July 9th Microbial genetics continued

chart....

Comparison of Bacterial Recombination Processes

Process	Donor must be?	Recipient must be?	Pili required?	Virus involved? (if yes, life cycle and mistake stage)	Which cell is changed genetically, and how?
Transformation	same species, dead	same species, competent	no	no	recipient: absorb donor genome and replaces old with new copy
Conjugation (plain)	F +	F - after: F +	yes	no	recipient receives F plasmid
Hfr Conjugation	F + incorporated into genome	F - after still F - but may have new genes	yes	no	genes closest to the incorporated F plasmid
Generalized Transduction	infected by lytic virus	injected with DNA from host 1 by a virus	no	yes, lytic	Random genome from host 1 inserted into host 2
Specialized Transduction	infected by lysogenic virus	injected with partial viral DNA and flanking genes	no	yes, lysogenic	Flanking genes where the viral DNA was inserted are more likely to be injected

act out bacterial sex skit....

transformation: donor must die and the genome breaks into random pieces.... recipient grab fragments... new 1 and 15, replace old 1 and 15

gained new ability that she did not have before? 

example: gained ability to juggle, used to have ability to walk on hands in order to gain others

Bacterial Conjugation

Fig. 7.34

      *Donor = F + (must have F plasmid)

     *does not matter which species

      * Recipient = F -

      * May be same or different species. 

      * F + cell makes sex pili

       * copy of F plasmid is sent along pili to F- cell

      * Recipient becomes F + (plasmid carrying donor)

NB If F plasmid joins R or T plasmid, more beneficial to recipient

FYI:

F Plasmid genes: how do they do that? 

* Synthesis of pili- traA, traL, traE, traK, traB, traV, traC, traW, traU, traF, traQ, traH, traG

* Surface exclusion- traS, traT

* Stabilization of mating pairs- traN, traG

* DNA transfer - traM, traY (exonuclease) traD, tral (helicase, traZ (exonuclease)....

Fig. 7. 34B

*** stop and draw this***

why is this so rare? what is the more common?

 Hfr conjugation

 "High frequency Conjugation"

Fig. 7. 35
* prior to conjugation:

• donor cell: 
• F plasmid incorporates into genome
• cell is now both F + and an Hfr donor

* during conjugation: entire circle begins to transfer.. will not be able to copy ALL of F plasmid because it start by sending part of the F plasmid genes and then ALL of the genome before even trying to send the rest of the F plasmid genes. however most will not be sent..

Results of Hfr Conjugation: 

• recipient gains:
•  partial F plasmid transferred (soon recycled)
• select genome genes from donor received
• ones beside plasmid genes are most likely to be transferred
• Does not become donor : partial transfer of F genes (time limits) 

 *Example picture:  if cells above were interrupted at the point seen, what would be the gene status of both cell A and B
(ie, F+ or -, Thr + or -

*** add to hand drawn diagram***

*** partial F plasmid copies are recycled***

**break**
http://www.ncwildlife.org/Portals/0/Regs/Documents/2011-12/2011-12_Hunting.pdf 

booklet with hunter's license about what parts of deer is usable and not usable...

*** group activity**

* Gene mapping using Hfr conjugation (stolen from genetics textbook)
*how do we know the DNA in bacteria is circular?
* Different strains of E. coli allow F plasmid insertion at different parts of the circular genome
   - Plasmid insertion point is Origin of transfer
* Use the Data in the chart below of which genes are transferred earlier or later in each strain to map the genes and tell where the O is for each strain..

Hfr Strain      Earliest                    Order of transfer                              latest
1                   Leu    Thr      Thi    Gal   Lac    Pro     Ton    Azi
2.                  Pro    Ton      Azi    Leu   Thr    Thi     Gal   Lac
3.                  Ton    Azi      Leu   Thr    Thi    Gal     Lac   Pro
4.                  Thr    Leu      Azi    Ton   Pro     Lac   Gal   Thi

Difference in strain 4? it sends the genes in the opposite order compared to the other three strains.

*** draws 4 diagrams of E. coli on blackboard**

The last two kinds of horizontal transfer

Transduction - two methods 

(usually same species because phages are usually species specific)

•  Generalized: 
• Lytic viruses
• mistake is made during ASSEMBLY stage: put wrong piece of DNA in the capsid
• RANDOM fragments of host I DNA injected into host 2
• Fig. 7. 33 bacteria makes hole with lysozyme and tail fibers ... Genome of host cell fragmented, most phages will be normal but some capsomeres come around a bacterial DNA fragment instead of a viral genome 
• **** hand draw process*** 1.attachment 2. penetration, host DNA cut up 3. synthesis of both proteins and viral DNA, very chaotic 4. proteins assemble around viral DNA and sometimes makes a mistake and assemble around host DNA fragments ( transducing phage, normal phages (most baby viruses are normal but some may be transducing phage)) 5. release
•  Specialized: 
• Lysogenic viruses
• Mistake in synthesis
• Specific genes on host 1 genome
• flanking insertion point of viral genes injected into host 2
• *** hand draw process***1. attachment 2. penetration of viral genes 3. integration 4. synthesis (some start at the right place and copy ALL of Viral genes but sometimes they start in the middle of the viral DNA and do not copy all ) makes all needed proteins. 5. Assembly of normal phages and transducing phages 6. Release... normal phage will go infect another cell... transducing phages (they go and infect another host but now this cell is getting something very different injected into it... attachment, Penetration: insertion of hybrid A and partial viral DNA; replace genes from inserted transducting phage and homologous genes in the host 2 cell  from host 1 cell and recycle partial viral DNA and old gene that was replaced....
•  

Tomorrow will be Diseases and disease types!