Molecular Genetic Analysis in Bacteria › psaxena › BIO366 › figures › chap_14.pdf · Molecular Genetic Analysis in Bacteria 14 FIND CHAP TOC Insert in phage φ105J19 CATATCAAGGAGGAATGAG

Molecular Genetic Analysis in Bacteria14 FIND CHAP TOC

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Table 14.1


tonB

E. coli X7800

Select ∆tonB mutations byplating on bacteriocins or phage

lacI lacPOZYAtrp

tonB lacI lacPOZYAtrp

Figure 14.1


Mate into cell containing one of thedeletions. Select Pro+ transconjugants

Select LacI+ recombinants

Step 2

Mutagenize cells containing F' factor. Lac operon deleted in chromosome. Select constitutive mutations

Chromosome has a tonB deletion extending into lacI

lac genes deleted

Step 1lacZYApro +la

cI

lacZYA pro +

lacI*

lacZYA

pro+

lacI*

Figure 14.2


∆his2236∆his2527 ∆his2527

His–

gndhisO G D C B H A F I E

2236 2527

∆his2236 ∆his2527

∆his2527

∆his2527

His+

∆his2236His–

∆his2236 ∆his2

52

7His–

∆his22

36

Figure 14.3


Step 2

Step 3 Plate on medium without histidine; incubate

Grow phage P22 in S. enterica serovarTyphimurium with one his deletion

His+ transductants

Step 1

Use phage to transduce bacteriawith the other his deletion

Figure 14.4


metG metG ∆his2527Met – His–∆his2236

metG+

metG ∆his2527

∆his2527

Met + His+∆his2236

Segregation

metG+

Met + His – Met – His –

∆his2236metG+ metG

Figure 14.5


Periplasmic space

Periplasmic space

Malr (cells do not lyse)

B

MalE

*

LacZ COOH

NH2 MalE*

LacZ COOH

NH2

MalE* LacZCOOH

NH2

MalE* LacZCOOH

NH2

MalE LacZCOOH

Mals (cells lyse)

NH2

A

MalELacZCOOH NH2

Mal

ELa

cZC

OO

H

NH

2

Mal

ELa

cZ

CO

OH

NH

2

Figure 14.6


secB+

Periplasmic space

Periplasmic space

OM

IMSignalsequence

SecBCOOH NH2SecB

secB mutant

COOH

NH2

OM

IM

B

A

Figure 14.7


A

APx

NH2

Periplasm

Color onXP

plates

Blue

OM

IM

COOH

x z

yNH2

Periplasm

OM

IM

B

APx z

yNH2

Periplasm Blue

OM

IM

C

D

x

yNH2

Periplasm Colorless

OM

IM

AP

Figure 14.8


pL

pR

pLp R

CI

CI

oriV

λ lysogen Cell with ColE1

ColE1λ DNA

λ att site

oriV cI λ ori

Plaques

RNA I

Phasmid contains

RNA I

λori

Plaques

λ lysogen with ColE1

Plaques

Phasmid with mutant RNA II not inhibited by RNA I

Integrase constitutive strain

PhasmidHarvest plasmid

svir2

RNA II

1 2

3

4

5

RNA I

Figure 14.9


C

Usvir2svir7

Gsvir11

Usvir12

CA

B

I

II

A

RNA IRNA I

Lysogens transformed with plasmid from phasmid mutantNo plasmid

Plaques

Phasmid

svir2

ori

RNase Hprocessing

RNA II

RNA I

svir11

Plaques

svir2 plasmid

No plaques

Plaques

svir11 plasmid

Plaques

No plaques

RNA IR

N A

Figure 14.10


“Patch” individualstrains with Tn5in plasmid (Kanr)

Incubate

Kanr

Patches which do not transfermay indicate that Tn5hopped into a tra gene

Replicate ontoplate containing

rifampin andkanamycin

Replicate onto plate spread with Rifr recipient bacteria

Incubate

Rifr

Figure 14.11


tra1

1 Clone one mutant traregion from a tramutant plasmid

2 Transform into a cellwith a differenttra mutant plasmid

3 Test for transferto a recipient. Iftransfer occurs,the mutationscomplement

Recipient

pKM101

tra2

tra1 tra2

tra1

Figure 14.12


A

B

C

D

E

F

Figure 14.13


Vegetativecell

spo0Aspo0K

Entry intosporulation

Polarseptation

Engulfment Cortexformation

Coatmaturation

Release

Stage 0 Stage II Stage III Stage IV Stage V–VI Stage VII

Dormantspore

spo0H spoIIAspoIIG

spoIIIAspoIIIE

spoIVCspoIVFcotA

Figure 14.14


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Table 14.2


KinABC

Signals (?)

KinABC~P

ATP

ADP

Spo0F

Spo0F~P

Spo0B

Spo0B~P

Spo0A

abrB

spoIIAspoIIEspoIIG

Antibiotics,degradative enzymes

SporulationSpo0A~P

Figure 14.15


KinABC

Kin~PABC

PhrA CSFRapA; RapBphosphatases

ATP

ADP

Spo0F

Spo0F~P

Spo0B

Spo0B~P

Spo0A

Spo0A~P

? Spo0Ephosphatase

Figure 14.16


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Table 14.3


pspo lacZ

LacZN C

ATG

mRNA

Transcriptionalfusion

PromoterTIRProtein

:::

spo genespoVG geneLacZ

Fusionjoint

+1

pspo ′lacZspo′

Spo′-′LacZN C

ATG

mRNA

Translationalfusion

PromoterTIRProtein

:::

spo genespo geneSpo-LacZ

Fusionjoint

+1

Figure 14.17


Insert in phageφ105J19

CATATCAAGGAGGAATGAG

spoIIAA translation initiation region

MATG

A

B

LCTT

CTGT

IATT

RCGA

LTTA

TACA

GGGC

EGAA

LCTC

In-frame fusion

DGAT

spoIIAA

PCCT

LCTA

EGAG

STCG

TACC

V GTC

VGTT

SAGC

LCTT

GGGA

IATT

DGAC

MATG

NATT

VGTC

KAAA

EGAA

STCT

VGTG

lacZ

LacZpspoIIA

pspoIIA

Cloned Cloned

Cmr

E

B. subtilischromosome A B C A B C D E

pspoVA

spo0A43spo0B136

spo0E11spo0F221

spo0H17spo0J93spo0K141

0.0160.023

0.0180.021

0.010.0480.013

Mutation

spo0

t1•5

0.0370.054

0.0150.050

0.0180.250.050

t4

22

22

222

No. ofdeterminations Mutation t1•5 t4

No. ofdeterminations

spoIIAA562spoIIAC1spoIIB131spoIID298spoIIE48spoIIG55

0.410.440.360.430.670.69

spoII

0.380.510.170.110.770.58

223233

spoIIIA65spoIIIB2spoIIIC94

0.180.650.24

spoIII0.220.130.23

424

spoIVA67spoIVB165spoIVC23

0.290.120.29

spoIV0.0800.190.15

333

spoVA89spoVB91spoVC134

0.440.490.61

spoV0.340.210.46

222

<

β-Galactosidase activity(units ml–1)

β-Galactosidase activity(units ml–1)

Figure 14.18


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Table 14.4


0–60 min

σA spoIIG

σH spoIIA

gpr80 min σE

σF

gerM

σE

gpr,spoIIIG

120 min

σF

spoIVC

σE

ssp

σG

150 min

cotA

σK

σG240 min

Figure 14.19


30 min

Polar septation activates σF

σF-dependent gene(spoIIR) triggers σE

activation

σE-dependent gene(unknown) activates σG

expression

σG-dependent gene(spoIVB) triggers σK

activation

σA spoIIG Pro-σE

Pro-σE

σFσH spoIIA IIAB

IIR

IVB

s?

80 min

σF

σE

spoIIIG

120 min

Pro-σK

σK

σE σG

150 min

σK σG240 min

σE

σF

Figure 14.20


Predivisional sporangium

IIAB phosphorylates IIAA

IIAA

IIAA-P

IIAB-ATP

Polar septationσFIIAB

P IIAA

Mother cell Forespore

IIE dephosphorylates IIAA-P

IIAA-P

IIAA

IIEPi

σF σFIIAB

P IIAA IIAAIIAB

IIE

Figure 14.21


Antagonisticinteractions

SpoIIAA

Active

Inactive

Active

Inactive

spoIIAA

SpoIIAB

Inactive

Active

target promoters

ON

OFF

spoIIAB

σF

”Anti-anti-sigma“ ”Anti-sigma“ ”Sigma F“

spoIIAC (sigF)pspoIIA

spoIIAoperon

A

B

Figure 14.22


A B′ lacI

pspac

pspoIIA

erm c

Figure 14.23


Spo0A spoIIGA

Protease

A

B

spoIIGB

pspoIIG

P σA

Pro-σE

Pro-σE

SpoIIGA

Septum formation andσF activation

SpoIIR activates SpoIIGA protease

Degradation of σE in forespore

σF

σE SpoIIR

Figure 14.24


IVFB IVB

σG

IVFA

BofA

Pro-σK

Mother cell

Forespore membranes

σK

Figure 14.25

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Molecular Genetic Analysis in Bacteria › psaxena › BIO366 › figures › chap_14.pdf · Molecular Genetic Analysis in Bacteria 14 FIND CHAP TOC Insert in phage φ105J19 CATATCAAGGAGGAATGAG