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Microbial Cell Structure and Function

01-20-15

I. Microscopy

• 2.1 Discovering Cell Structure: Light Microscopy

• 2.2 Improving Contrast in Light Microscopy

• 2.3 Imaging Cells in Three Dimensions

• 2.4 Probing Cell Structure: Electron Microscopy

Microbial Size

http://learn.genetics.utah.edu/content/begin/cells/scale/




Microscopy for Different Size Scales

• Different microscopes are required to resolve various cells and subcellular structures

2.1 Discovering Cell Structure: Light Microscopy

• Compound light microscope uses visible light to illuminate cells

• Many different types of light microscopy:

– Bright-field

– Phase-contrast

– Dark-field

– Fluorescence

Animation: Light Microscopy

light_microscopy.html

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• Bright-field scope– Specimens are visualized

because of differences in contrast (density) between specimen and surroundings

• Two sets of lenses form the image – Objective lens and ocular lens– Total magnification = objective

magnification ocular magnification

– Maximum magnification is ~2,000

Ocularlenses

Objective lens

Stage

Condenser

Focusing knobs

Light source

Specimen onglass slide

Visualized

image

Eye

Ocular lens

Intermediate image

(inverted from that

of the specimen)

Objective lens

Specimen

Condenser lens

Light source

None

100, 400,

1000

10

10, 40, or

100 (oil)

Magnification Light path


• Resolution: the ability to distinguish two adjacent objects as separate and distinct

– Resolution is determined by the wavelength of light used and numerical aperture of lens

– Limit of resolution for light microscope is about 0.2 m

2.2 Improving Contrast in Light Microscopy

• Improving contrast results in a better final image

• Staining improves contrast

– Dyes are organic compounds that bind to specific cellular materials

– Examples of common stains are methylene blue, safranin, and crystal violet

Animation: Staining

Animation: Microscopy & Staining Overview

staining.html

staining.html

microscopy_staining.html

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I. Preparing a smear

II. Heat fixing and staining

III. Microscopy

Spread culture in thin

film over slide

Pass slide through

flame to heat fix

Dry in air

Flood slide with stain;

rinse and dry

Place drop of oil on slide;

examine with 100

objective lens

Slide Oil

2.2 Improving Contrast in Light Microscopy

• Differential stains: the Gram stain

• Differential stains separate bacteria into groups

• The Gram stain is widely used in microbiology

– Bacteria can be divided into two major groups: Gram-positive and Gram-negative

– Gram-positive bacteria appear purple and Gram-negative bacteria appear red after staining

Step 1

Result:

All cells purple

Result:

All cells

remain purple

Result:

Gram-positive

cells are purple;

gram-negative

cells are colorless

Result:

Gram-positive

(G+) cells are purple;

gram-negative (G-) cells

are pink to red

Flood the heat-fixed

smear with crystal

violet for 1 min

Add iodine solution

for 1 min

Decolorize with

alcohol briefly

— about 20 sec

Counterstain with

safranin for 1–2 min

G+

G-

Step 2

Step 3

Step 4

2.2 Improving Contrast in Light Microscopy• Phase-Contrast Microscopy

– Phase ring amplifies differences in the refractive index of cell and surroundings

– Improves the contrast of a sample without the use of a stain– Allows for the visualization of live samples– Resulting image is dark cells on a light background

• Dark-Field Microscopy– Light reaches the specimen from the sides– Light reaching the lens has been scattered by specimen– Image appears light on a dark background – Excellent for observing motility

2.2 Improving Contrast in Light Microscopy• Fluorescence Microscopy

– Used to visualize specimens that fluoresce• Emit light of one color when illuminated with

another color of light

– Cells fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye like DAPI

– Widely used in microbial ecology for enumerating bacteria in natural samples

bright-field fluorescence DAPI-stained

2.3 Imaging Cells in Three Dimensions

• Differential Interference Contrast (DIC) Microscopy– Uses a polarizer to create two distinct beams

of polarized light

– Gives structures such as endospores, vacuoles, and granules a three-dimensional appearance

– Structures not visible using bright-field microscopy are sometimes visible using DIC

2.3 Imaging Cells in Three Dimensions• Confocal Scanning Laser Microscopy (CSLM)

– Uses a computerized microscope coupled with a laser source to generate a three-dimensional image

– Computer can focus the laser on single layers of the specimen

– Different layers can then be compiled for a three-dimensional image

– Resolution is 0.1 m for CSLM

2.4 Electron Microscopy• Electron microscopes use electrons instead

of photons to image cells and structures

• Two types of electron microscopes:

– Transmission electron microscopes (TEM)

– Scanning electron microscopes (SEM)

Electronsource

Evacuatedchamber

Sampleport

Viewingscreen

Cytoplasmic membrane

DNA(nucleoid)

Septum Cell wall

2.4 Electron Microscopy

• Transmission Electron Microscopy (TEM)– Electromagnets function as lenses– System operates in a vacuum– High magnification and resolution

(0.2 nm)– Enables visualization of structures at

the molecular level– Specimen must be very thin (20–60 nm) and

stained

Animation: Electron Microscopy

electron_microscopy.html

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2.4 Electron Microscopy

• Scanning Electron Microscopy (SEM)

– Specimen is coated with a thin film of heavy metal (e.g., gold)

– An electron beam scans the object

– Scattered electrons are collected by a detector and an image is produced

– Even very large specimens can be observed

– Magnification range of 15–100,000

II. Cells of Bacteria and Archaea

• 2.5 Cell Morphology

• 2.6 Cell Size and the Significance of Being Small

2.5 Cell Morphology

• Morphology = cell shape

• Major cell morphologies

– Coccus (pl. cocci): spherical or ovoid

– Rod: cylindrical shape

– Spirillum: spiral shape

• Cells with unusual shapes

– Spirochetes, appendaged bacteria, and filamentous bacteria

• Many variations on basic morphological types

Coccus

Rod

Spirillum

Spirochete

Stalk Hypha

Budding and

appendaged bacteria

Filamentous bacteria

2.5 Cell Morphology

• Morphology typically does not predict physiology, ecology, phylogeny, etc. of a prokaryotic cell

• Selective forces may be involved in setting the morphology

– Optimization for nutrient uptake (small cells and those with high surface-to-volume ratio)

– Swimming motility in viscous environments or near surfaces (helical or spiral-shaped cells)

– Gliding motility (filamentous bacteria)

2.6 Cell Size and the Significance of Being Small

• Size range for prokaryotes: 0.2 µm to >700 µm in diameter– Most cultured rod-shaped

bacteria are between 0.5 and 4.0 µm wide and <15 µm long

– Examples of very large prokaryotes• Epulopiscium fishelsoni

• Thiomargarita namibiensis

• Size range for eukaryotic cells: 10 to >200 µm in diameter

r = 1 m

r = 2 m

r = 1 m

r = 2 m

Surface area (4r2) = 12.6 m2

Volume ( r3) = 4.2 m3

Surface area = 50.3 m2

Volume = 33.5 m3

34

SurfaceVolume

= 3

SurfaceVolume

= 1.5


• Surface-to-Volume Ratios, Growth Rates, and Evolution – Advantages to being

small • Small cells have more

surface area relative to cell volume than large cells (i.e., higher S/V)

– Support greater nutrient exchange per unit cell volume

– Tend to grow faster than larger cells


• Lower Limits of Cell Size– Cellular organisms < 0.15 µm in diameter are

unlikely– Open oceans tend to contain small cells (0.2–0.4

µm in diameter)

II. The Cytoplasmic Membrane and Transport

• 2.7 Membrane Structure

• 2.8 Membrane Functions

• 2.9 Nutrient Transport

2.7 Membrane Structure

• Cytoplasmic membrane:

– Thin structure that surrounds the cell

– 6–8 nm thick

– Vital barrier that separates cytoplasm from environment

– Highly selective permeable barrier

• Allows concentration of specific metabolites

• Excretion of waste products

• Composition of Membranes– General structure is phospholipid bilayer

• Contain both hydrophobic and hydrophilic components

– Can exist in many different chemical forms as a result of variation in the groups attached to the glycerol backbone

– Hydrophobic fatty acids point inward

– Hydrophilic portions remain exposed to external environment or the cytoplasm


Animation: Membrane Structure

membrane_structure.html

membrane_structure.html

Fatty acids

Glycerol

Phosphate

Ethanolamine

Fatty acids

Glycerophosphates

Hydrophilicregion

region

region

Hydrophobic

Hydrophilic

Fatty acids

Phospholipids

6–8 nm

Integral

membrane

proteins

Hydrophilic

groups

Hydrophobic

groups

Phospholipid

molecule

Out

In

2.7 Membrane Structure• Cytoplasmic Membrane

– Embedded proteins– Stabilized by hydrogen bonds and hydrophobic interactions– Mg2+ and Ca2+ help stabilize membrane by forming ionic

bonds with negative charges on the phospholipids– Somewhat fluid


• Membrane Proteins

– Outer surface of cytoplasmic membrane can interact with a variety of proteins that bind substrates or process large molecules for transport

– Inner surface of cytoplasmic membrane interacts with proteins involved in energy-yielding reactions and other important cellular functions

– Integral membrane proteins

• Firmly embedded in the membrane

– Peripheral membrane proteins

• One portion anchored in the membrane

EsterEther

ArchaeaBacteriaEukarya

2.7 Membrane Structure • Archaeal Membranes

– Ether linkages in phospholipids of Archaea

– Bacteria and Eukarya that have ester linkages in phospholipids

– Archaeal lipids lack fatty acids, have isoprenes instead

– Major lipids are glycerol diethers and tetraethers

– Can exist as lipid monolayers, bilayers, or mixture

isoprene

Glycerol diether

CH3 groups

Isoprene unit

Biphytanyl

Diglycerol tetraethers

Crenarchaeol

Out Out

In In

Glycerophosphates

Phytanyl

Membrane protein

Biphytanyl orcrenarchaeol

2.8 Membrane Function

• Permeability Barrier

– Polar and charged molecules must be transported

– Transport proteins accumulate solutes against the concentration gradient

• Protein Anchor

– Holds transport proteins in place

• Energy Conservation

Permeability barrier:Prevents leakage and functionsas a gateway for transport ofnutrients into, and wastes outof, the cell

Protein anchor:Site of many proteins thatparticipate in transport, bioenergetics, and chemotaxis

Energy conservation:Site of generation and use of theproton motive force

2.9 Nutrient Transport

• Carrier-Mediated Transport Systems – Show saturation effect – Highly specific– Highly regulated

Rate

of

so

lute

en

try

Transporter saturated

with substrate

Transport

Simple diffusion

External concentration of solute

Out In

Transportedsubstance

1

2

3


• Transport systems in prokaryotes– Simple transport• Driven by the energy in the proton

motive force

– Group translocation• Chemical modification of the

transported substance driven by phosphoenolpyruvate

– ABC system• Periplasmic binding proteins are

involved and energy comes from ATP

• All require energy in some form, usually proton motive force or ATP


• Three transport events are possible: uniport, symport, and antiport– Uniporters transport in one direction across the

membrane

– Symporters function as co-transporters

– Antiporters transport a molecule across the membrane while simultaneously transporting another molecule in the opposite direction


• Simple Transport:

– Lac Permease of Escherichia coli

• Lactose is transported into E. coli by the simple transporter lac permease, a symporter

• Activity of lac permease is energy driven

• Other symporters, uniporters, and antiporters

Out

In

Sulfatesymporter

Potassiumuniporter

Phosphatesymporter

Sodium-protonantiporter

Lac permease(a symporter)

PE

Direction of P transfer

Enz HPr

Nonspecific components

Enz

IIa

Enz

IIb

Specific components

Cytoplasmic

membrane

Enz

IIC

Out

In

Glucose 6–P

Directionof glucosetransport

Glucose

3.5 Transport and Transport Systems• The Phosphotransferase System in E. coli

– Type of group translocation: substance transported is chemically modified during transport across the membrane

– Best-studied system

– Moves glucose, fructose, and mannose

– Five proteins required

– Energy derived from phosphoenolpyruvate

3.5 Transport and Transport Proteins

• ABC (ATP-Binding Cassette) Systems

– >200 different systems identified in prokaryotes

– Often involved in uptake of organic compounds (e.g., sugars, amino acids), inorganic nutrients (e.g., sulfate, phosphate), and trace metals

– Typically display high substrate specificity

– Contain periplasmicbinding proteins

IV. Cell Walls of Bacteria and Archaea

• 2.10 Peptidoglycan

• 2.11 LPS: The Outer Membrane

• 2.12 Archaeal Cell Walls

2.10 Peptidoglycan

Peptidoglycan

– Rigid layer that provides strength to cell wall

– Polysaccharide composed of

• N-acetylglucosamine and N-acetylmuramic acid

• Amino acids

• Lysine or diaminopimelic acid (DAP)

• Cross-linked differently in gram-negative bacteria and gram-positive bacteria

N-Acetylglucosamine N-Acetylmuramic acid

N-Acetyl

group

Lysozyme-

sensitive

bondPeptide

cross-links

L-Alanine

D-Glutamic acid

Diaminopimelic

acid

D-Alanine

Glyc

an

te

tra

pe

ptid

e

Polysaccharide

backbone

Peptides

Escherichia coli(gram-negative)

Staphylococcus aureus(gram-positive)

Interbridge

Pe

pti

de

bo

nd

s

Glycosidic bondsX

Y

2.10 Peptidoglycan

• Gram-Positive Cell Walls

– Can contain up to 90% peptidoglycan

– Common to have teichoic acids (acidic substances) embedded in the cell wall

• Lipoteichoic acids: teichoic acids covalently bound to membrane lipids

Peptidoglycancable

Teichoic acid Peptidoglycan Lipoteichoicacid

Cytoplasmic membrane

Wall-associatedprotein

2.11 LPS: The Outer Membrane• Gram-Negative Cell Walls

– Total cell wall contains ~10% peptidoglycan

– Most of cell wall composed of outer membrane (aka lipopolysaccharide [LPS] layer)• LPS consists of core polysaccharide and

O-polysaccharide

• LPS replaces most of phospholipids in outer half of outer membrane

• Endotoxin: the toxic component of LPS

O-specificpolysaccharide

Core polysaccharide

Lipid A Protein

Lipopolysaccharide (LPS)

Phospholipid

Lipoprotein

8 nm

Out

Cell

wall

Outer

membrane

Periplasm

Cytoplasmic

membrane

In

Porin

Peptidoglycan

2.11 LPS: The Outer Membrane

• Porins: channels for movement of hydrophilic low-molecular weight substances

• Periplasm: space located between cytoplasmic and outer membranes

– ~15 nm wide

– Contents have gel-like consistency

– Houses many proteins

Periplasm

Cytoplasmic

membrane

Outer membrane

2.12 Archeal Cell Walls

• No peptidoglycan

• Typically no outer membrane

• Pseudomurein

– Polysaccharide similar to peptidoglycan

– Composed of N-acetylglucosamine and N-acetyltalosaminuronic acid

– Found in cell walls of certain methanogenicArchaea

• Cell walls of some Archaea lack pseudomurein

Lysozyme-insensitive

N-Acetylglucosamine

N-Acetyltalosaminuronicacid

N-Acetylgroup

Peptidecross-links

2.12 Archaeal Cell Walls

• S-Layers

– Most common cell wall type among Archaea

– Consist of protein or glycoprotein

– Paracrystallinestructure

V. Other Cell Surface Structures and Inclusions

• 2.13 Cell Surface Structures

• 2.14 Cell Inclusions

• 2.15 Gas Vesicles

• 2.16 Endospores

2.13 Cell Surface Structures

• Capsules and Slime Layers

– Polysaccharide layers

• May be thick or thin, rigid or flexible

– Assist in attachment to surfaces

– Protect against phagocytosis

– Resist desiccation


• Fimbriae

– Filamentous protein structures

– Enable organisms to stick to surfaces or form pellicles

Flagella

Fimbriae


• Pili– Filamentous protein structures

– Typically longer than fimbriae

– Assist in surface attachment

– Facilitate genetic exchange between cells (conjugation)

– Type IV pili involved in twitching motility

Virus-coveredpilus

-carbon

Polyhydroxyalkanoate

2.14 Cell Inclusions

• Carbon storage polymers– Poly--hydroxybutyric acid

(PHB): lipid – Glycogen: glucose

polymer

• Polyphosphates: accumulations of inorganic phosphate

• Sulfur globules: composed of elemental sulfur

• Magnetosomes: magnetic storage inclusions

Sulfur

Vegetative cell

Developing spore

Sporulating cell

Mature spore

2.16 Endospores• Endospores

– Highly differentiated cells resistant to heat, harsh chemicals, and radiation

– “Dormant” stage of bacterial life cycle

– Ideal for dispersal via wind, water, or animal gut

– Only present in some gram-positive bacteria

VI. Microbial Locomotion

• 2.17 Flagella and Motility

• 2.18 Gliding Motility

• 2.19 Microbial Taxes

2.17 Flagella and Swimming Motility

• Flagellum (pl. flagella): structure that assists in swimming

– Different arrangements: peritrichous, polar, lophotrichous

– Helical in shape

Animation: The Prokaryotic Flagellum

prokaryotic.html

prokaryotic.html

Rod

C Ring

MS Ring

Motprotein

Mot proteinMot protein Fli proteins(motor switch)

45 nm

Cytoplasmicmembrane

Basalbody

Rod

C Ring

MS Ring

P Ring

Periplasm Peptidoglycan

L Ring

HookOutermembrane(LPS)

Flagellin

Filament

MS

L

P

15—20 nm


• Flagellar structure

– Consists of several components

– Filament composed of flagellin

– Move by rotation

Polar

Peritrichous

CW rotation

CCW rotation

Unidirectional flagella

Cellstops,reorients

Reversible flagella

Bundledflagella(CCW rotation)

Tumble—flagellapushed apart(CW rotation)

Flagella bundled(CCW rotation)

CW rotation

CW rotation


• Flagella increase or decrease rotational speed in relation to strength of the proton motive force

• Differences in swimming motions– Peritrichously

flagellated cells move slowly in a straight line

– Polarly flagellated cells move more rapidly and typically spin around

No attractant present:Random movement

Attractant present:Directed movement

Tumble

Run

Tumble

Run

Attractant

2.19 Chemotaxis and Other Taxes• Taxis: directed movement in response to

chemical or physical gradients– Chemotaxis: response to chemicals

• Best studied in E. coli

• Bacteria respond to temporal, not spatial, difference in chemical concentration

• “Run and tumble” behavior

• Attractants sensed by chemoreceptors

2.19 Chemotaxis and Other Taxes

• Measuring chemotaxis– Measured by inserting a capillary tube containing

an attractant or a repellent in a medium of motile bacteria

– Can also be seen under a microscope

Time

Repellent

Control

Attractant

Repellent

Control Attractant

Cells p

er

tub

e