Physics and Nanotechnology to Study Bacterial Cells m B m B - - PowerPoint PPT Presentation

Jaan Männik

Saturday Morning Physics Lecture

• Mar. 11, 2017

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Physics and Nanotechnology to Study Bacterial Cells

Impact of physics on studies of living systems

• 2. Application of physics based theories to explain life

processes.

• 1. Application of physics based methods and techniques

to experiments with living systems

Bacteria – the unseen majority

• Majority of Earth’s biomass are bacteria and archea. They

are

• ne
• f

the main determinants how biosphere functions

• There are estimated 10 times more bacterial cells in

human body than our own cells

• Recognizing bacteria and other microorganisms

as disease agents have lead human life span to increase twofold over past 100 years. This gain may not be permanent.

Standard microbiology toolbox

The standard microbiology tools are not suitable to follow:

An agar plate Cell culture tubes

• 2. Molecular process in individual cells in real time.
• 1. How bacteria behave in complex environments

Lab-on-a-chip based tools

• Well controlled environment for the cells which physical and

chemical characteristics can be controlled and manipulated

• Compatible

with high resolution microscopy

• f

cells (including super-resolution imaging, SEM)

Advantages of lab-on-a-chip platform

2 m

• In situ biochemical analysis of cells
• Automation
• F. K. Balagadde et al Science 309 (2005) 137.
• J. W. Hong et al Nat. Biotech. 21 (2003) 1179

Device to extract bacterial DNA

Applications of lab-on-a-chip in studies of cells

• S. Park et al.

Science 301 (2003) 188

• N. Q. Balaban et al.

Science 305 (2004) 1622

Molecules Cells Populations Organisms

• Y. Mercy et al.

PNAS 104 (2007) 11889

Sequencing Antibiotic resistance studies Ecology, evolution Bacteria and tissue interactions

D.Huh et al. Science 328 (2010) 1622

Understanding how bacterial cells move in small pores using bio- mimetic lab-on-a-chip devices

Bacterial movement in channels and pores

• Most bacteria in different environments live in pores 10 m

and smaller

Can’t do it in patient mouth …

Experimental setting

• Monitor bacterial movement through channels (constrictions) using

fluorescent microscopy

100 m

channel chamber

• Fabricate on the single chip, large number of differently sized and

shaped channels.

Microfabrication cleanroom

Microchip fabrication

Use electron beam or photolithography to write pattern of channels Develop resist Reactive ion etch Si wafer Lift-off resist Close channels Repeat the process for different channel heights

1 2 3 4

PMMA Si

5

Si PDMS coated glass coverslip electron beam channels

Drill access holes

Channels

1 µm

• On single chip, channel width is made to vary between ≈300 nm to 5 m

to using a RIE cryoetch process

1 µm 1 µm

D

Side view Bacterium

Fluorescence

Most materials/objects do not fluoresce when excited with blue light (many materials fluoresce when exited with UV light) Exception – jellyfish Aequorea victoria UV excitation Excitation with blue light allows to selectively observe engineered molecules such as GFP and have very little background from other molecules.

Fluorescent proteins

• Insert genetic code for GFP molecule into bacterial

genome or in plasmid (short circular DNA that many bacteria carry). Bacteria will synthesize the protein. GFP (Green Fluoresccent Protein)

• Shine blue light on bacteria that express GFP. They will

shine green light back. There is little background at green wavelength region. Blue light

Gram-negative and Gram-positive bacteria

Bacteria Gram-negative bacteria Gram-positive bacteria cell wall lipid membrane

cytoplasm cytoplasm

Mollicutes (no cell wall)

Escherichia coli Bacillus subtilis

Two bacterial species studied

• E. coli RP437

1 µm

1 µm

D

Escherichia coli Gram-negative bacterium Bacillus subtilis Gram-positive bacterium

1 µm

D

• Superfically looking the two bacterial species are similar but on

molecular level they are different than humans are from roundworms.

Bacterial swimming

• E. coli with fluorescently labeled flagella
• H. C. Berg group, Harvard University

Wikipedia

• E. coli RP437

1 µm

Introduction: Bacterial motility

• Bacterial flagellar motor is a rotary motor superficially similar to DC

electrical motor

• Rotation speed controlled membrane potential and pH gradient across

inner membrane

• Bacterial sensory system (chemotaxis receptors through signaling

cascade) control direction of rotations

Bacterial motility in channels

channel chamber chamber

W = 1.2 m

• E. coli RP437

0.5 1.0 1.5 2.0 2.5 10 20 30

<v> [m/s] W [m]

• E. coli and B. subtilis bacteria are

motile in channels which are only 30-40% wider than their diameter

• In smaller channels bacteria lose

their ability to swim but ..

Growth and division is bacterial solution to penetrate narrower channels

• E. coli

channel chamber chamber

• B. subtilis

W = 0.6 m

Different layout of channels

• More details of cells

visible in the microscope

• Channel ceiling soft;

Bacteria can deform it

Bacterial movement in sub-micron channels

• B. subtilis grows to filaments that buckle and finally divide
• B. subtilis
• E. coli
• Growth in confinement alter drastically E.coli shapes

Re-emergence of regularly shaped bacteria

5 hrs 49 hrs 0 hrs

10 µm

• Over period 1-2 days regular rod-shaped bacteria replace initial

population of aberrantly shaped bacteria

Modes of penetration for different channel widths

• E. coli but not B. subtilis bacteria are able to grow through channels

which widths are smaller than their diameters

Mechanical properties and propagation in narrow channels

• Cell-wall has high Young modulus but is easily bendable (think of

inflated balloon)

• The thicker the cell wall the higher the Young modulus and the

higher osmotic pressure can bacterium maintain in its interior

D W

channel wall channel wall

• E. coli
• B. subtilis

Posm=2-3 atm

• A. Boulbitch et al PRL 85 (2000) 5246

Posm=26 atm

• V. R. F. Matias et al Mol. Microbiol 56 (2005) 240

cell wall 3 nm cell wall 30-40 nm

Pressure actuated valves to mechanically manipulate cells and sub-cellular structures

• Characterize the mechanical properties of bacterial cells

glass PDMS pressure channel channel for bacteria

• Use it as tool to manipulate sub-cellular structures in a

bacterial cell

Mechanical limits of bacterial deformations

• Bacteria break open at isolated weak spots at cell poles

Step # pmax = 2 atm

pmax

pmax = 3 atm

• Bacteria can be stretched up 25% elastically

Short vs long time response of E. coli

1 2 3

• Elastic deformation after entry in shallow channel
• Slow cellular response after prolonged stay in channel

1 2 3

Summary on lab-on-a-chip and squeezing measurements

• Lab-on-a-chip platform allows to create biomimetic environments

where cell behavior can be studied using high resolution optical microscope and bioanalytical tools.

• E. coli and B. subtilis are well adapted to swim in small channels

despite their long flagella. Both species retain ability to swim in channels which only 30% exceed their body diameter

• Surprisingly, bacteria can get through even narrower channels! For

that they use growth and division.

• Bacterial growth is robust despite drastic changes in their shapes
• Mechanical properties of bacteria determine how small channels

they can penetrate.

Understanding how cells are built from molecules up using quantitative high resolution microscopy and modelling

Escherichia coli as living “hydrogen atom”

Escherichia coli E. coli

• Bacteria present the simplest systems to understand how

the cellular processes unfold using basic physics principles

Jacque Monod, “What is Valid for E. coli is also valid for the elephant”

1 m

What is known

1 m

FtsZ MatP tetramer SlmA tetramer

2 nm

• Functions of about 70% genes to some degree
• 50% of protein structures (most based on homology)

What is not known: from genes to cell

How nanometer-scale proteins, DNA and lipids come together and form the micro-size cell?

1 m DNA lipid polysaccharide protein

?

Cellular organization in bacteria

• Bacterial cells are highly organized despite

their apparent simplicity

Bag with soup of molecules Assembly of molecular machines

Wikipedia

envelope

• How are chromosomes and cell division

apparatus organized in a bacterial cell?

• How cell division proteins position relative

to nucleoids?

Chromosome organization

Genetic information is tightly packed in the cell

Escherichia coli circular chromosome 4.6106 basepairs 1.6 mm

1 μ

• Thousand fold compaction of DNA in the cell

HupA-mCherry labelled chromosome

Nucleoid

1 μ

• DNA spreads just to a fraction of cell volume (~50%) – the

nucleoid (not to be confused with nucleus)

HupA-mCherry labelled chromosome

nucleoid

• There is no membrane surrounding the nucleoid

cytosol

What compacts DNA?

Depletion Forces

• Molecules move so that to minimize excluded volume. This

leads to appearance of an average force (depletion force) that pushes larger molecules together.

D

Volume gained (Vgained) by small molecules if two large molecules come together

• Alternative view: smaller molecules exert (osmotic) pressure to

big ones which pushes them together. abundant small molecules (crowders) few large molecules

Alignment and compaction of DNA strands as result of depletion forces

• DNA strands are pushed together and aligned

Depletion forces compact DNA and lead to phase separation

1 μ

Nucleoid contains DNA and

• ccludes most proteins and ribosomes

Cytosol phase is protein and ribosome rich

• Proteins and ribosomes are crowders. They push and

compact DNA. In doing so DNA and proteins phase separate from each other (like water and ice do)

Dream of a bacterium is to make from

• ne cell two cells (Jacque Monod)
• To divide bacterium needs to duplicate its DNA

Bacterium living up its dream

DNA within nucleoid is dynamic

• Some chromosomal regions occupy well-defined locations

in the cell

• How exactly DNA is folded in the nucleoid not yet understood.

Modelling replicating chromosome in a cylindrical confinement

• Model DNA as self-avoiding chain of 100 nm blobs that

moves under thermal force (Langevin thermostat)

Ter

• riC
• riC

100 nm blob Partially replicated chromosome

Dynamics of replication terminus region

• Replication terminus region positions itself in the cell

center as a result of entropic force

Ter

• riC
• riC

Ter

starting configuration equilibrium configuration

Cell division and

• rganization cell division

apparatus

Cell division in bacteria

chromosome (nucleoid) Z-ring constriction

• Cell division proteins assemble to a ring-like structure, the Z-

ring, that separates two daughters from each other.

Z-ring FtsZ ring

FtsZ monomer FtsZ filament

• H. P. Erickson et al., Microbiol. Mol. Biol. Rev. 74 (2010) 504
• FtsZ filaments act as a scaffold. This scaffold recruits

about 30 other proteins to the ring (the fellowship of the ring).

• Assembly of FtsZ filaments is the first step in cell division
• D. W. Adams, J. Errington, Nat. Rev. Micro. 7 (2009) 642

Z-ring

Assembly of the Z-ring

Green, Red – FtsZ pointing up and down Patchy band model Continuous cohesive ring Patchy band

• S. Holden et al PNAS 2014 (111) 4566
• How filaments are exactly organized in the Z-ring is not

known

Positioning of cell division proteins

• Fellowship of the ring locates within 2% accuracy in the

center of the cell

Go to the right place !!!

PDB: 1FSz

First view

• There is a little control freak inside every cell who knows

where things need to go and it takes care of it.

• Self-assembly of molecules to macromolecular

structures driven by diffusion, intermolecular binding, conformational entropy, excluded volume effect etc.

Second view

PDB: 1FSz

Let’s get together at the middle

Two known molecular systems in E. coli to localize its Z-ring:

• The Min system
• Nucleoid occlusion mechanism

• 2
• 1

1 2

• 2
• 1

1 2

x [m] y [m]

Min operon: minC, minD, minE

Min oscillations in E. coli cells

• MinD, MinE and MinC proteins oscillate between two poles
• f rod-shaped E. coli.

MinD::GFP

• Their time-averaged concentration is high at the cell poles.

This defines inhibitory zone for Z-ring localization.

• K. C. Huang et al PNAS 100 (2003) 12724

Reaction-diffusion system:

Min system: Turing instability in nature

• , , …

Nucleoid occlusion

• Nucleoid occlusion: presence of nucleoid produces

inhibition for the Z-ring assembly

• D. W. Adams, J. Errington, Nat. Rev. Micro. 7 (2009) 642

Nucleoid occlusion factor SlmA

• SlmA binds to specific sequences of DNA that are missing in

replication terminus region.

N.K. Tonhat et al EMBO J. 30 (2011) 154

• SlmA is inhibitor for Z-ring formation.

Terminus Origin

• H. Cho et al PNAS 108 (2011) 3773

Terminus

• Replication terminus region positions itself in the center of

the cell. No inhibition of Z-ring formation at the cell center. SlmA DNA

What happens to cells that lack these positioning systems?

WT slmA minC minC

a minicell

slmA

Evidence for divisome localization mechanisms independent of the Min system and SlmA in Escherichia coli, M. W. Bailey, P. Bisicchia, B. T. Warren, D. J. Sherratt and J. Männik, PLoS

• Genet. 10 (2014) e1004504.

How can E . coli cells without known positioning systems still divide about its middle?

• 2

2 0.0 0.5 1.0

Intensity (arb. units) Length along the long axes [m]

Xz Xn

phase DAPI GFP

• 0.4
• 0.2

0.0 0.2 0.4

• 0.4
• 0.2

0.0 0.2 0.4

Xz/L

Xn/L slmA min

Localization of Z-ring in cells w/o known positioning systems

• Z-ring localizes the center of the nucleoid rather than to

nucleoid free regions.

• 0.30 -0.15

0.00 0.15 0.30 20 40 60 counts Xz-Xn [m]

slmA min

= 66 nm

slmA min

Positive regulation

• A positive signal that guides localization of cell division

proteins from the cell center to the nucleoid center.

time [min] Length along long axes [m]

Z-ring Chromosome

Length along long axes [m]

slmA min 2 m

• 4
• 2

2 4 6 1 2

I [arb. units] Length along long axes [m]

nucleoid Z-ring

Realization of the positive signal

ΔslmA ΔminC ΔzapA ΔslmA ΔminC ΔzapB

Misplaced Misplaced

slmA min matP

• 1.0
• 0.5

0.0 0.5 1.0 20 40 60 counts Xz-Xn [m] N=123

• 1.0
• 0.5

0.0 0.5 1.0 20 40 60 80 counts Xz-Xn [m] N=218

• 1.0
• 0.5

0.0 0.5 1.0 20 40 60

counts Xz-Xn [m]

N=145

= 202 nm = 150 nm = 222 nm

• ZapA, ZapB and MatP proteins are required for the

positive signal

Summary on organization of bacterial cells

• Min system and nucleoid occlusion are two known systems

for positioning of bacterial Z-ring via a negative regulation.

• ZapA, ZapB and MatP proteins are needed for the positioning.
• Replication terminus region can spontaneously position itself

in mid-cell as a result of entropic force. This spontaneous process appears ultimately to control the placement of cell division proteins.

• We

have found a positive regulatory mechanism that promotes Z-ring formation at the vicinity of the Ter region of the chromosome.

Thanks

Matthew Bailey, UTK George Siopsis, UTK Dan Castillo, UTK Jaana Männik, UTK Paola Bisicchia, Oxford Univ. David Sherratt, Oxford Univ. Piet de Boer, Case Western Reserve Univ. Alex Dajkovic, Univ. of Paris 5 Rodrigo Lamothe-Reyes, McGill Univ. Cees Dekker, Delft University of Tech. Conrad Woldringh, Univ. of Amsterdam Arieh Zaritsky, Ben Gurion University NSF CAREER award

CNMS User Program: 2015-231 UTK start-up funding

Thank you for your attention!

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