How E. Coli find their middle

Xianfeng Song

Sima Setayeshgar

Outline

Introduction to E. coli Two systems regulate division site placement

Nucleoid occlusion Min proteins

Experiments: Qualitatively understand Min Proteins in E. coli

Model: How and why Min Proteins work in E. coli?

About E. coli

E. coli is a bacterium commonly found in the intestinal tracts of most vertebrates.

Studied intensively by geneticists because of its small genome size, normal lack of pathogenicity, and ease of growth in the laboratory.

Size: about 0.5 microns in diameter and 1.5 microns long

From: cwx.prenhall.com

E. coli life cycle

Typical lifecycle

E. coli cell division

Division accuracy: .50 +/- .02 Placement of FtsZ ring: .50 +/- .01

Two systems regulate division site placement Nucleoid occlusion

Min proteins Including MinC, MinD, and MinE

Min proteins (Experiments) MinC

Inhibits FtsZ ring formation

MinD MinD:ATP recruits MinC

to membrane MinE

Binds to MinD:ATP in membrane and induces ATP hydrolysis

Black: MinC Red: MinD Blue: MinE

Min proteins (Experiments)

Without Min proteins, get minicelling phenotype (Min-)

If MinC is over-expressed, get filamentous growth (Sep-)

Min proteins oscillate from pole to pole

Hal

e et

al.

(200

1)

MinD-GFPMinD-GFP

MinE ring caps MinD polar region

MinE ring is membrane bound.

Ring appears near cell center and moves toward pole.

Hal

e et

al .

(200

1)

MinE-GFPMinE-GFP

Wavelength of oscillations is ~10 microns.

Ha l

e et

al .

(200

1)

MinE-GFPMinE-GFP

Filamentous cell has “zebra stripe” pattern of oscillations

MinD-GFPMinD-GFP

Raskin and

de Boer (1999)

Phenomenology of Min oscillations MinD polar regions grow as end caps. MinE ring caps MinD polar region. Oscillation frequency:

[MinE] frequency , [MinD] frequency .

Filamentous cell has “zebra stripe” pattern of oscillations.

In vivo oscillations require MinD and MinE but not MinC.

How and Why Min-proteins oscillate (Modeling effort) Howard et al. (2001)

Very simple 1D model MinE is recruited by cytoplasmic MinD to membrane MinD polar region fails to reform at poles. (not agree with experi

ment) Huang and Wingreen (2003)

MinE is recruited by membrane-bound MinD-ATP MinD aggregation on the membrane follow a one-step process

Kruse et al. (2005) Consider protein diffusion on the membrane. MinD aggregation on the membrane follow a two-step process: fir

st attach to membrane, then self-assembles into filament Meinhardt and de Boer (2001)

Requires new protein synthesis.

Model from Huang and Wingreen (2003)

Equations for model from Huang and Wingreen (2003)

membranein ATP:MinD:MinE

membranein ATP:MinD

cytoplasmin MinE

cytoplasmin MinD

de

d

E

D

dedeEdEde

dt

d

EdEdedeEEE

dt

d 2D

ATPDdeddDDEdEd

dt

d:)]([

s

ss

ss

ED

deE

dDD

2

3

3

m 5.2

1 8.0;

m 16.0

m001.0;

m025.0

DD

ATPDdeddDD

ATPADP

ADPDATPDD

ATPD

dt

d

:

::

2:

)]([

D

ATPADP

ADPDdedeADPDD

ADPD

dt

d

:

:2: D

Result: MinD/E movie

MinE

MinDMinD

Result: MinD end caps and MinE ring

Mechanism for growth of MinD polar regions (Huang and Wingreen, 2003) MinD ejected from old end cap

diffuses in cytoplasm.

Slow nucleotide exchange implies uniform reappearance of MinD:ATP.

Capture of MinD:ATP by old end cap leads to maximum of cytoplasmic MinD:ATP at opposite pole.

Result: Frequency of oscillations ~ [MinE]/[MinD]

No oscillations for [MinE] too high, or for [MinD] too low.

Minimum oscillation period 25s.

4 micron cell 4 micron cell

Result: “Zebra stripe” oscillations in long cells

Stripes form with wavelength of ~10 microns

Oscillation makes E. Coli divide accurately The oscillation make MinD has smaller avera

ge concentration at the middle and higher concentration at the ends.

MinC can only be bounded to memberane if it is recruited by MinD-ATP

MinC can Inhibits FtsZ ring formation, thus inhibits the division

Linear stability analysis around homogeneous solution

tikziii etx 0),(

Red curve corresponds to a normal cell

Predictions of model (Huang and Wingreen, 2003)

Delay in MinD:ATP recovery is essential. (Verified by Joe Lutkenhaus – nucleotide exchange rate is slow, ~2 / s.)

Rate of hydrolysis of MinD:ATP by MinE sets oscillation frequency.

Diffusion length of MinD before rebinding to membrane sets oscillation wavelength.

Recent development Helical morphology of MinD polymers.

Some recent modeling effort (mainly focus on cleaning some details, no breakthrough) the fluctuation effects on the min proteins (Howard, et al, 2003), The new model considering the membrane diffusion and more reactions

(Kruse, et al, 2005) min-protein oscillations in round bacteria (Huang and Wingreen, 2004)

Open questions

MinE ring moving in reverse.

The helical structure of MinD polymers

Conclusions

Although bacteria such as E. coli is a very simple creature, it is also very complicated system.

To understand this system, physicist can help a lot.

Thanks to Doc. Wingreen kindly let us to use his slides.

Thank you

Evidence from in vitro studies

A. Phospholipid vesicles

B. MinD:ATP binds to vesicles and deforms them into tubes

C. MinD:ATP polymerizes on vesicles

D. Diffraction pattern indicates

well-ordered lattice of MinD:ATP

E. MinE induces hydrolysis of MinD:ATP and disassembly of tubes

Hu

Hu et al.

et al. (2002) (2002)

Min proteins in spherical cells:Neisseria gonorrhoeae

Wild typeWild typeMinDMinDNgNg

--

Sze

to

Sze

to e

t al

et a

l . (2

001)

. (2

001)

Min-protein oscillations in nearly round cells

In E. coli, Min oscillations “target” MinD to poles

Why does E. coli need an oscillator?

In B. subtilis, minicelling is prevented by MinCD homologs, but polar regions are static.

Marston Marston et alet al. (1998). (1998)

How E. coli find its middle Proteins are too small to see the caps’ curvature Subtilis have local proteins fixed at two ends, but

E. coli does not have