Download Structure and function of the divisome in E. coli – the factory of cell

Structure and function of the
divisome in E. coli – the factory of
cell division
Course:Physics of the living cell
Author:Guy Alis
Lecturer:Prof. Mario Feingold
BGU 2016
Prokaryotes – simplest cells – no nucleus, only
ribosomes, cell wall - bacteria, archaea (‫)חיידקים קדומים‬
E. coli - Cw – cell wall, N – nucleoid (DNA), R - ribosomes
The Prokaryotic Cell
Bilayer (Membrane)
E coli is Gram-Negative
Scanning electron micrographs of gram-positive and
gram-negative bacteria,respectively. note differences in
surface texture. Each cell in the TEMs is about 1µm
wide
Nutrition and cell chemistry
C-carbon;
O-oxygen;
N-nitrogen;
H-hydrogen;
P-phosphorus
(‫;)זרחן‬
Se-selenium
(‫;)מינרל‬
Introduction
• The cell cycle includes 3 stages
• 1)lag
• 2)constriction
• 3)generation
The Microbial Growth Cycle
Death Phase
If incubation continues after a population reaches the stationary
phase, the cells may remain alive and continue to metabolize, but
they will eventually die.
The rate of cell death is much slower than the rate of exponential
growth.
The Mathematics of Exponential
Growth
N  N0 2
n
g=100minutes
t
g
n
N  N0 2
t
g
N-final cell number
N0-the initial cell number
N-number of generations during
the period of exponential growth
g=107minutes
A photomicrograph of cells of E.Coli treated in such a
way as to make the nucleoid visible.A single cell is
about 3µm and the nucleoid about 1µm long
Bacterial Cell Division-who cares?
•Designing methods to control
microbial growth,in particular,
bacterial growth. This methods
are used to treat a lifethreatening infectious disease or
simply to disinfect a
surface.Cancer is uncontrolled
cell growth.
Cell Growth and Binary Fission
Cells elongate to
approximately
twice their
original length
and then form a
partition that
constricts the
cell ito two
doughter cells.
Cell Growth and Binary Fission
This partition is
called septum
and results from
the inward
growth of the
cytoplasmic
membrane and
cell wall from
opposing
directions;
The Zring
The Zring
• Fission (‫ )בקוע‬is coordinated by a protein
complex called the divisome
• The assembly of which is directed by the
conserved tubulin (cytoskelton component)
homologue FtsZ.
Before The Zring
Elongation mode of
peptidoglycan synthesis.
Insertion of peptidoglycan
precursors in to the
existing peptidoglycan
layers.
Needed for
efficient
invagination
( ‫התקפלות‬
‫)כלפי פנים‬
Of the outer
membrane
Forms the
Z ring plane
and
attracts
other
divisome
proteins
The Zring
• First, FtsZ undergoes polymerization into filaments
that form a defined ring- like structure (Z ring) at the
site of cytokinesis.
• Membrane-associated FtsZ-interacting proteins, such
as FtsA and ZipA in E.Coli,tether the Z ring to the cell
envelope, which results in an initial complex called
the proto-ring.
ZipA(pink) and
FtsA(blue) tether
FtsZ(green)
protofilament and
polymer bundles
to the
membranes using
flexible linkers
Overlay of the
cytoplasmic membrane
(red) and the Zring
(green-yellow)
(b)FtsZ - membrane tethering
(c)Cyro-electron tomography of
FtsZ and FtsA
(d)Flouorescence microscopy and
differential interference contrast
microscopy of FtsZ and FtsA
The Zring
After about 40%
of generation
time, the FtsZring is formed.
Connects
the FtsZ ring
to the
cytoplasmic
membrane
and
stabilizes it
Same as
FtsA but has
a relatively
stronger
bound to
the
membrane
and is less
concentrate
d
The Zring-Role of FtsA in Formation of
the Divisome
• It tethers the FtsZ
filaments to the
membrane
• Along with ZipA, it is
required for recruitment
of all the downstream
division proteins.
• FtsA is much more
conserved in evolution
than ZipA,it has more
direct role in the
recruitment of
downstream proteins.
The Zring-Role of FtsA in Formation of
the Divisome
• Two FtsA
molecules
arranged in a
filament
• MreB is similar to
conventional
actin
The Zring
Appearance and
breakdown of the
FtsZ ring during the
cell cycle of E.Coli.
Phase
contrast
Stained
cells
Ftsz
Ring
iPALM
images of
vertical
E.coli
not yet
formed
start to
forms as
segregate cell elongates
breakdown,
division
The Zring
iPALM
images of
horizontal
E.coli
Trapped cell , GFP
images of FtsZ
Fts Proteins and Cell Division
•In a cell of E.coli about
10,000 FtsZ molecules
polymerize to form the
ring.
•The ring attracts other
divisome proteins
•ZipA is an anchor that
connects the FtsZ ring
to the cytoplasmic
membranes
The cytoskeletal protein MreB is an actin analog that
winds as a coil through the long axis of a rod-shaped
cell, making contact with the cytoplasmic membrane
in several locations . These are sites of new cell wall
synthesis.
MreB protein flourescence
The Zring
• During the seconds stage,the proto-ring
recruits enzymes that are involved in cell wall
(septum) synthesis, such as FtsI, and proteins
that probably coordinate signalling between
these enzymes and the proto-ring, such as
FtsN and FtsBLQ, thereby forming the mature
divisome.
FtsA(blue) recruits
FtsN(purple).The
periplasmic domain of
FtsN is targeted to
peptidoglycan,which
reinforces its loclazation
and potentially stimulates
Ftsl and the synthesis of
septal peptidoglycan
After The Zring
About one fifth of a
generation time later (overall
60% generation time since
the beginning), the proteins
Ftsk up to AmiC assemble
onto the ring, and
constriction of the cell is
initiated .
Last division
proteins to arrive
at the
ring,triggers
septation,forming
the mature
divisome
Encodes the
membrane
components of
theABC transporter
Encodes the ATPase
components of the
ABC transporter
Dedicated to
peptidoglycan
synthesis
during cell
elongation only
Localize to
the ring
and
promotes
the
integrity of
the Z ring
The Zring
• The divisome then constricts the cytoplasmic
membrane, which in concert with the
synthesis of new peptidoglycan( ‫קיפול כלפי‬
‫ )פנים‬at the division septum and invagination
of the outer membrane achieves cytokinesis
and cell separation.
Translocates DNA
away from the
septum. May play a
role in fusion of the
invaginating
membrane to
complate cytokinesis
The Zring
In the absence of AmiB and AmiC, AmiA is still
able to carry out the seplitting of the septum
The Z ring is targeted to the site of
division by related but distinct
mechanisms.
Phase Constrast
For M9 (poor media) Ƭg≈100 minutes
For LB (rich media) Ƭg ≈20 minutes
Double Stranded DNA
From DNA to proteins – information
flow
• when the cell divides DNA is replicated – DNA polymerase
• - DNA contains genes that code for proteins
• - when activated, RNA polymerase builds an RNA copy,
messenger RNA (mRNA) – transcription
• - mRNA goes to the ribosome – there transfer RNA (tRNA)
helps copying the mRNA into a protein – translation. Each 3
mRNA bases code for 1 amino acid
• - polypeptide folds into 3D structure, sometimes
chaperones help
DNA Replication
DNA replication is a semi conservative process in all cells –
the two resulting double helices have consist of one new
strand and one parental strand
Events at the DNA replication fork
Synthesized a
short strech of
RNA (11-12
nucleotides
Unwinds a
double
helix
Catalyze
(‫ )מזרזים‬the
addition of
nucleotides
Nucleic
acid
molecule
Extension of a
DNA chain by
adding a
deoxyribonucleoside
triphosphate
at the 3’ end.
DNA helicase
unwinding a
double helix
Events at the DNA replication fork
Descrete
3’-OH
Keeps the double helix
locally open while the
replication fork
continues
Continueous
3’-OH
Why is a primer required for
DNA replication?
Bidirectional Replication and the
Replisome
Circular DNA.
The origin of the replication is also known as OriC
the origin of the end of replication is also known as
TerC.
Notice the change of the inner and outer color of
the circumference of the replicated DNA strainds
?
How can Escherichia coli carry out cell
division in less time than
it takes to duplicate its chromosome?
Answer: superposition of
replication forks
References
QUIZ