Recombination in bacteriaRecombination in bacteria

I.I. Bacterial ReviewBacterial ReviewII.II. ConjugationConjugationIV.IV. Bacteriophage geneticsBacteriophage genetics

A.A. Phage cyclePhage cycleB.B. Plaque assayPlaque assayC.C. Phage crossPhage cross

V.V. TransductionTransductionA.A. Generalized transductionGeneralized transductionB.B. CotransductionCotransduction

I. Bacterial ReviewI. Bacterial ReviewA.A. Can be grown both in liquid and agar medium, Can be grown both in liquid and agar medium,

colonycolony = visible cluster of cells = visible cluster of cellsB.B. Antibiotic Antibiotic resistant mutantsresistant mutants = able to grow in the = able to grow in the

presence of an antibioticpresence of an antibioticC.C. Nutritional mutantsNutritional mutants

PrototrophsPrototrophs = wildtype cells can synthesize nutrients from = wildtype cells can synthesize nutrients from simple molecules present in the growth mediasimple molecules present in the growth media

AuxotrophsAuxotrophs = can’t synthesize an essential nutrient and = can’t synthesize an essential nutrient and can’t grow unless that nutrient is supplied in the mediumcan’t grow unless that nutrient is supplied in the medium

Minimal mediumMinimal medium = contains only inorganic salts, energy = contains only inorganic salts, energy source and carbon atomssource and carbon atoms

Nonselective medium = all wild type cells form coloniesNonselective medium = all wild type cells form colonies Selective mediumSelective medium = medium that allows growth of only one = medium that allows growth of only one

type of celltype of cell

Selective plating:Selective plating:

Allows the desired mutant to Allows the desired mutant to reproduce but not wild-type reproduce but not wild-type genotypesgenotypes antibiotic resistance (mutants able antibiotic resistance (mutants able

to grow in presence of antibiotic)to grow in presence of antibiotic)• StrStrrr (mutants that are resistant to (mutants that are resistant to

streptomycin)streptomycin) minimal medium supplemented minimal medium supplemented

with specific nutrientwith specific nutrient• MetMet-- auxotroph is able to grow if auxotroph is able to grow if

minimal medium has methionineminimal medium has methionine

CNA (Columbia Naladixic Acid) Agarselective for Gram-positive bacteria

II. ConjugationA. Lederberg & Tatum’sexperiment 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)B. F plasmid (F factor)

Ability to transfer based on presence of Fertility Ability to transfer based on presence of Fertility factor, now known as factor, now known as F plasmidF plasmid~12% the size of the bacterial chromosome~12% the size of the bacterial chromosome sex pili genessex pili genes surface exclusion protein genes, preventing F+ surface exclusion protein genes, preventing F+

conjugating with F+conjugating with F+ transfer origintransfer origin

EpisomeEpisome – F plasmid can remain as a free – F plasmid can remain as a free plasmid plasmid oror be integrated into the bacterial be integrated into the bacterial chromosomechromosome

1). Properties of F plasmid:1). Properties of F plasmid:

Can be replicated inside the cellCan be replicated inside the cell Cells with F plasmid (F+) have sex piliCells with F plasmid (F+) have sex pili F+ and F- cells can conjugateF+ and F- cells can conjugate Transfer of information is one-way from donor (F+) to Transfer of information is one-way from donor (F+) to

recipient (F-)recipient (F-)Strain AStrain A Strain BStrain B

F+ x F-F+ x F-(donor) (recipient)(donor) (recipient)

F+ cannot conjugate with other F+ cellsF+ cannot conjugate with other F+ cells F+ can become integrated into the host chromosome F+ can become integrated into the host chromosome

(rare event) – F+ carries one or more (rare event) – F+ carries one or more insertion sequenceinsertion sequence elementselements (IS) (IS)

F may leave genome, taking copies of some genes F may leave genome, taking copies of some genes (F’)(F’)

F pili promotecell to cell contact

host chromosomeF 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 frequencyof 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 C. Mapping the E. coliE. coli chromosome using chromosome using interrupted matinginterrupted mating

A. A. Interrupted matingInterrupted mating – used a blender to separate – used a blender to separate bacterial cells that were in the act of conjugation, without bacterial cells that were in the act of conjugation, without killing themkilling 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.

sλ

Gal

Lac T1 A2

Whether or not bacteria could grow Whether or not bacteria could grow depended upon their genotypesdepended upon their genotypes

i.e. a cell that is Azs can’t grow on i.e. a cell that is Azs can’t grow on azide plates…a cell that is Azr canazide plates…a cell that is Azr can

T

F-

% 100 100 90 70 40 25 15Time 8 8 9 11 18 25 26 90

T L A2 T1 Lac Gal λ S

Rate of transfer

To determine gene order, To determine gene order, colonies were picked from colonies were picked from previous plates and previous plates and restreaked on plates that restreaked on plates that had azide or had azide or bacteriophage T1, or on bacteriophage T1, or on minimal platesminimal plates

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

Conclusions:Conclusions:

there was a point of origin (O), there was a point of origin (O), because transfer occurs from because transfer occurs from a fixed point on the donor a fixed point on the donor chromosomechromosome O first, F lastO first, F last

determined the gene order determined the gene order based on the gradient of based on the gradient of transfertransfer

chromosome was circularchromosome was circular the F is integrated at different the F is integrated at different

points and different directionspoints and different directions

Last first

STRAINSTRAIN

11 GG EE BB DD NN AA

22 PP YY LL GG EE BB

33 XX TT JJ FF PP YY

44 BB EE GG LL YY PP

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.

MARKERS Hfr 1 Hfr 2 Hfr 3

R 10 20 -

I 40 - 5

U 25 5 -

E - 60 -

C - 45 -

S 55 - 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, TransformationConjugation, Transduction, Transformationrecombination occur in asexual prokaryotes via:recombination occur in asexual prokaryotes via:

A. Phage Cycle:1) Virus binds to host

cell2) Tail sheath causes

core penetration of cell wall

3) DNA in head is extruded

Once inside cell:a) All processes haltedb) Degradation of host

DNA initiated

c) Phage DNA replicated, transcribed & translated using host machinery

d) Virus parts assembled

e) Host cell ruptures

IV. Bacteriophage geneticsIV. Bacteriophage genetics

B. Plaque assay & the Phage crossPhage crossPlaques are clear zones formed in a lawn of cells due to lysis by phage. Different phages produce distinct plaque morphologies

1)1) Two parental strains: Two parental strains: hh- - rr++ X h X h+ + r r --

h+ only infects strain#1h+ only infects strain#1 h- infects both strainsh- infects both strains r+ small plaquer+ small plaque r- large plaquer- large plaque

Double infection Double infection Phage lysate analyzed, looked at different plaque typesPhage lysate analyzed, looked at different plaque typesRF = RF = (h(h++rr++) + (h) + (h--rr--))

total plaquestotal 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. TransductionV. TransductionA.A. Generalized TransductionGeneralized Transduction:: Small fraction of DNA from the Small fraction of DNA from the

donor bacterial strain are carried by the phagedonor bacterial strain are carried by the phage Only a few genes are carriedOnly a few genes are carried Any host marker can be transducedAny host marker can be transduced

Phage infects the recipient, transferring the DNA fragment, Phage infects the recipient, transferring the DNA fragment, creating a merozygotecreating a merozygote

Remains in cytoplasm (Remains in cytoplasm (abortive transductionabortive transduction)) Transduced bacterial genes may be incorporated into the bacterial Transduced bacterial genes may be incorporated into the bacterial

chromosome (chromosome (complete transductioncomplete transduction))

B. B. cotransductioncotransduction

If 2 genes are close together along the If 2 genes are close together along the chromosome, a bacteriophage may chromosome, a bacteriophage may package a single piece of the package a single piece of the chromosome that carries both genes and chromosome that carries both genes and transfer that piece to another bacteriumtransfer that piece to another bacteriumThe likelihood of this occurring depends The likelihood of this occurring depends upon upon how close togetherhow close together they are they areCan map genes usingCan map genes using cotransductioncotransduction……

Mapping genes using cotransductionMapping genes using cotransduction

Select for the transduction of one gene and then Select for the transduction of one gene and then monitor whether or not another gene is monitor whether or not another gene is cotransduced along with itcotransduced along with itDonor: Donor: argarg++ metmet++ strstrss (infected w/P1 and lysate (infected w/P1 and lysate mixed w/recipient)mixed w/recipient)Recipient: Recipient: argarg-- metmet-- strstrrr

1.1. Plated on minimal media w/arg and strep but Plated on minimal media w/arg and strep but no no met, met,

2.2. Pick each of the colonies and re-streak on plates Pick each of the colonies and re-streak on plates without met and without argwithout met and without arg..

3.3. Calculate cotransduction frequency: Calculate cotransduction frequency: # growing on media (no arg)# growing on media (no arg)

total # coloniestotal # colonies