How to switch from "On" to
"Off" and back again with
OxyR, DNA modification and
a replication fork.
Please note this is an edited version of the presentation. If
you attended the meeting and would like a copy of the
original drop me an email!
Marjan van der Woude
DEPARTMENT OF BIOLOGY
Centre for Immunology and Infection
STOMP
Stochastic dynamical modelling of gene networks in prokaryotes
… microbiologists, mathematicians and physical
scientists, to use stochastic dynamical modelling for
"real-life" problems pertaining to bacterial response
and behaviour - specifically, contribute to
understanding stochastic effects in gene regulation
in response to environmental stresses, in communal
living .. and in gene transfer ..
Outline
• Background
• OxyR and Dam-dependent agn43
phase variation
• Natural variation on the agn43 theme
• Concluding thoughts
Phase variation
2.
1.
1. Graded level of expression. Response to stimulus
within population, stochastic. Non-heritable.
2. Phase variation. Population with both On and Off.
Heritable.
Phase variation:
Heritable yet reversible gene expression states
Cell division
s io
iv i
ll d
iv i
ns
Ce
ll d
s io
Ce
ns
Switch frequency
restreak
restreak
lacZ reporter
Phase variation
Very prevalent among all types/genera of bacteria
Different molecular mechanisms
• slipped strand mispairing, site specific
recombination, DNA methylation
• probably all have a stochastic element
• some incorporate environmental signals, others do not
Phase variable genes code for different classes of
proteins, including:
•
•
•
•
Adhesins
Outer membrane proteins
Transporters
Restriction/modification genes
Biological significance of phase variation?
Prevalent theories:
- Evade the immune system
Benefits population- survival
Co-existence serotypes
- Adhesins:
Facilitates bacterial dispersal
-“Contingency” genes
Swarming in soil bacteria
Model system:
Dam-dependent Phase Variation of Ag43
Wt=
Off and On
100% ON
culture
• Outer membrane protein in E. coli
• Variable roles attributed - adherence, biofilm,
persistence (UTI)…
• 0-3 alleles in E. coli isolates
• Gene agn43 [aka flu]
• Expression phase varies, requiring Dam and OxyR.
aggregated
The DNA binding protein: OxyR
•
•
•
•
•
•
Is a repressor of agn43 transcription
Binds DNA as a dimer of dimers
Global regulator for protections agains oxidative stress
Oxidation state does not affect agn43 repression
Amount in cell: approx 150 molecules (Ishihama, pers comm)
Probably just a few binding sites in genome (non-oxidative
stress conditions)
Dam
The enzyme: Dam
(Deoxyadenosine methyltransferase)
• Methylates adenine at GATC
sequences
• Processive enzyme; both Hemi- and
Unmethylated DNA is substrate
• No de-methylating enzyme in E. coli
• Is integral part of E coli biology and
not part of restriction /modification
system
• Is always present; 100-300
molecules per cell?
Unmeth GATC
Meth GATC
Unmethylated
Hemi-methylated
Methylated
The agn regulatory region
Questions:
How is heritability conferred?
What event determines whether a switch takes place?
Can we identify parameters to predict occurrence?
Summary of key data:
OFF
•
•
•
•
ON
dam mutant locked OFF
oxyR mutant locked ON
DNA methylation state correspond to expression state
In vitro
• OxyR binds unmethylated agn43 region but not
methylated agn43 region
• OxyR binding sequesters region from Dam
• OxyR represses in vitro transcription
The 2 Basic Principles of the agn43 ON and OFF states:
1. DNA methylation-dependent OxyR binding
2. OxyR-dependent methylation protection
GATC-II
GATC-I
GATC-III
+1
-35
-10
agn
lacZ
OxyR binding site
Dam
Dam
OxyR represses: OFF
OxyR can not bind:ON
Key aspects:
Memory conferred by DNA methylation state
No feedback from expression
No known environmental signals incorporated
The model explains a stable On and Off phase,
but what is molecular event (or sources?) of
stochastic behaviour and the switch?
Events in the cell?
?
OFF
ON
KEY
OxyR
Dam
Methyl
group
DNA
replication
DNAs
Old strand
Newly synthesized strand
OFF
Two roles of methylation at agn43
• Decrease OxyR binding affinity
100
90
% bound Ag43 DNA
80
70
60
Non-meth:
50
40
30
Hemi-meth:
20
10
Top strand methylated
0
10
15
20
nanograms of OxyR
25
30
Bottom strand methylated
0
5
10
15
20
25
30
nanograms of OxyR (wt)
• Enhance promoter activity
oxyR mutant
11027 MU
dam oxyR mutant
81 MU
Significance OxyR binding affinity
Role of each GATC and binding site
WTK12
OFF
Altered switch
frequency
x
GATC mutants
ON
x
(NA locked Off)
x
(NA locked Off)
WTRS218
x
x
x
x
x
Altered switch
frequency
PV depends on global Dam and OxyR
concentration/ratio
Locked OFF
dam mutant
OxyR overproducer
Locked ON
oxyR mutant
Dam overproducer
Dam
Dam
Dam
OFF cells
Dam
Dam
ON cells
Haagmans et al, 2000
Wallecha et al 2002
Bias competition to OFF
OFF
UM
ON
M
HM
To Model:
a DNA-binding protein
a DNA-modifying enzyme
effect of DNA replication.
- Analysis of plasmid encoded GFP after signal amplification, FACS
- Implicates additional factor in Off phase.
- In addition to known Off, On intermediate states invoked, one with
“nonspecific transcription” [“intermediate”].
- Model simulations: intermediate states are required “buffers” that
facilitate inheritance of parental expression state.
How do following fit?
• OxyR Affinity
• Concentration
• RNA pol role
• Dam processivity
• Availability locally
• Lim: intermediate
states
OxyR half site
OxyR half site
Molecular Rules Dam-dependent PV?
ON
agn family
OxyR
Pap family
Lrp, needs PapI ( ) )
OFF
David Low
Known Dam-dependent PV
Bacterial species
Affected moiety /phenotype
Campylobacter jejuni
Escherichia coli
LOS modification
Fimbriae (type 1, CS18)
Fimbriae (Pap, S, F1845, Clp)
Outer membrane protein
Fimbriae (Pef)
DNA modification
fimbriae LKP
LOS modification
DNA R/M
LPS modification
Flagella
Membrane lipid composition
Outer membrane proteins
Haemoglobin receptors
Capsule
Fimbriae (MR/P)
Capsule
Metabolism
DNA R/M
Salmonella enterica Tm
Haemophilu influenzae
Helicobacter pylori
Neisseria meningitidi s
Proteus mirabilis
Streptococcus pneumoniae
OxyR and Dam
Class(es) of regulated
gene/operon
enzyme
structural, regulatory
structural, regulatory
structural
sructural, regulatory
enzyme
structural
enzyme
enzyme
enzymes
structural
enzyme
structural
structural
enzyme
structural, regulatory
structural
enzyme
enzyme
Molecular
Mechanism
SSM
Recomb
DNA methyl
DNA methyl
DNA methyl
SSM
SSM
SSM
SSM
SSM
SSM
SSM
SSM
SSM
SSM
Recomb
Recomb
SSM
SSM
Lrp and Dam
after van der Woude and Baumler, 2004
Defining the molecular rules: why?
• Bacterial genera with dam, oxyR and lrp include
Yersinia,
Haemophilus,
Vibrio,
Neisseria,
Legionella
• Dam may interact with other regulators
• Work to predict from genome sequence:
o PV prediction (vaccine!)
o systems insight
• Use for synthetic biology?
•
•
•
•
Renata Kaminska
Sarah Broadbent
Mark Davies
Ruth Verstraten, Sophie Foppolo
Matthew Lakins and other previous
lab members
Support from
Previous funding
NSF, Dupont, EU-IRG
DEPARTMENT OF BIOLOGY
Centre for Immunology and Infection
GATC point mutations: only site
specific methylation state is affected
GATG
AATC
AATC
agctcaataatagaataaaacgatcaatatcTattttatcgatcgtttatatcgatcgataagct
motif
atat t
a ctat
atag t
a ctat
Gelshift analysis with
purified OxyR
100
90
Percent shifted
80
70
60
OxyR binding to mutant
DNA is comparable to
binding to unmethylated
wild type DNA.
50
40
30
WT unmethylated DNA
20
10
GATC-I, II, III mutant DNA
0
0
20
40
60
OxyR(C199S) AU AU
OxyR(C199S)
80