Conjugation FIND CHAP TOC

5 Conjugation FIND CHAP TOC

Figure 5.1

bla IS46

Rep 35 korB BamHI HpaI-8 EcoRl Sall-1 kilA BgIII-6 korA Pstl BgIII-5 traA Hpal-1 5 mucB 30 HpaI-7

SmaI-2 traB mucA Kpnl-1 BgIII-4

pKM101 Hpal-2 traC eex 35.4 kb Sall-2 traD 10 HpaI-3 25 kilB KpnI-2 BgIII-3 traE HpaI-6 HpaI-4 SmaI-1 traF BgIII-1 HpaI-5 BgIII-2 15 traG stb 20 KpnI-3 nuc traK fip traJ oriT traI traH 5 Conjugation FIND CHAP TOC

Box 5.1

A T-DNA oriT B oriT n oc Cut out a piece of the leaf r vi t ra I/ t r Bacterium-to-plant b transfer Float in plate containing an o Agrobacterium strain r

Replication within / with engineered r Ti e Ti plasmid Agrobacterium p plasmid

a c c R Incubate on plate Regenerating / a c containing plant plants c Bacterium-to-bacterium regeneration transfer medium

t ra R/ tr aM

oriT/tra Excise germinating Kanamycin- shoots and transplant resistant shoots to plates containing kanamycin

Transgenic plant 5 Conjugation FIND CHAP TOC

Box 5.1 cont.

C Target cell

Pilus (VirB2)

Outer membrane

Inner membrane

Cytoplasm Coupling protein (VirD4) Relaxase (VirD2)

T-DNA 5 Conjugation FIND CHAP TOC

Figure 5.2

Donor Recipient

MPF structure Formation of mating pairs Coupling protein

Coupling protein signals to relaxase

o r i

Relaxase Single-strand nick at oriT and strand displacement

3′OH

Helicase

3′OH primes Relaxase DNA Pol

Strand transfer, closing of circle by relaxase, replication in recipient

Relaxase

Primase

Separation of mating pair

Donor Transconjugant 5 Conjugation FIND CHAP TOC

Figure 5.3

Relaxase

Tyr-OH Donor riT o cell Chromosome

Covalent bond between relaxase and DNA B

Relaxase 5′ O P O Tyr oriT Recipient cell iT Donor 3′ OH r o cell

Relaxase Tyr OOP Recipient cell 5 Conjugation FIND CHAP TOC

Figure 5.4

Variable length depending on pilus type

Outer membrane Subunit C LepB peptidase assembly

Inner membrane

Pilin complex N Pilin Leader N peptide

C Propilin 5 Conjugation FIND CHAP TOC

Figure 5.5 A Genetic organization of tra region

P P mRNA traJ traY

oriT traM traJ traY traX finO

Antisense RNA PfinP

B Immediately after entry into cell

J Activates P traY Pili and transfer functions

oriT traM traJ traY traX finO

C After plasmid establishment

FinO FinP blocks translation: no TraJ FinP

oriT traM traJ traY traX finO

FinP FinO

FinO FinO stabilizes FinP 5 Conjugation FIND CHAP TOC

Figure 5.6

IS3 γ δ 0

90 10

IS 3 a r t I S 2

80 20

100 kb

70 30

o r 60 40 iT 50

oriV 5 Conjugation FIND CHAP TOC

Table 5.1

�� 5 Conjugation FIND CHAP TOC

Figure 5.7

r r r Amp Amp Amp

Clone pieces randomly into oriT a nonmobilizable oriT cloning vector Transform into cells containing a self-transmissible plasmid

Ampr Camr

oriT Ampr Camr Mate with recipient; only oriT-containing plasmids are mobilized

Camr r Amp

Select Ampr transconjugants

Ampr

oriT 5 Conjugation FIND CHAP TOC

Figure 5.8

Tra F

Self-transmissible plasmid encodes tra functions that allow cell contact

Coupling protein signals to relaxase Relaxase

Nick made at oriT of mobilizable plasmid

Mobilizable plasmid transferred

Mobilizable plasmid is replicated in the recipient 5 Conjugation FIND CHAP TOC

Figure 5.9 I II

Self- transmissible Mobilizable plasmid plasmid

Recipient strain

III

Step 1

I II

Step 2

III 5 Conjugation FIND CHAP TOC

Figure 5.10

IS2

Homologous recombination

Chromosome IS2

IS2 F IS2 5 Conjugation FIND CHAP TOC

Figure 5.11

Hfr F–

The F plasmid encodes tra functions, including pili

or iT

A nick at oriT initiates transfer

Replication occurs in the donor as one strand is transferred

The transferred fragment undergoes recombination in the recipient

Hfr F– 5 Conjugation FIND CHAP TOC

Figure 5.12

Hfr Arg– Trp+ × F– Arg+ Trp–

arg + F arg trp+ trp

Formation of mating pair

+ arg trp + arg trp+ trp

DNA transfer from Hfr donor to F– recipient

arg F trp trp+ trp+

Recombination between homologous regions of incoming and recipient DNA

arg F arg+

+ trp trp+ Hfr F– Recombinant: Arg+ Trp+ 5 Conjugation FIND CHAP TOC

Table 5.2 ��

�� 5 Conjugation FIND CHAP TOC

Figure 5.13 Donor Recipient

∆lac-pro argH5 arg + 90 8 90

25 25 44 44 trp trpA3 + his hisG1 +

Pro– His– Arg– Trp–, Rifr

arg

trp+ arg+ his trp + his

Plate: Arginine plus tryptophan: His+ Tryptophan plus histidine: Arg+ Arginine plus histidine: Trp+ 5 Conjugation FIND CHAP TOC

Figure 5.14