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

2.1 Discovering Cell Structure: Light Microscopy

Bright-field scope
Specimens are visualized
because of differences in
contrast (density) between
specimen and surroundings

Ocular
lenses

Two sets of lenses form the

image
Objective lens and ocular lens
Total magnification = objective
magnification ocular
magnification
Maximum magnification is
~2,000

Objective lens
Stage
Condenser
Focusing knobs
Light source

Specimen on
glass slide

Magnification Light path

100, 400,
1000

10

Visualized
image
Eye

Ocular lens
Intermediate image
(inverted from that
of the specimen)

10, 40, or
100 (oil)

Objective lens
Specimen

None

Condenser lens

Light source

2.1 Discovering Cell Structure: Light Microscopy

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: Microscopy & Staining Overview

Animation: Staining

I. Preparing a smear

Spread culture in thin

film over slide

Dry in air

II. Heat fixing and staining

Pass slide through

flame to heat fix

Flood slide with stain;

rinse and dry

III. Microscopy

Slide

Oil

Place drop of oil on slide;

examine with 100
objective lens

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 Gramnegative bacteria appear red after staining

Step 1

Flood the heat-fixed

smear with crystal
violet for 1 min

Result:
All cells purple

Step 2

Add iodine solution

for 1 min

Result:
All cells
remain purple

Step 3

Decolorize with
alcohol briefly
about 20 sec

Result:
Gram-positive
cells are purple;
gram-negative
cells are colorless

Step 4

G-

Result:
Gram-positive
(G+) cells are purple;
gram-negative (G-) cells
are pink to red

Counterstain with
safranin for 12 min
G+

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 threedimensional 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)

Electron
source

Evacuated
chamber
Sample
port

Viewing
screen

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 (2060 nm) and
stained
Cytoplasmic
membrane

Septum Cell wall

DNA
(nucleoid)

Animation: Electron Microscopy

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
15100,000

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

Spirochete

Stalk

Rod

Hypha

Budding and
appendaged bacteria

Spirillum
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 m to >700 m
in diameter
Most cultured rod-shaped
bacteria are between 0.5 and
4.0 m wide and <15 m long
Examples of very large
prokaryotes
Epulopiscium fishelsoni
Thiomargarita namibiensis

Size range for eukaryotic cells:

10 to >200 m in diameter

2.6 Cell Size and the Significance of Being Small

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

r = 1 m
r = 1 m

Surface area (4r2) = 12.6 m2

Volume (43 r3) = 4.2 m3

Surface
=3
Volume
r = 2 m

r = 2 m
Surface area = 50.3 m2
Volume = 33.5 m3

Surface
= 1.5
Volume

2.6 Cell Size and the Significance of Being Small

Lower Limits of Cell Size
Cellular organisms < 0.15 m in diameter are
unlikely
Open oceans tend to contain small cells (0.20.4
m 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
68 nm thick
Vital barrier that separates cytoplasm from
environment
Highly selective permeable barrier
Allows concentration of specific metabolites
Excretion of waste products

2.7 Membrane Structure

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

Glycerol

Fatty acids
Phosphate
Ethanolamine

Hydrophilic
region
Fatty acids

Hydrophobic
region
Hydrophilic
region
Glycerophosphates

Fatty acids

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
Out
Phospholipids
Hydrophilic
groups
68 nm
Hydrophobic
groups

In

Integral
membrane
proteins

Phospholipid
molecule

2.7 Membrane Structure

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

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
Ester

Ether
isoprene

Bacteria
Eukarya

Archaea

Glycerol diether

CH3 groups
Isoprene unit
Biphytanyl

Diglycerol tetraethers

Crenarchaeol

Out

Out

Glycerophosphates
Phytanyl

Biphytanyl or
crenarchaeol

Membrane protein

In

In

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:

Protein anchor:

Energy conservation:

Prevents leakage and functions

as a gateway for transport of
nutrients into, and wastes out
of, the cell

Site of many proteins that

participate in transport,
bioenergetics, and chemotaxis

Site of generation and use of the

proton motive force

2.9 Nutrient Transport

Carrier-Mediated Transport Systems

Rate of solute entry

Show saturation effect

Highly specific
Highly regulated

Transporter saturated
with substrate

Transport

Simple diffusion

External concentration of solute

2.9 Nutrient Transport

Transport systems in
prokaryotes

In

Out

Simple transport

Driven by the energy in the proton

motive force

Group translocation

Transported
substance

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

1
2
3

2.9 Nutrient Transport

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

2.9 Nutrient Transport

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
Sulfate
symporter

Potassium
uniporter

Phosphate
symporter

Sodium-proton
antiporter

Lac permease
(a symporter)

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
Glucose
Energy derived from
Out
phosphoenolpyruvate
Cytoplasmic
membrane
Nonspecific components

Specific components

Enz
IIC

PE

Enz

HPr

Enz
IIa

Direction of P transfer

Enz
IIb

In
Glucose 6P

Direction
of glucose
transport

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 periplasmic
binding 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 Nacetylmuramic acid
Amino acids
Lysine or diaminopimelic acid
(DAP)
Cross-linked differently in gramnegative bacteria and grampositive bacteria

N-Acetylmuramic acid

N-Acetyl
group

Peptide
cross-links

Lysozymesensitive
bond
L-Alanine
D-Glutamic acid
Diaminopimelic
acid
D-Alanine

Glycan tetrapeptide

N-Acetylglucosamine

Polysaccharide
backbone
Interbridge
Peptides

Escherichia coli
(gram-negative)

Staphylococcus aureus
(gram-positive)

Peptide bonds

X
Glycosidic bonds

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
Wall-associated Teichoic acid
Peptidoglycan Lipoteichoic
to membrane lipids protein
acid

Peptidoglycan
cable

Cytoplasmic membrane

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-specific
polysaccharide

Core polysaccharide
Lipid A

Protein

Out

Lipopolysaccharide
(LPS)
Porin
Outer
membrane

8 nm

Cell
wall
Phospholipid
Periplasm

Peptidoglycan
Lipoprotein

Cytoplasmic
membrane

In

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 Nacetyltalosaminuronic acid
Found in cell walls of certain methanogenic
Archaea

Cell walls of some Archaea lack pseudomurein

N-Acetyltalosaminuronic
acid
Lysozyme-insensitive

N-Acetylglucosamine

Peptide
cross-links

N-Acetyl

group

2.12 Archaeal Cell Walls

S-Layers
Most common cell
wall type among
Archaea
Consist of protein or
glycoprotein
Paracrystalline
structure

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

2.13 Cell Surface Structures

Fimbriae
Filamentous protein structures
Enable organisms to stick to surfaces or form
pellicles

Flagella

Fimbriae

2.13 Cell Surface Structures

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
Viruscovered
pilus

2.14 Cell Inclusions

Carbon storage polymers

-carbon

Poly--hydroxybutyric acid
(PHB): lipid
Glycogen: glucose
polymer

Polyphosphates:
accumulations of
inorganic phosphate
Sulfur globules:
composed of elemental
sulfur
Magnetosomes: magnetic
storage inclusions

Polyhydroxyalkanoate

Sulfur

2.16 Endospores

Endospores

Highly differentiated cells resistant

heat, harsh chemicals, and radiation
Vegetative cell
Dormant stage of
bacterial life cycle
Ideal for dispersal
Developing spore
via wind, water,
or animal gut
Only present
Sporulating cell
in some
gram-positive
bacteria
Mature spore

to

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

2.17 Flagella and Swimming Motility

1520 nm

L
P

Filament
Flagellin

MS

Flagellar structure

Hook

Outer
membrane
(LPS)

L Ring
Rod

Consists of several
components
Filament composed of
flagellin
Move by rotation

P Ring

Periplasm

Peptidoglycan
Rod
MS Ring
MS Ring

Basal
body
C Ring
Cytoplasmic Mot protein Fli proteins Mot protein
(motor switch)
membrane
45 nm

C Ring

Mot
protein

2.17 Flagella and Swimming Motility

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

Tumbleflagella
pushed apart
(CW rotation)
Bundled
flagella
(CCW rotation)

Flagella bundled
(CCW rotation)

Peritrichous

Reversible flagella

CCW rotation

CW rotation

Unidirectional flagella

CW rotation

Polar

Cell
stops,
reorients

CW rotation

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
Tumble
Tumble
Run

Run

No attractant present:
Random movement

Attractant present:
Directed movement

Attractant

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

Cells per tube

Control
Attractant
Repellent

Attractant

Control
Repellent
Time