1. LECTURE 8: GENETICS OF BACTERIA & THEIR VIRUSES I
CH5 key concepts
working with microorganisms
bacterial conjugation

2. CHAPTER 5: KEY CONCEPTS
fertility factor (F) permits bacterial cells to transfer DNA to other bacteria cells through conjugation
F can be integrated or cytoplasmic
when integrated, F can transfer host chromosome markers through conjugation
bacteriophages can transfer DNA from one bacterial cell to another in two ways ...
generalized transduction is the transfer of randomly incorporated bacterial chromosome fragments
specialized transduction is the transfer of specific genes near phage integration sites
these methods of gene transfer facilitate construction of detailed maps of bacterial genomes

3. WORKING WITH MICROORGANISMS
so far... recombination & mapping in eukaryotes
now... prokaryotes & viruses  resolution
3 ways to incorporate & recombine DNA in bacteria:
1. conjugation – plasmid-mediated transfer
2. transformation – absorb from environment
3. transduction – bacteriophage-mediated transfer 

4. WORKING WITH MICROORGANISMS

5. WORKING WITH MICROORGANISMS
binary fission
known rate ~ °C
liquid medium
plating ...
serial dilutions ...
1 cell  ~ 107 cells visible colonies
or undiluted ...
lawn
use both methods

6. WORKING WITH MICROORGANISMS
strains
prototrophs = wild type
grow on minimal medium
auxotrophs = mutants
do not grow on minimal medium
nutrition
carbon source
resistant mutants

7. BACTERIAL CONJUGATION
do bacteria have genetic exchange & recombination ?
Lederberg & Tatum, 1946
Escherichia coli (E. coli)
single circular “chromosome”
haploid

8. BACTERIAL CONJUGATION
experiment ... contact requirement ?
2 strains, > 1 mutation
no colonies on A or B ...
 no spontaneous back or reversion mutations
BUT… some colonies (10-7) on mixed ...
 prototrophs from recombination

9. BACTERIAL CONJUGATION
experiment ... contact ?
selective filter prevents cell contact
no growth (prototrophs) on minimal medium
 contact required for recombination

10. BACTERIAL CONJUGATION
Hayes, 1953
genetic transfer in bacterial “crosses” unidirectional
donor & recipient strains ...
not really sex ( & ) as strains donate unequally

11. BACTERIAL CONJUGATION
fertility factor – F plasmid
F+ donor & F– recipient strains
F+ x F–  both F+
unidirectional rolling circle plasmid replication
F DNA transferred through a pore in the pilus

12. BACTERIAL CONJUGATION
the F plasmid can integrate into the host chromosome
generates a high frequency recombinant strain ... Hfr

13. BACTERIAL CONJUGATION
Hfr transfers part of the host genome during conjugation
Hfr x F–  F– rarely converted to Hfr or F+
isolate & purify Hfr from F+ for mapping

14. BACTERIAL CONJUGATION
Hfr x F–  recombination of donor genes in host

15. BACTERIAL CONJUGATION
Hfr x F–  recombination of donor genes in host
terms: exogenote and endogenote

16. BACTERIAL CONJUGATION
Wollman & Jacob, 1957 – gradient of transfer
selective marker – donor is strs & recipient is strr
origin of replication is transferred first

17. BACTERIAL CONJUGATION
mapping in E. coli by interrupted-mating
donor genes recombined into host genome

18. BACTERIAL CONJUGATION
interrupted-mating
selective markers
donor is strs
recipient is strr
origin of replication transferred first
1st transferred markers most frequent in exconjugants

19. BACTERIAL CONJUGATION
mapping in E. coli by interrupted-mating
distance measured in time (min)

20. BACTERIAL CONJUGATION
bacterial chromosome is circular
integration of F factor is pseudo-random
integration in either orientation

21. BACTERIAL CONJUGATION
bacterial chromosome is circular
integration of F factor is pseudo-random
integration in either orientation

22. BACTERIAL CONJUGATION
F factor integrates by recombination
pairing regions of homology (hatched)
episome = plasmid with free & integrated states

23. BACTERIAL CONJUGATION
F plasmid = episome
F+ & Hfr replicate during transfer
F+a+ x F–a–  F+a–
~10–3 F+a+  Hfr a+
Hfr a+ x F–a–  F–a– (exogenote lost) or F–a+ (exogenote incorporated)

24. BACTERIAL CONJUGATION
so far, genetic transfer only
recombination of Hfr exogenote & F– endogenote ...

25. BACTERIAL CONJUGATION
exogenote enters cell ... merozygote = partial diploid
single recombination event (3x, 5x, ...) nonviable
double recombination event (4x, 6x, ...) viable

26. BACTERIAL CONJUGATION
exogenote enters cell ... merozygote = partial diploid
single recombination event (3x, 5x, ...) nonviable
double recombination event (4x, 6x, ...) viable

27. BACTERIAL CONJUGATION
exogenote enters cell ... merozygote = partial diploid
single recombination event (3x, 5x, ...) nonviable
double recombination event (4x, 6x, ...) viable

28. BACTERIAL CONJUGATION
gradient of transfer  bridge spontaneously breaks
 early marker transfer more likely than late
Hfr leu+ arg+ met+ strs x F– leu– arg– met – strr 
leu arg met+
MORE LIKELY 
LESS LIKELY

29. BACTERIAL CONJUGATION
determination of gene order by gradient of transfer
Hfr leu+ arg+ met+ strs x F– leu– arg– met – strr 
of those markers transferred...
leu arg met+
Hfr F–
leu– arg – met –

30. BACTERIAL CONJUGATION
determination of gene order by gradient of transfer
Hfr leu+ arg+ met+ strs x F– leu– arg– met – strr 
of those markers transferred... which also recombine?
leu arg met+
Hfr F–
leu– arg – met –

31. BACTERIAL CONJUGATION
determination of gene order by gradient of transfer
Hfr leu+ arg+ met+ strs x F– leu– arg– met – strr 
met+ = 100%
arg+ = 60%
leu+ = 10%
leu arg met+
Hfr F–
leu– arg – met –

32. BACTERIAL CONJUGATION
bias in recovery of markers
gradient of transfer used for determination of gene order only
 to determine map distances, select late marker to ensure transfer of all relevant genes ... high resolution mapping

33. BACTERIAL CONJUGATION
high-resolution mapping by recombinant frequency

34. BACTERIAL CONJUGATION
high-resolution mapping by recombinant frequency

35. BACTERIAL CONJUGATION
high-resolution mapping by recombinant frequency

36. BACTERIAL CONJUGATION
high-resolution mapping by recombinant frequency

37. Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325
4th Ed., CH10, pp. 349 – 355

38. Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325
4th Ed., CH10, pp. 349 – 355

39. Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325
4th Ed., CH10, pp. 349 – 355

40. Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325
4th Ed., CH10, pp. 349 – 355

41. BACTERIAL CONJUGATION
marker transfer by episomes ... F'
a) integrated F  Hfr
b) imprecise excission of F
c) incorporation of genes
d) transfer to F––

42. BACTERIAL CONJUGATION AND RECOMBINATION MAPPING: PROBLEMS
in Griffiths chapter 5, beginning on page 179, try questions #1-3, 5-10, 12, 13, 15, 22, 23, 25-27
begin with the solved problems on page 177 if you are having difficulty
look at the way Schaum’s Outline discusses conjugation (pp ) and mapping (pp )
try Schaum’s Outline questions and on page 361