DNA uptake during transformation of Bacillus subtilis
description
Transcript of DNA uptake during transformation of Bacillus subtilis
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DNA uptake during transformation of Bacillus subtilis
Look here throughout for the names of the people who did the work!
Public Health Research Institute
and
Department of Microbiology and Molecular GeneticsUniversity of Medicine and Dentistry
Newark, New Jersey
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Not another transformation talk!
Edvard Munch
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ComK is the Master Regulator of Competence
PrcomK
+
Genes for DNA uptake
+
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Time
Transform
ationG
row
th
Competence Develops in Stationary Phase
T0
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comK Synthesis Begins in Stationary Phase
Time
Gro
wth
T0 Com
K
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Competence gene expression exhibits heterogeneity
Only 10-20% of the cells express competence!
This shows a culture with a GFP fusion to a competence protein(ComK-GFP)
Jeanette Hahn
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DNA Uptake: take home lessons
1. DNA uptake is mediated by two subsets of proteins that probably form complexes.
2. The first subset provides for access across the wall. It involves a pilus-like system.
3. The second subset includes three proteins that mediate membrane transport. These proteins may constitute an atypical ABC transporter.
(All the proteins to be discussed are under ComK control).
4. DNA uptake probably takes place preferentially at the cell poles and at least some competence proteins are localized at the poles.
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TRANSFORMATION PATHWAY
Integration
Binding
Fragmentation
Transport and degradation
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Proteins needed for DNA binding:
comE operonEA
DNA binding protein
(EC)
Major pilin-like protein
comK
comC
peptidase
C
comK
ATP binding protein
comG operonGA GB GCGD
GE
GF
GG
Integra
l membr
ane p
rotei
n
Type 4 pilin
-like pro
teins
comK
Mark Albano, Jeanette Hahn, Sandhya Mohan
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ComGComCComEA
NucANucA is the fragmentation nuclease. It is an integral membrane protein.
ComEA is a DNA receptor. It is an integral membrane protein.
But why are the ComG/ComC proteins needed for DNA binding?
Roberta Provvedi, Young Sook Chung
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The ComG/ComC proteins are needed to provide access to ComEA
NucAComEAComG proteins
Wall
Hypothesis: The ComG proteins form a complex (a pseudopilus) that may traverse the wall.
Roberta Provvedi
OUT
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F
prepilin peptidase cleavage
hydrophobic core
Type IV pilins and pilin-like proteins
Neisseria pili Klebsiella pseudopilus(Sauvonnet et al. EMBO J. (2000)19:2221)
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The protoplast supernatant
Gram positive cell(B. subilis)
lysozyme
osmoprotectant
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ComGC monomer is oxidized, cleaved and translocated
HS
SH
BdbDCComC
Integral membrane protein
In protoplast supernatant
s-s
S-S
What are the roles of ComC, BdbD and BdbC?
NH2
COOH
Roberta Provvedi, Ines Chen, Young Sook Chung & Sierd Bron lab
Does ComGC form a higher order structure?
OUT
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ComC is an integral membrane peptidase that converts pre-ComGC, pre-ComGD, pre-ComGE and pre-ComGG to their mature forms
Young Sook Chung
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ComC is needed for the translocation of ComGC
Young Sook Chung
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MNEKGFTLVEMLIVLFIISILLLITIPNVTKHNQTIQ
KKGCEGLQNMVKAQMTAFELDHEGQTPSLAD
LQSEGYVKKDAVCPNGKRIIITGGEVKVEH
ComGC has an intramolecular disulfide bond
Young Sook Chung
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bdbD
ComK
BdbD and BdbC are thiol-disulfide oxidoreductases required for competence
bdbC
Sierd Bron lab, Mark Albano
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BdbD and C are needed to stabilize ComGC, probably by introducing the disulfide bond
Protoplast supernatant
Membrane
WT
bd
bD
bd
bD
xbd
bC
bd
bC
bd
bC
xbd
bC
com
GA
12
WT
bd
bD
bd
bD
xbd
bC
bd
bC
com
GA
12
bd
bC
xbd
bC
Ines Chen, Roberta Provvedi, Sierd Bron Lab
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1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
BD3002 (bdbDC)
BD3355 (bdbD)
BD2999 (bdbC)
BD2528
control1mM Cystine5mM Cystine
Phenotypic suppression of bdbD and bdbC mutants
Ines Chen
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S
SBdbD
BdbC
S
SBdbD
Added oxidizing agents (GSSG or cystine)
S
SBdbD
Reduced ComGC (unstable)
Oxidizing agents
BdbDC act as oxidoreductase partners to introduce a disulfide bond in ComGC
Oxidized ComGC (stable)
OUT
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ComGC in protoplast supernatant
+ ME -ME
ComGC forms a multimeric complex stabilized by disulfide bonds
kDa
46
29
2015
5
com
G
com
G
Ines Chen, Roberta Provvedi
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wt A B C D E F G GA(ATP)
All the comG genes are necessary for ComGC complex formation
ComGC in protoplast supernatantInes Chen
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The ComC, BdbDC and ComG proteins are sufficient as well as necessary for complex
formation
Strain has Pspac-comG, Pspac-bdbDC, Pspac-comC and is comK.
- + - + IPTGßSH- - - -+ + + +
Ines Chen, Petrina Boucher
comGC monomer
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Processing and Assembly of ComGC multimer
s-s
s-s
ComG proteinsHS
SH
BdbDCComC
s-s
S-S
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NucAComEAComGC
Wall
ComGC does form a multimeric complex, providing access to ComEA
Does the ComGC multimer traverse the wall and provide a passageway for DNA, as shown?
Is the structure dynamic? Does its disassembly draw DNA into contact with ComEA?
OUT
What is the composition and structure of the complex?
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Three membrane proteins needed for DNA transport
EC
FA
Out
In
Putative channel protein
ATP binding protein
EA
DNA binding protein
Gordon Inamine, Roberta Provvedi, Arturo Londoño, Irena Draskovic, Jeanette Hahn
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Dual role of ComEA
EC
FA
EAOut
In
Putative channel protein
“QQGGGGSVQSDGG” linker
(When linker is deleted, DNA binding still occurs, but no transport. So ComEA is needed for both steps)
DNA binding may induce bending and presentation of DNA to the channel
Gordon Inamine, Roberta Provvedi
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Role of ComFA
EC
FA
EAOut
In ATP binding protein
3. Integral membrane protein, accessible to protease only from inside.
4. Walker A site essential for function.
1. Required for transport, not binding.
2. Resembles DEAD family helicases and PriA.
ComFA may be needed to drive DNA translocation, for gating the channel or as a helicase.
Arturo Londoño
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Topology of ComEC: absolutely required for transport
N-loop
1 2 3 4 5
Irena Draskovic
lacZ
phoA
Both
OUT
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ComEC has an intramolecular disulfide bond
WT comECWT comEC
- + - + DTT
Irena Draskovic
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Cys is used here as a natural crosslinker. This disulfide bond does not occur in vivo.
Oligomerization of ComEC
+ NEM0’ 10’ 30’ 60’
EC monomer
Dimer
0’ 10’ 30’ 60’
Irena Draskovic
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Which cysteine residues are responsible for the in vivo intramolecular disulfide bond
and for the in vitro cross-linking?
Irena Draskovic
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ComEC has 8 cysteine residues
N-loop
Irena Draskovic
OUT
C2 C3
C1 C4
C5
C8
C7
C6
Each of the Cys residues has been converted to Ser.
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C2/C3wt KO+NEC+N- + - + - + - + DTT
ECN
EC(-SH)EC(-S-S-)
ECN is stable; no shift in mobility
C2 and C3 form an internal disulfide bond
The intramolecular -S-S- bond is in the N-loop
ECC2/C3 is degraded
Irena Draskovic
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ComEC Disulfide Bond
N-loop
S S
1 2 3 4 5
Introduced by BdbDC
Irena Draskovic
C S mutations in the remaining 6 Cys residues do not affect stability, transformation or the mobility shift.
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BdbD and C are needed to introduce the N-loop disulfide bond
wt bdbDC comG comC
DTT - + - + - + - +
EC
EC
(-SH)
(-S-S-)
Irena Draskovic
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Dimerization assay
0 10 20 60 0 10 20 60 0 10 20 60 0 10 20 60 wt C6/7S C8S C6/7/8S
Conclusion: ComEC dimerizes using contacts near cysteines (6), 7 and 8.
Irena Draskovic
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N-loop
S S
1 2 3 4 5
Irena Draskovic
Disulfide bond (in N-loop) and the oligomerization helix
C8
C7
C6
482 483 494
xxxxCCxxxxxxxxxxCx
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N-loop
BPD
Metallo-ß-lactamase domain
Protein motifs in ComEC
S S
1 2 3 4 5
Irena Draskovic
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What is the BPD (Binding Protein Domain) motif?
2. Probably involved in protein-protein interactions, most likely with the ATPase component.
1. Present in the permease component of ABC transporters.
3. Several point mutations in the BPD of comEC inactivate function without affecting protein stability.
Irena Draskovic
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BPD mutants
ComEC RAA - - - G - - - - - S - - - - - RV
- + - + - + - + - + - + wt RE RL AD AS GP
100% 2% 4% 36% 88% 6%
E - D - - - PL - S
Transformation frequenciesIrena Draskovic
DTT
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What is a metallo-ß-lactamase domain?
5. Several point mutations, introduced in this domain of comEC, lose function without affecting stability.
6. Possible roles in ComEC: Cell-wall remodeling? Non-transforming strand nuclease?
2. Includes enzymes that use water for nucleophilic attacks on covalent bonds. Usually with dinuclear Zn(II) centers.
3. Substrates often esters with associated negative charges.
4. Artemis: Involved in V(D)J recombination and double strand break repair. Processively degrades ssDNA (5’3’)!!!
1. Present in a large family of proteins.
Irena Draskovic
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N A A A
ß-lactamase domain mutants
Motif 1 2 3
Motif 4 5
LIDTG HADQDHIG H
WILTGD KVGHH
LhDsG HxHxDHxG C/H
hhxsGD hhxxH
2% 3% 4% 4%
Irena Draskovic
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ß-lactamase ß-lactamase domain domain
BPDBPD
4 33
N-loopN-loop N-loopN-loop
Cartoon of ComEC channel
112 2
5 5
OUT
Irena Draskovic
ccc
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ComEC-10His Contacts ComEA
Competent cells +/- DNA
Crosslink (DSP)
Solubilize membrane proteins
Isolate membranes
Pull-down ComEC (Ni2+-resin)
EC
EA
- + - + DNA - - + + His tag
unbound EC
unbound EA
Irena Draskovic
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1. Three components: ligand binding (ComEA), polytopic membrane permease (ComEC) and ATPase (ComFA).
2. ComEC is a dimer (so is ComEA).
4. The BPD domain plays a role.
5. The ATP binding site of ComFA is essential for transport.
3. ComEC has 5 transmembrane segments per monomer.
Could these three proteins constitute an (atypical) ABC transporter?
ECFA
Out
InPutative channel protein
ATP binding protein
EADNA binding protein
6. ComEA and ComEC contact one another.
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But, transport may not be driven directly by ATP hydrolysis.
Perhaps ComFA plays a signaling role (channel gating?) or is needed as a helicase, while PMF drives transport.
Inhibitor data from the Konings lab (Groningen) suggests that transport is driven by the pH component of PMF.
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permease (EC)permease (EC)
receptor receptor (EA)(EA)
ABC cassette ABC cassette (FA)(FA)
DNA uptake
Irena Draskovic
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permease (EC)permease (EC)
receptor receptor (EA)(EA)
ABC cassette ABC cassette (FA)(FA)
NucANucA
DNA uptake
Irena Draskovic
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permease (EC)permease (EC)
ABC cassette ABC cassette (FA)(FA)
receptor receptor (EA)(EA)
NucANucA
DNA uptake
Irena Draskovic
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permease (EC)permease (EC)
ABC cassette ABC cassette (FA)(FA)
receptor receptor (EA)(EA)
NucANucA
DNA uptake
Irena Draskovic
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permease (EC)permease (EC)
ABC cassette ABC cassette (FA)(FA)
receptor receptor (EA)(EA)
NucANucA
DNA uptake
ATPATP ADPADPATPATP ADPADP
Irena Draskovic
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permease (EC)permease (EC)
ABC cassette ABC cassette (FA)(FA)
receptor receptor (EA)(EA)
NucANucA
DNA uptake
Irena Draskovic
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permease (EC)permease (EC)
ABC cassette ABC cassette (FA)(FA)
receptor receptor (EA)(EA)
NucANucA
Nuclease?Nuclease?
DNA uptake
Irena Draskovic
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permease (EC)permease (EC)
ABC cassette ABC cassette (FA)(FA)
receptor receptor (EA)(EA)
NucANucA
Nuclease?Nuclease?
DNA uptake
Irena Draskovic
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Transformation at the poles?
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ComGA localization
Jeanette Hahn
Localization of comGA-gfp
Localization of comGA-gfp-atp
Localization of comGA-myc (IF)
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Localization of Pspac-comGA-gfp
ComK is required for localization
comK-
comK+
What is role of localization-Assembly at poles?Jeanette Hahn
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GFP-ComK
ComK is itself at the poles and probably also associated with the nucleoid.
Peter Prepiak
What is the role of localization? Assembly of DNA uptake machinery and transformation at the poles?
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Co-localization of ComGA-CFP and ComFA-YFP
ComFA-YFP
ComGA-CFP
Jeanette Hahn
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Competence proteins tend to assemble preferentially at the poles.
Hypotheses:
Transformation occurs preferentially at the poles.
Is the recombination machinery assembled at the poles?
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Regulation DNA uptake
Jeanette Hahn
Mireille Ansaldi
Kursad Turgay
Leendert Hamoen
Mark Albano
Pablo Tortosa
Flavia Piazza
Marjan Persuh
Lauren Logsdon-Peterson
Tran Thu Hoa
Peter Prepiak
Thi Yen Linh Ho
Soo Jeong Cho
Young Sook Chung
Ines Chen
Irena Draskovic
Mark Albano
Jeanette Hahn
Roberta Provvedi
Arturo Londoño
Gordon Inamine
Sandhya Mohan
Tatjana Trcek
Fred Breidt
Roopesh Kumar Pundhir
Reinhard Breitling
Mathieu Bergé
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Collaborations
Sierd Bron et al, Groningen
BdbDC:
Optical trap:
Berenike Meier and Michael Sheetz, Columbia University
Leendert Hamoen, Wiep Klaas Smits and Oscar Kuipers, Groningen
Action at the comK promoter:
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Competence Growth Arrest
Time
T0
Bul
k cu
lture
gro
wth
Com
petent cell growth
2 hours
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109
ß-lactamase ß-lactamase domain domain
BPDBPD
543
2
53
32
N-loopN-loop N-loopN-loop
11
7 7
8
6
10
97
7
8
6
ComEC channel
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Helical wheel projection of ComEC helix 6, looking toward the cytosol
HelixDraw
78
6
Irena Draskovic