of what two cells produce an Hfr cell?
F+ and F- cells
What different cells are needed to perform chromosome mapping using conjugation?
F+ and Hfr- cells
In chromosome mapping using conjugation, what cells are rare? why?
Hfr cells because you will rarely have the whole chromosome integrated (F+ --> Hfr conjugation technique)
What was the first experiment to prove transformation?
Griffith's discovery of transformation in STREPTOCOCCUS in 1927
What was the second experiment to prove transformation?
Lederberg and Tatum's experiment in 1946
Explain Davis' experiment #3 on transformation... (6)
METHOD A 1) auxotrophic strain A 2) RESULT --> no growth in minimal medium METHOD B 3) U-tube that contains strain A and strain B separately with airflow 4) RESULTS --> both strain A and Strain B exhibit no growth in minimal medium METHOD C 5) auxotrophic strain B 6) RESULT --> no growth in minimal medium
Can all bacteria perform transformation?
NO --> only bacteria that have competent-induced proteins can perform a transformation
Explain the process of bacterial conjugation... (7)
STEP 1 1) the F Pillai of the F+ donor cell makes contact with the F- recipient cell and pull the cells together 2) genes on the F factor direct the synthesis of the conjugation bridge 3) one strand of DNA is cleaved at the origin of replication of the F factor STEP 2 4) rolling-circle replication transfers one strand of the F factor into the recipient cell 5) replication of the F factor occurs in both cells (one strand is synthesized in each cell) during transfer STEP 3 6) transfer of the F factor is completed, yielding two F+ bacteria 7) (1) F+ donor cell contains F factor, donor chromosome, and a pilus attached to the outside --> F- recipient cell contains recipient chromosome (they make contact) --> (2) conjugation bridge provides a channel between cells --> (3) rolling-circle replication in donor cell produces a bacteria which contains an F factor and the recipient chromosome (F+); replication in recipient cell produces a bacteria which contains the donor chromosome and an F factor (F+)
Explain Lederberg and Tatum's experiment #2 on transformation... (9)
STRAIN A 1) their requirements (auxotrophs): thr, leu, thi, met+, bio+ 2) wash and spread cells 3) RESULTS --> in minimal medium there was no growth STRAIN B 4) auxotrophs: thr+, leu+, thi+, met, bio 5) wash and spread cells 6) in minimal medium, no growth STRAIN A AND B 7) combine all auxotrophs 8) wash and spread cells 9) in minimal growth, phototrophic colonies appeared
Which of the three parasexual processes in bacteria is this? cell contact required? NO --> sensitive to DNase? yes
TRANSFORMATION
Hfr Cell
a cell in the F factor --> appears when the F factor is integrated into the bacterial chromosome
In the history of studying genetics, after the studies of peas, what did they move to for their model organism?
bacteria and viruses
What is bacteriophage also known as?
bacteriophage = virus
How can you make cells competent in the lab?
by chemical treatment or by electroporation to open up those pores to allow transformation
Auxotroph
can't synthesize something due to some type of mutation --> have to supplement that media with one of those essential metabolites
The genetics of bacteria and their viruses mark a transition from ____________ to ________________
classical genetics to molecular genetics
What should be one of the first things to do if you identify a mutant mutation in E. coli?
conjugate mapping
What can chromosome mapping using conjugation allow us to do?
conjugate mapping can allow us to find the location of a mutation in a chromosome
What is the natural method of genetic exchange for E. coli?
conjugation
Which of the three parasexual processes in bacteria is this? cell contact required? YES --> sensitive to DNase? NO
conjugation
What is the outcome of an unsuccessful bacterial transformation?
degradation of donor DNA fragment
How can the F+ --> Hfr conjugation technique help you map chromosomes?
different Hfr strains are used to map different regions of the chromosome
What would happen over time if you had a mixture of F+ and F- cells (the donor and recipient cells)?
eventually all of those cells would become F+ cells
In experiment #1 on transformation, what was the expected result in method D, when a mixture of type IIR bacteria and heat-killed type IIIS bacteria are injected into the mouse? What was the actual result?
expected mouse to live because when heat-killed type IIIS bacteria was injected, the mouse lived --> mouse ended up dying
What is the significance of "competence" or competent cells?
for bacterial transformation, the nucleus is needed to degrade donor DNA and make it single-stranded, so that single-stranded DNA is guided into the cell and incorporated into the recipient chromosome by these competent-induced proteins --> these cells are required for transformation (bacteria cannot perform transformation without them)
What conclusion was made on experiment #3 on transformation?
genetic exchange requires direct contact between bacterial cells
How is the F+ --> Hfr conjugation technique helpful for geneticists?
they can use this technique to do interruptive mating studies --> these mating studies are used to do E. coli or bacterial mapping using this technique
What happens if DNA is sensitive to DNase? What parasexual process exhibits this characteristic?
if DNA is sensitive to DNase, it is going to be degraded in the presence of DNA --> transformation (free DNA) is sensitive to DNase treatments
In bacterial conjugation, how do you know which cell is the F+?
if the cell has pillai, it is the F+
Sheath of Bacteriophage T4
injects DNA into bacteria
What structures would be found in an F- cell?
just the chromosome
What type of virus is bacteriophage T4?
lyctic
What is the major contribution of bacteria and viruses to genetics?
many basic concepts of genetics were first deduced from studies of bacteria and viruses
Describe the phenotypes in bacteria...
mutated bacterial genes produce altered phenotypes
Parasexual Processes
processes that are similar to the sexual processes in eukaryotes --> bacteria do not go through mitosis and meiosis, but recombination does occur and it's important in their evolution and is a part of these parasexual processes
In bacteria, what is important for their evolution and is also a part of their parasexual process?
recombination
Why are competence cells studied in lab?
scientists study methods for making cells competent, either by chemical competency or electroporation --> they are opening up these pore membranes so that the free DNA can be taken up by the cell (transformation)
What process is needed for the formation of an F' plasmid?
sexduction
What parasexual process does E. coli use as their mechanism of genetic exchange?
they cannot perform transformation naturally, so they use conjugation
Why is transduction not sensitive to DNase and does not require cell contact?
that bacterial DNA is contained within that virus (the bacteriophage) so it is protected
What technique allows us to do E. Coli or bacterial mapping?
the F+ --> Hfr Conjugation technique
How can chromosome mapping occur by using conjugation?
the F+ --> Hfr conjugation technique can be used to do interruptive mating studies, which can be used to map genes --> because of this natural interruption, it allows us to do our mapping
If you were looking at an image, how could you tell which cell is the donor cell for conjugation?
the cell that has surface appendages is the donor cell
Griffith's Experiment on Transformation
the first experiment to prove transformation --> shows that there was a mixture (some type of exchange) between the type IIR and the heat-killed type IIIS that was able to kill the mouse
What happens if there is only a single crossover event in recombination for bacteria?
the recipient chromosome will become an unstable linear chromosome which will eventually degrade --> you need two crossover events for recombination to be successful, meaning the integrity of that circular chromosome is maintained so it'll be able to replicate
Sexduction
the reverse process of integration (combination of F+ and F- cells to produce Hfr cells), the F factor is excised out of the bacterial chromosome --> instead of being F- or F+ cell, it is an F' (prime) cell because part of the bacterial chromosome has been incorporated into the F Factor
Lederberg and Tatum's Experiment on Transformation
the second experiment to prove transformation --> using the ability of different bacterial strains to be grown on plates that are in culture
Bernard Davis' U-Tube Experiment on Transformation
the third experiment to prove transformation and also the first experiment that displayed conjugation --> showed some contact required conjugation
Prototroph
the wild type, if you give them a carbon source, they can synthesize all other metabolites that they need
What is the importance of the 3 different experiments on transformation?
these experiments were critical for confirming or discovering transformation and that parasexual processes occur
How do bacteria exchange genetic information?
through three different parasexual processes (mechanisms)
What is the only parasexual process that occurs in ALL bacteria?
transduction
Which of the three parasexual processes in bacteria is this? cell contact required? NO --> sensitive to DNase? NO
transduction
What is required for recombination in bacteria?
two pairs of crossover events to occur successfully
Chromosome Mapping Using Conjugation
when new genes are mutated in the chromosome, one of the first things you do is conjugate mapping to find the location of that mutation --> then, more precise mapping can be done with transformation or transduction
Heteroduplex
when the single-stranded donor DNA is mixed with the circular chromosome from the recipient --> formed by successful bacterial transformation
