Recombination in bacteria I.Bacterial Review II.Conjugation IV.Bacteriophage genetics A. Phage cycle B. Plaque assay C. Phage cross V.Transduction A. Generalized transduction B. Cotransduction

I. Bacterial Review A.Can be grown both in liquid and agar medium, colony = visible cluster of cells B.Antibiotic resistant mutants = able to grow in the presence of an antibiotic C.Nutritional mutants Prototrophs = wildtype cells can synthesize nutrients from simple molecules present in the growth media Prototrophs = wildtype cells can synthesize nutrients from simple molecules present in the growth media Auxotrophs = can’t synthesize an essential nutrient and can’t grow unless that nutrient is supplied in the medium Auxotrophs = can’t synthesize an essential nutrient and can’t grow unless that nutrient is supplied in the medium Minimal medium = contains only inorganic salts, energy source and carbon atoms Minimal medium = contains only inorganic salts, energy source and carbon atoms Nonselective medium = all wild type cells form colonies Nonselective medium = all wild type cells form colonies Selective medium = medium that allows growth of only one type of cell Selective medium = medium that allows growth of only one type of cell

Selective plating: Allows the desired mutant to reproduce but not wild-type genotypes antibiotic resistance (mutants able to grow in presence of antibiotic) antibiotic resistance (mutants able to grow in presence of antibiotic) Str r (mutants that are resistant to streptomycin) minimal medium supplemented with specific nutrient minimal medium supplemented with specific nutrient Met - auxotroph is able to grow if minimal medium has methionineMet - auxotroph is able to grow if minimal medium has methionine CNA (Columbia Naladixic Acid) Agar selective for Gram-positive bacteria

II. Conjugation A. Lederberg & Tatum’s experiment illustrated that DNA was transferred from one bacterium to another. Strain #1: B - M - P + T + Strain #2: B + M + P - T - These colonies are due to the transfer of genetic material between two strains by conjugation.

Bacterial sex – Sex Pili required for a good time!!!

B. F plasmid (F factor) Ability to transfer based on presence of Fertility factor, now known as F plasmid ~12% the size of the bacterial chromosome sex pili genes sex pili genes surface exclusion protein genes, preventing F+ conjugating with F+ surface exclusion protein genes, preventing F+ conjugating with F+ transfer origin transfer origin Episome – F plasmid can remain as a free plasmid or be integrated into the bacterial chromosome

1). Properties of F plasmid: Can be replicated inside the cell Can be replicated inside the cell Cells with F plasmid (F+) have sex pili Cells with F plasmid (F+) have sex pili F+ and F- cells can conjugate F+ and F- cells can conjugate Transfer of information is one-way from donor (F+) to recipient (F-) Transfer of information is one-way from donor (F+) to recipient (F-) Strain A Strain B F+ x F- F+ x F- (donor) (recipient) F+ cannot conjugate with other F+ cells F+ cannot conjugate with other F+ cells F+ can become integrated into the host chromosome (rare event) – F+ carries one or more insertion sequence elements (IS) F+ can become integrated into the host chromosome (rare event) – F+ carries one or more insertion sequence elements (IS)  F may leave genome, taking copies of some genes (F’)

F pili promote cell to cell contact host chromosome F factor F+ F- F+F-

2)F plasmid replication: F replicates through rolling circle replication and it is transferred to a recipient cell via temporary cytoplasmic bridge between two cells. A copy remains in the donor cell. F+

3. Intigration of F (Hfr)  On rare occasions, the F plasmid is integrated into the circular chromosome, and there is genetic recombination… this can then be transferred to a recipient cell and incorporated into the recipient's genome. Hfr = high frequency of recombination. Still only partial transfer, not 100%

i.e. prior to mating, the recipient was lac - and pro -, however after a short time period of mating, the recipient is now lac +, but still pro - after a longer mating, the cell is lac + and pro + (lac is always transferred first, pro later) Chromosome transferred in a linear manner…

C. Mapping the E. coli chromosome using interrupted mating A. Interrupted mating – used a blender to separate bacterial cells that were in the act of conjugation, without killing them Hypothesis: the time it takes for genes to enter a recipient cell is directly related to their order along the bacterial chromosome Interrupted matings would lead to various lengths of the Hfr chromosome being transferred to the recipient.

s λ Ga l La c T1T1 A2A2 Whether or not bacteria could grow depended upon their genotypes i.e. a cell that is Azs can’t grow on azide plates…a cell that is Azr can T F- % Time TLA 2 T 1 LacGalλS Rate of transfer To determine gene order, colonies were picked from previous plates and restreaked on plates that had azide or bacteriophage T1, or on minimal plates

Gradient of transfer: Frequency of inheritance corresponds to the order of transfer Genes are mapped according to time of appearance of recombinants Circular, low resolution map is made by combining maps from different Hfr donors

Conclusions: there was a point of origin (O), because transfer occurs from a fixed point on the donor chromosome there was a point of origin (O), because transfer occurs from a fixed point on the donor chromosome O first, F last O first, F last determined the gene order based on the gradient of transfer determined the gene order based on the gradient of transfer chromosome was circular chromosome was circular the F is integrated at different points and different directions the F is integrated at different points and different directions

Last first

STRAIN 1GEBDNA 2PYLGEB 3XTJFPY 4BEGLYP ORDER OF TRANSFER Random reshuffling of genes??? Pattern: L next to G, G next to E, L next to B ORDER OF GENES ON ORIGINAL F+: XTJFPYLGEBDNA

gene order: CRUISE Using the E. coli map (that is based on 100 minutes), identify the location of the origin of both Hfr 1 & Hfr 3. The Hfr2 origin has already been identified. MAP ORDER USING Hfr 2 FIRST, then you can map the Hfr 1 & 3 relative to that. MARKERSHfr 1Hfr 2Hfr 3 R1020- I40-5 U255- E-60- C-45- S55-20 R U I S E C 1 3

If leu+ str-r recombinants are desired from the cross: Hfr leu+ str-s x F- leu- str-r, on what kind of medium should the matings pairs be plated? Plate on minimal medium that lacks leucine (select for leu+) but contains streptomycin (selects for Str-r)

Formation of a “Partial diploid” The F “pops out” often taking a piece of the chromosome with it…becoming an F’ [F prime], in the process creating a stable “partial diploid” = MEROZYGOTE D. The F’ state and Merozygotes

Conjugation, Transduction, Transformation recombination occur in asexual prokaryotes via:

A. Phage Cycle: 1)Virus binds to host cell 2)Tail sheath causes core penetration of cell wall 3)DNA in head is extruded Once inside cell: a)All processes halted b)Degradation of host DNA initiated c)Phage DNA replicated, transcribed & translated using host machinery d)Virus parts assembled e) Host cell ruptures IV. Bacteriophage genetics

Phage cross B. Plaque assay & the Phage cross Plaques are clear zones formed in a lawn of cells due to lysis by phage. Different phages produce distinct plaque morphologies 1)Two parental strains: h - r + X h + r -  h+ only infects strain#1  h- infects both strains  r+ small plaque  r- large plaque Double infection Phage lysate analyzed, looked at different plaque types RF = (h + r + ) + (h - r - ) total plaques total plaques

Plaque phenotypes produced by progeny of the cross h-r+ x h+r- Four plaque phenotypes can be differentiated: 2 parental types, (h-r+ and h+r-) and 2 recombinant types (h+r+ and h-r-) Thus, phage genomes can be mapped by analysis of RF Double Infections:

V. Transduction A.Generalized Transduction: Small fraction of DNA from the donor bacterial strain are carried by the phage Only a few genes are carried Only a few genes are carried Any host marker can be transduced Any host marker can be transduced Phage infects the recipient, transferring the DNA fragment, creating a merozygote Remains in cytoplasm (abortive transduction) Remains in cytoplasm (abortive transduction) Transduced bacterial genes may be incorporated into the bacterial chromosome (complete transduction) Transduced bacterial genes may be incorporated into the bacterial chromosome (complete transduction)

B. cotransduction If 2 genes are close together along the chromosome, a bacteriophage may package a single piece of the chromosome that carries both genes and transfer that piece to another bacterium The likelihood of this occurring depends upon how close together they are Can map genes using cotransduction…

Mapping genes using cotransduction  Select for the transduction of one gene and then monitor whether or not another gene is cotransduced along with it Donor: arg + met + str s (infected w/P1 and lysate mixed w/recipient) Recipient: arg - met - str r 1.Plated on minimal media w/arg and strep but no met, 2.Pick each of the colonies and re-streak on plates without met and without arg. 3.Calculate cotransduction frequency: # growing on media (no arg) total # colonies