dna uptake during transformation of bacillus subtilis look here throughout for the names of the...

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

Not another transformation talk!

Edvard Munch

ComK is the Master Regulator of Competence

PrcomK

+

Genes for DNA uptake

+

Time

Transform

ationG

row

th

Competence Develops in Stationary Phase

T0

comK Synthesis Begins in Stationary Phase

Time

Gro

wth

T0 Com

K

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

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.

TRANSFORMATION PATHWAY

Integration

Binding

Fragmentation

Transport and degradation

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

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

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

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)

The protoplast supernatant

Gram positive cell(B. subilis)

lysozyme

osmoprotectant

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

ComC is an integral membrane peptidase that converts pre-ComGC, pre-ComGD, pre-ComGE and pre-ComGG to their mature forms

Young Sook Chung

ComC is needed for the translocation of ComGC

Young Sook Chung

MNEKGFTLVEMLIVLFIISILLLITIPNVTKHNQTIQ

KKGCEGLQNMVKAQMTAFELDHEGQTPSLAD

LQSEGYVKKDAVCPNGKRIIITGGEVKVEH

ComGC has an intramolecular disulfide bond

Young Sook Chung

bdbD

ComK

BdbD and BdbC are thiol-disulfide oxidoreductases required for competence

bdbC

Sierd Bron lab, Mark Albano

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

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

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

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

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

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

Processing and Assembly of ComGC multimer

s-s

s-s

ComG proteinsHS

SH

BdbDCComC

s-s

S-S

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?

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

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

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

Topology of ComEC: absolutely required for transport

N-loop

1 2 3 4 5

Irena Draskovic

lacZ

phoA

Both

OUT

ComEC has an intramolecular disulfide bond

WT comECWT comEC

- + - + DTT

Irena Draskovic

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

Which cysteine residues are responsible for the in vivo intramolecular disulfide bond

and for the in vitro cross-linking?

Irena Draskovic

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.

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

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.

BdbD and C are needed to introduce the N-loop disulfide bond

wt bdbDC comG comC

DTT - + - + - + - +

EC

EC

(-SH)

(-S-S-)

Irena Draskovic

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

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

N-loop

BPD

Metallo-ß-lactamase domain

Protein motifs in ComEC

S S

1 2 3 4 5

Irena Draskovic

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

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

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

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

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

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

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.

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.

permease (EC)permease (EC)

receptor receptor (EA)(EA)

ABC cassette ABC cassette (FA)(FA)

DNA uptake

Irena Draskovic




NucANucA

DNA uptake

Irena Draskovic




NucANucA

DNA uptake

ATPATP ADPADPATPATP ADPADP

Irena Draskovic




NucANucA

DNA uptake

Irena Draskovic




NucANucA

Nuclease?Nuclease?

DNA uptake

Irena Draskovic

Transformation at the poles?

ComGA localization

Jeanette Hahn

Localization of comGA-gfp

Localization of comGA-gfp-atp

Localization of comGA-myc (IF)

Localization of Pspac-comGA-gfp

ComK is required for localization

comK-

comK+

What is role of localization-Assembly at poles?Jeanette Hahn

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?

Co-localization of ComGA-CFP and ComFA-YFP

ComFA-YFP

ComGA-CFP

Jeanette Hahn

Competence proteins tend to assemble preferentially at the poles.

Hypotheses:

Transformation occurs preferentially at the poles.

Is the recombination machinery assembled at the poles?

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

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:

Competence Growth Arrest

Time

T0

Bul

k cu

lture

gro

wth

Com

petent cell growth

2 hours

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

Helical wheel projection of ComEC helix 6, looking toward the cytosol

HelixDraw

78

6

Irena Draskovic

dna uptake during transformation of bacillus subtilis look here throughout for the names of the...

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