Microbial Cell Structure and
Function
(Chapter 2: pp. 75-87)
22 February 2022
mby_251_basic_principles_of_microbiology | up | BGM | ThabisoMotaung
In today’s lecture
i.
The Cell Membrane and Wall
ii.
Cell surface structures
iii.
Inclusion bodies
iv.
Microbial locomotion
Learning outcomes
I.
The Cell Membrane and Wall
i.
Major functions of the cytoplasmic membrane and cell wall.
ii.
Structural differences between GPB and GNB cytoplasmic membranes and cell walls.
iii. Major chemical components in the outer membrane of GNB.
iv. Functions of porins in a gram-negative cell wall.
v. Most common type and functions of cell walls in Archaea.
Learning outcomes
I.
Cell surface structures
i.
Attachment structures, genetic exchange, and twitching motility.
ii.
Structurally and functionally differentiate fimbriae from pili.
iii. Functions of hami.
II.
Inclusion bodies
i.
Organic and inorganic cell inclusions and their functions
ii.
Formation of magnetosomes
iii. Importance of gas vesicles to photosynthetic prokaryotes
iv. Endospore formation stages, endospore structural characteristics, dehydration
mechanisms, and the importance of dehydration
III.
Microbial locomotion
i.
Major types of prokaryotic cell movement
Learning outcomes
Check Your Understanding
• Use MINIQUIZZes to test yourself.
• Very important to enhance your understanding of
the concepts.
I. The Cell Membrane and Wall
Page 75-85
Cell structure and functions

The cytoplasmic membrane (CM)
•
Selective permeable boundary between cytoplasm and immediate surrounding.
•
Small ions cannot freely pass due to their charges.
•
If compromised cell contents leak out and cell dies.
•
Does not confer specific shape or rigid support to the cell.
•
•
These are roles of the cell wall.
Due to fluid nature membrane:
•
Allows for lateral movement of phospholipids and proteins.
•
Also serves as gateway for transport of nutrients into & waste out of the cell.
•
Can be energized by separating protons to the outside of the membrane from hydroxyl ions
on the inside – proton motive force (PMF).
•
ATP (ATP synthase: ADP + Pi) (Chapter 3).
Major functions of the cytoplasmic membrane are summarized in Figure 2.4.
Cell structure and functions
 CM of bacteria
•
Phospholipid bilayer:
—
Hydrophobic –fatty acids (linked to glycerol)
— Hydrophilic –glycerol with phosphate and a functional
group- ethanolamine
•
Phospholipid bilayer contains proteins:
— Integral proteins – embedded in the membrane
—
Peripheral proteins – loosely attached
—
Hydrophobic domains span the membrane
— Hydrophilic domains contact environment or cytoplasm
Fig. 2.1. Phospholipid bilayer
membrane.
Cell structure and functions
Phytanyl
 CM of Archaea
•
Basic structural properties same as bacteria.
•
Archaea contain ether linkages between the
glycerol and a hydrophobic side chain.
•
Membranes composed of carbon isoprene
units that combine to form phytanyl side
chains.
•
Together, the glycerol and phytanyl form a
glycerol diether (archaeol).
If archaeols are not joint at
hydrophobic ends, a lipid bilayer is
formed.
Fig. 2.3. Major lipids of Archaea
and the architecture of archaeal
membranes.
Cell structure and functions
Biphytanyl
 CM of Archaea
•
Basic structural properties same as bacteria.
•
Archaea contain ether linkages between the
glycerol and a hydrophobic side chain.
•
Membranes composed of carbon isoprene
units that combine to form phytanyl side
chains.
•
Together, the glycerol and phytanyl form a
glycerol diether (archaeol).
If archaeols are joint at
hydrophobic ends, a lipid
monolayer is formed.
Fig. 2.3. Major lipids of Archaea
and the architecture of archaeal
membranes.
Cell structure and functions
Crenarchaeol
 CM of Archaea
•
Basic structural properties same as bacteria.
•
Archaea contain ether linkages between the
glycerol and a hydrophobic side chain.
•
Membranes composed of carbon isoprene
units that combine to form phytanyl side
chains.
•
Together, the glycerol and phytanyl form a
glycerol diether (archaeol).
A lipid monolayer is
composed of 5- and 6-carbon
rings.
Fig. 2.3. Major lipids of Archaea
and the architecture of archaeal
membranes.
Transporting Nutrients into the Cell
 Cytoplasmic membrane is an effective barrier to leakage.
 Cells need to import nutrients and export waste products.
•
To fuel metabolism and support growth.
•
Active transport:
•
Solutes accumulate against the concentration gradient.
―
Simple transport
―
Group translocation
― ABC transport systems
Transporting Nutrients into the Cell
 Simple transport
— Driven by the energy in the proton motive
force.
— Symport reactions
— Antiport reactions
Fig. 2.6. Classes of transport systems.
Transporting Nutrients into the Cell
 ABC transporters:
(Figure 2.6; pp 78;
https://www.youtube.com/watch?v=LwSKgrdomPM)
•
Three components:
1. Periplasmic substrate-binding proteins
— Extremely high substrate affinity.
2. Transmembrane protein channel.
3. ATP-hydrolyzing protein.
— Supplies the energy required to drive substrate
transport.
Fig. 2.6. Classes of transport systems.
Transporting Nutrients into the Cell
 Group translocation
•
Substrate transported is modified.
•
Glucose group translocation
—
•
Phosphorylation from phosphoenolpyruvate (PEP).
Phosphotransferase system
(Figure 2.6; pp 78, https://www.youtube.com/watch?v=653U2JW2TRw)
•
•
HPr and Enzyme I: non-specific cytoplasmic proteins
•
Enzyme II: specific for each sugar
—
Enz IIa: cytoplasmic
—
Enz IIb: lies on inner surface of membrane
—
Enz IIc: integral
Energy-rich organic compound drives the transport event.
•
Phosphoenolpyruvate
Fig. 2.6. Classes of
transport systems.
Cell structure and functions
 The bacterial cell wall:
• Provides overall strength to the cell.
• Also helps maintain the cell shape and rigidity.
• Protect cells against osmotic lysis.
— Certain antibiotics target bacterial cell wall biosynthesis.
— This leaves the cell susceptible to osmotic lysis.
— Humans lack cell walls and are thus not affected by antibiotics.
Cell structure and functions

The bacterial cell wall contains peptidoglycan:
•
Divides bacteria into gram negatives (GNB) and gram positives (GPB)
(not present in Archaea).
•
Sensitive to lysozyme due to β(1,4).
—
An enzyme that cleaves the glycosidic bond.
—
Present in human secretions.
—
Function a major line of defense.
GPB cell wall (purple)
GNB cell wall (colorless/pink)
90% Peptidoglycan
10% Peptidoglycan
Figure 2.7. Cell envelopes of bacteria.
Cell structure and functions
 The bacterial peptidoglycan
•
Composed of glycan tetrapeptide
— Two amino sugars, N-acetylglucosamine (G)
and N-acetylmuramic acid (M).
— A tetrapeptide (L-ala-D-glu-DAP-D-al) attached
to M.
— In GNB amino acids are present as cross-
linkers.
Figure 2.8. Structure of the repeating
unit in peptidoglycan, the glycan
tetrapeptide.
Cell structure and functions
 The bacterial peptidoglycan
•
Composed of glycan tetrapeptide
•
Two amino sugars, N-acetylglucosamine (G)
and N-acetylmuramic acid (M).
•
A tetrapeptide (L-ala-D-glu-DAP-D-al) attached
to M.
•
In GNB amino acids are present as cross-
linkers.
•
In GPB crosslinks contain interbridge.
•
E.g., glycine interbridge.
Figure 2.9. Peptidoglycan structure in
the cell wall.
Cell structure and functions
 The bacterial peptidoglycan
•
Archaea lack peptidoglycan.
•
Lack a polysaccharide-containing cell wall.
•
•
Instead have an S-layer.
•
S-layer prevents osmotic lysis.
Certain methane-producing Archaea:
•
Contain a polysaccharide called pseudomurein
•
N-acetylglucosamine and N-
acetyltalosaminuronic acid (replaces the Nacetylmuramic acid of peptidoglycan).
•
Glycosidic bonds between the sugar derivatives
are β-1,3.
•
Amino acids are all of the L stereoisomer.
Figure 2.11. Pseudomurein.
Cell structure and functions
GNB cell wall
 The bacterial outer membrane (OM)
•
Represents a second lipid bilayer of the cell.
•
Also contains polysaccharides linked to lipids, hence
is called lipopolysaccharide (LPS).
•
Acts as a permeability barrier.
•
•
Protects against penetration of cytoplasmic membrane.
Anchored to the peptidoglycan layer by the Braun
lipoprotein
Figure 2.7. Cell envelopes of bacteria.
Cell structure and functions
GNB cell wall
 The structure of the LPS:
•
Three covalently linked regions.
•
Lipid A–KDO–core–O-specific
Highly variable among
species
Figure 2.13. Structure of bacterial lipopolysaccharide.
Comprises the
endotoxin
complex
Figure 2.7. Cell envelopes of bacteria.
Cell structure and functions
Out
 The structure of the LPS
•
LPS
Three covalently linked regions
•
Lipid A–KDO–core–O-specific
In
Figure 2.12. The gram-negative bacterial cell
envelope.
Highly variable among
species
Figure 2.13. Structure of bacterial lipopolysaccharide.
Comprises the
endotoxin complex
(toxicity to animals)
Anchors OM to
peptidoglycan layer
Cell structure and functions
 The periplasm:
•
Exists between outer surface of CM and
Out
inner surface of OM.
LPS
•
~15 nm.
•
House cellular proteins (periplasmic
proteins) from diffusing away from the
cell.
—
Reach periplasm via CM-associated protein-
exporting system.
—
Hydrolytic enzymes, binding proteins,
chemoreceptors, and structural proteins.
In
Figure 2.12. The gram-negative bacterial cell
envelope.
Cell structure and functions
 The porins in OM
•
OM is permeable to small molecules due to porins
—
Transmembrane proteins of 3 identical polypeptides.
•
Function as entrance-exit channels of solutes.
•
Non-specific porins
—
Water-filled channels through which hydrophilic substances
can pass.
•
Specific porins
— Contain binding site for one or a group of structural related
substances.
Figure 2.12. The gramnegative bacterial cell
envelope.
IV. Cell Surface Structures
Page 87-89
Cell structure and functions
 Surface polysaccharides
•
Assist in attachment to solid surfaces.
•
Biofilm – a group of adherent cells glued
together by an extracellular matrix
EPS
containing polymeric exopolysaccharides
(EPS).
•
Scanning electron micrograph of biofilm
formation by Vibrio cholerae O1 strain TSI-4/R
Source: FEMS Microbiology Letters. doi:
10.1111/j.1574-6968.1999.tb08786.x.
EPS – extracellular macromolecules
excreted as tightly bound capsule or
loosely attached slime layer.
•
Biofilms
Major cause of device-related hospital-acquired
infections.
Cell structure and functions
 Capsules and slime layers
•
Surface polysaccharides assist in attachment to solid surfaces.
•
Also:
— Act as a virulence factor by contributing to bacterial pathogenicity.
•
•
—
Assist bacteria in evading host immune response.
—
Protect cells from desiccation (trap water).
Capsules:
Figure 2.16. Bacterial capsules and
slime formation.
—
Polysaccharide structure that covers the outer layer of the cell wall.
—
Organized in a tight matrix that excludes small particles and is tightly attached.
Slime layers (pseudo-capsule):
—
Unorganized layer of extracellular material that covers bacteria cells.
— More easily deformed and loosely attached.
Cell structure and functions
 Fimbriae and Pili
Pellicle
• Thin filamentous structures made of
proteins extending from the cell surface.
• Fimbriae:
•
Enable cells to stick to surfaces or to form
pellicles.
— Pellicles are a biofilm floating at the air-liquid
interface.
Figure 2.17. Fimbriae.
Cell structure and functions
 Fimbriae and Pili
•
Pellicle
Pili:
•
Similar to fimbriae but longer and few on the cell surface.
•
Can be receptors for certain types of bacterial viruses
(bacteriophages).
•
Facilitate genetic exchange between cells during conjugation
(sex pili).
•
Enable adhesion of pathogens to host tissues.
•
Support twitching motility (Type IV pili)/ movement along a
solid surface.
Figure 2.18. Pili.
Cell structure and functions
 Hami:
•
Pellicle
Present in uncultivated archaea SM1 Euryarchaeon
(Grappling archaea).
•
Molecular grappling hooks, which resemble type IV pili in
Bacteria.
•
Facilitate the formation of a dense biofilm network that may
help trap nutrients in their deep subsurface habitat.
Archaeal cocci of the SM1 biofilm with numerous hami
Source: Front. Microbiol. https://doi.org/10.3389/fmicb.2014.00397
Hami attaching to a filamentous bacterium
Source: Front. Microbiol.
https://doi.org/10.3389/fmicb.2014.00397
V. Inclusion Bodies
Page 89-95
Cell inclusions and functions
 Cell inclusions:
•
Function as energy reserves and or carbon reservoirs or have
special functions.
•
Synthesized by cells when there is an excess of organic and
inorganic compounds.
•
Enclosed in a thin membrane and partitioned in a cytoplasm.
•
Carbon Storage Polymers
•
Poly-b-hydroxybutyric acid (PhB) and glycogen
Cell inclusions and functions
 Cell inclusions:
• Inorganic cell inclusions:
•
•
Polyphosphate, Sulfur, and Carbonate Minerals.
Magnetic storage inclusions (Magnetosomes):
•
Comprise magnetic nanoparticles surrounded by lipid bilayer membrane.
•
Produced by magnetotactic bacteria,
— Ubiquitously present aquatic sediments (low in O2 levels).
•
Small motile compass needles:
•
Passively align and swim along the Earth's magnetic field lines
•
And into the sediments where O2 levels are low.
•
Considered a mechanism of motility.
Cell inclusions and functions
 Cell inclusions:
• Gas vesicles
•
Conical-shaped structures made of a protein shell only
permeable to gases.
•
Mostly found in planktonic (free-living) bacteria and archaea.
•
Provide buoyancy to planktonic cells by decreasing their overall
cell density.
•
Considered a mechanism of motility.
Cell inclusions and functions
 Endospores:
• Highly differentiated and stress tolerant cells.
• Function as survival structures and a dormant stage of
bacterial life cycle.
• Easily dispersed (e.g., wind, air, etc.).
•
Wide distribution of endospore-forming bacteria.
Cell inclusions and functions
 Endospores: Formation and Germination
•
Lack of nutrients
Activation
•
Germination
•
Outgrowth
Asymmetric division
(Forespore)
Endospore can remain
dormant for long periods of
time until conditions are
favorable.
Peptidoglycan dissolved
(Forespore engulfment)
Cortex formation & coat
deposition
Dehydration and maturation
(programmed cell death and
endospore release)
Cell inclusions and functions
 Endospores: Structure and Features
• Several layers which make it resistant
to harsh conditions.
•
Outermost – exosporium
•
Second layer – spore coat
•
Third layer – cortex
•
Inside the cortex – core
Cell inclusions and functions
 Endospores: Structure and Features
•
Differs structurally from vegetative cells.
•
Dipicolonic acid accumulates in the core.
•
Also contain large amounts of Ca2+
— Complexed with dipicolinic acid
•
Calcium–dipicolinic acid (DPA) complex:
— Bind free water within the endospore.
— Helps dehydrate developing endospore.
— Helps stabilize DNA against heat denaturation.
Cell inclusions and functions
 Endospores: Structure and Features
• Contain small acid-soluble spore proteins (SASPs).
— Only made during the sporulation process.
— Protec DNA from UV radiation, desiccation, and dry heat.
— Function as a carbon and energy source for the outgrowth of a new
vegetative cell.
VI. Microbial locomotion
Page 94-99
Cell locomotion
 Flagella and flagellation:
•
Long and thin appendages ~15–20 nm wide.
•
Arranged in a variety of ways on the cell surface.
•
Rotate to propel the cell.
•
Direction of the rotation.
•
•
Cell moves forward, backward, or tumbles in place.
Requires significant energy directly from a proton
motive force.
Peritrichous
Polar
Lophotrichous
Figure 2.31. Bacterial flagella.
Cell locomotion

Flagella and Functions (pp 93-94):
https://www.youtube.com/watch?v=B7PMf7bBczQ
•
Flagellin – principal component of bacterial flagella, forms the filament.
•
Hook – connects the filament to the flagellum motor in the base.
•
Flagellum motor – rotating machine anchored in the CM and cell wall.
•
Rod passing through a set of rings
•
L (outer) ring anchored in the OM and cell wall.
•
P ring is anchored in the peptidoglycan.
•
MS and C rings located in the CM and cytoplasm, respectively.
•
Only the inner pair of rings is present in GPB.
Figure 2.34. Structure and
function of the flagellum in
gram-negative
bacteria.
Cell locomotion
 Archaella and Functions (pp 97-99):
https://www.youtube.com/watch?v=vJpYS1XjxCk
•
Flagella and archaella are functionally similar.
•
Motors are powered in fundamentally different ways
•
Rotation of the archaellum is driven by the hydrolysis of ATP.
•
This suggests that swimming motility evolved separately in Bacteria and Archaea.
Figure 2.36. Archaella.
•
Archaellum have small diameter compared to the flagellum.
•
Several different proteins make up the filament.
•
Archaellum considered a rotating type IV pilus capable of both clockwise and
counterclockwise rotation.
•
Energy for rotation comes from ATP hydrolysis
Cell locomotion
 Surface motility:
•
Not all swimming bacteria are flagellated.
•
Ability to actively translocate on surfaces without involving external
appendages.
•
Found in environments including in biofilms, microbial mats, and soil.
•
Twitching motility:
•
Type VI pili – repeated extension and retraction.
•
Energy required comes from ATP hydrolysis.
•
Allows cells to move together in groups – facilitated by Type VI pili and EPS.
•
Figure 2.38a, pp 98.
Cell locomotion
 Gliding motility:
•
A continuous and smooth form of movement along the long axis of a
cell.
•
Helical intracellular track made of proteins.
— Run in a continuous loop around the cell.
— Associated with tracks are gliding motors driven by the proton motive force.
— Adhesion proteins that help cells grab onto surfaces.
— Cell is propelled forward over the surface.
—
Rotate around its axis as it moves forward.
— Figure 2.38b, pp 98.
Recap

The Cell Membrane and Wall

Cell surface structures

Inclusion bodies

Microbial locomotion
Homework/self study:
1) Structure of the gram-positive bacterial cell wall (pp 81).
Teichoic and lipotechoic acids in GPB cell wall structure (Figure 2.10).
2) ABC transporters and Phosphotransferase system:
https://www.youtube.com/watch?v=LwSKgrdomPM
https://www.youtube.com/watch?v=653U2JW2TRw
Figure 2.6; pp 78.
3) Flagella and Functions (pp 95-96).
https://www.youtube.com/watch?v=B7PMf7bBczQ
4) Archaella and Functions (pp 97).
https://www.youtube.com/watch?v=vJpYS1XjxCk
Microbial Growth and Control
(Chapter 4: pp. 144-180)
28 February 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Feeding the Microbe
II.
Dynamics of Microbial Growth
III.
Measuring Microbial Growth
IV.
Environmental Effects on Microbial Growth
V.
Controlling Microbial Growth
Learning outcomes

Feeding the Microbe
•


Macro and micronutrients and their importance to the cell.
Dynamics of Microbial Growth
•
Binary fission and the microbial growth cycle.
•
Different microbial growth phases.
•
Quantitative aspects of microbial growth.
•
Mathematics of bacterial growth and specific growth rate.
•
Consequences of growth.
Measuring Microbial Growth
•
Total cell count.
•
Methods for viable counts and applications.
•
Optical density and its relationship to cell numbers.
Learning outcomes


Environmental Effects on Microbial Growth
•
The impact of temperature, pH, osmolarity, and oxygen.
•
Different classes of organisms in relation to environmental effects.
•
Mechanisms of adaption to environmental effects.
Controlling Microbial Growth
•
The effect of temperature on the heat killing of microorganisms
•
Effect of antimicrobial Agents on growth
•
Different types of antimicrobial agents
•
Antimicrobial agent susceptibility assays
Learning outcomes
Check Your Understanding
• Use MINIQUIZZes to test yourself.
• Very important to enhance your understanding of
the concepts.
I. Feeding the Microbe
Page 145-146
Cell Nutrition
 All microbes require a core set of nutrients.
 Macronutrients are required in large amounts.
 Micronutrients are required in minute amounts.
 Nutrients are important to the cell’s chemical make up.
•
All living things require the same basic elements.
•
Hydrogen, carbon, nitrogen, phosphorus, sulfur and selenium.
•
Water (75% wet weight) and macromolecules.
•
Macromolecules: proteins; nucleic acids, lipids, polysaccharides.
•
Proteins and RNA are most abundant
•
DNA contributes a very small percentage of cell’s dry weight.
Feeding the Microbe: Cell Nutrition
 Macronutrients:
• Carbon:
― Main element in all classes of macromolecules.
― Organic compounds e.g. amino acids, fatty acids, organic acids, sugars, and
nitrogen bases assimilated by bacteria.
• Nitrogen:
― Typical bacterial cell is ~13% nitrogen (by dry weight)
― Key element in proteins, nucleic acids
Feeding the Microbe: Cell Nutrition
 Macronutrients
•
Other macronutrients:
― P: nucleic acids and phospholipid synthesis.
― S: amino acids (cysteine, methionine), vitamins (thiamine, biotin and co-
enzyme-A).
― K: Required for enzyme activity.
― Mg: Stabilizes membranes, ribosomes and nucleic acids. Also required for
enzyme activity.
― Ca: Stabilizes cell walls and provide heat stability to endospores.
― Na: Required for growth of some microbes.
Feeding the Microbe: Cell Nutrition
 Micronutrients
• Metals
— Fe: cellular respiration. Siderophores bind iron from minerals and
transport it into the cell.
— Trace metals: (Cr, Co, Cu, Mn, Mo, Ni, Se, W, V, Zn): components of
enzymes.
• Growth factors
— Required by some microbes in small quantities.
— Vitamins, amino acids, purines and pyrimidines.
II. Dynamics of Microbial Growth
Page 154-161
Binary Fission and Budding
 Microbial growth is an increase in the number of cells in a
population.
 Microbial growth is a result of cell division.
•
Binary fission:
—
•
Cell elongates twice the original size then separates into two cells.
Budding:
— Cell divides as a result of unequal cell growth.
—
Simple budding, budding from hyphae, division of stalked organisms,
polar growth without differentiation of cell size.
—
pp 161; Fig. 4.18.
Figure 4.8. Binary fission in a rod-shaped
bacterium.
Quantitative Aspects of Microbial Growth
 When a cell divides into two cells, it is said to have one
generation.
 The time it takes for this to happen is called the generation time.
 The total cells numbers and mass doubles per generation time.
— 1 cell  21  22  23  24  25   2n
n = number of generations

Cell numbers in a bacterial culture can quickly become very
large.

Most bacteria have shorter generation times than eukaryotic
microbes.
—
15-20 minutes in E. coli under optimum lab conditions.
Figure 4.8. Binary fission in a rod-shaped
bacterium.
Quantitative Aspects of Microbial Growth

Exponential (logarithmic) growth: cells in a bacterial
population double at a constant time interval.

Logarithmic (log10) scale as a function of time.
—
Semilogarithmic graphs allow the plotting of very small to very large cell
numbers.
—
The exponential growth phase of a culture can be visualized as a straight
line.
—
Initial number of cells in culture vs. number after exponential growth is
expressed by N = N02n, where:
N = the final cell number
N0 = the initial cell number
n = the number of generations during the period of exponential growth
Figure 4.11. The rate of growth of
a microbial culture.
Quantitative Aspects of Microbial Growth
 The equation can be expressed in terms of n:
N = N02n
log N = log N0 + n log 2
log N – log N0 = n log 2
log N – log N0 / log 2 = n
log N – log N0 / 0.301 = n
3.3 (log N – log N0) = n
Figure 4.11. The rate of growth of
a microbial culture.
Quantitative Aspects of Microbial Growth

Generation of an exponentially growing population expressed as g = t/n where
t is the duration of exponential growth
n is the number of generations during the period of exponential growth
 The generation time can thus be calculated as follows:
Consider N = 108, N0 = 5 x 107, and t = 2, Then
n = log N – log N0 / log 2
n = (8- 7.69)/ 0.301
n = 1; thus, g (generation time) = t/n = 2/1 = 2h
 Specific growth rate (k):
— Rate at which the population is growing at any instant.
— Expressed in units of reciprocal hours (h–1).
Specific growth rate (k):
k = 0.693/g.
Quantitative Aspects of Microbial Growth
 Consequences of exponential growth
•
Cell numbers in laboratory cultures can be
extremely high (>109 cells/ml).
•
Rapid spoilage of food products: spoilage of
pasteurized milk at room temperature.
Figure 4.11. The rate of growth of
a microbial culture.
The Microbial Growth Cycle

Exponential growth is only part of the microbial growth cycle.

Cells cannot grow exponentially indefinitely.
—
Batch culture where growth occurs in an enclosed vessel (e.g., flask).
The Microbial Growth Cycle

Exponential growth is only part of the microbial growth cycle.

Cells cannot grow exponentially indefinitely.
—

Batch culture where growth occurs in an enclosed vessel (e.g., flask).
Growth cycle: lag, exponential, stationary, and death.
Figure 4.10. Typical growth curve for a bacterial population.
The Microbial Growth Cycle

Exponential growth is only part of the microbial growth cycle.

Cells cannot grow exponentially indefinitely.
—

Batch culture where growth occurs in an enclosed vessel (e.g., flask).
Growth cycle: lag, exponential, stationary, and death.
Lag phase
• Cells prepare themselves for growing
in new medium/environment (HOW?).
• It can be long, short or absent
(WHEN?).
Exponential/log phase
• Cells in healthiest state
• Cells grow at the fastest rate
Stationery phase
• Growth rate is zero (no net increase
or decrease in cell number) (WHY?).
• Energy metabolism and biosynthetic
processes at reduced rate.
Death phase
• Cells eventually die
• Do all cells die?
Figure 4.10. Typical growth curve for a bacterial population.
Continuous Culture


A continuous culture is an open system.
—
Fresh medium is continuously added .
—
Used medium and cells are harvested at the same time.
—
Steady state i.e., the culture volume stays constant.
Fresh medium
from reservoir
Flow-rate
regulator
Sterile air or
other gas
Gaseous
headspace
Culture
vessel
Experimental uses of continuous culture
Culture
— Cells can be kept in exponential growth phase for long periods.
Overflow
— Repetition of experiments with reproducibility of cell population density.
—
Used in studies of microbial ecology and evolution.
—
Direct enrichment and isolation of bacteria from nature.
Effluent containing
microbial cells
Figure 4.13. Continuous culture device
(chemostat).
Biofilm Formation
 A population of cells enmeshed in a polysaccharide matrix that
is attached to a surface.
 Stages:
1) Attachment of planktonic cells to a surface
—
Mediated by flagella, fimbriae, or pili
2) Colonization of the surface
—
Produce sticky extracellular polysaccharides (EPS)
3) Development
—
Cells in the biofilm begin to change their metabolism
4) Dispersal
—
Cells disperse from mature biofilm and colonize new sites.
 Biofilms and Humans: pp 160.
III. Measuring Microbial Growth
Page 150-154
Microscopic Counts

Microbial cells are enumerated by microscopic observations.

Achieved by use of counting chambers.
To calculate number
per milliliter of sample:
12 cells  25 large squares  50  103
Ridges that support coverslip
Coverslip
Number/mm2 (3  102)
Sample added here. Care must be
taken not to allow overflow; space
between coverslip and slide is 0.02 mm
( 501 mm. Whole grid has 25 large
squares, a total area of 1 mm2 and
a total volume of 0.02 mm3.
Microscopic observation; all
cells are counted in large
square (16 small squares):
12 cells. (In practice, several
large squares are counted and
the numbers averaged.)
Number/mm3 (1.5  104)
Number/cm3 (ml) (1.5  107)
Figure 4.4. Direct microscopic counting procedure using the Petroff–Hausser counting chamber.
Microscopic Counts
 Limitations of microscopic counts
Petroff–Hausser counting chamber.
— Cannot distinguish between live and dead
cells without special stains.
— Small cells can be overlooked.
— Cell suspensions of low density hard to count.
— Motile cells need to immobilized.
— Debris in sample can be mistaken for cells.
Viable Counts
 Viable cell
•
One that is able to divide and form offspring.
•
Counted using viable/plate count method:
—
Each viable cell will grow and divide to yield one colony.
— Colony numbers are a reflection of cell numbers.
—
The spreadplate and the pour-plate method (Fig. 4.5; pp 151).
—
Usually after first performing a serial dilution.
—
Used in: food, dairy, medical, and aquatic microbiology; wastewater and
other water analyses.
Viable Counts
 Viable cell
•
One that is able to divide and form offspring.
•
Counted using viable/plate count method:
— Each viable cell will grow and divide to yield one colony.
—
Colony numbers are a reflection of cell numbers.
—
The spreadplate and the pour-plate method (Fig. 4.6; pp 151).
—
Usually after first performing a serial dilution.
—
Used in: food, dairy, medical, and aquatic microbiology; wastewater and
other water analyses.
Figure 4.6. Procedure for viable
counting using serial dilutions of the
sample and the pour-plate method.
Viable Counts
 The great plate count anomaly
• The discrepancy between cell
numbers from natural
environments that form colonies
on agar media and the numbers
countable by microscopic
examination.
Viable Counts
 The great plate count anomaly
•
Why is this?
— Microscopic methods count dead cells,
whereas viable methods do not.
— Different organisms may have vastly
different requirements for growth.
Turbidimetric Method
 Spectrophotometer is used to estimate
cell numbers based on the density of a
liquid culture.
— Suspension of cell looks turbid because the cells
scatter light passing through the suspension.
—
Require preparation of a standard curve that
relates cell numbers (microscopic or viable count)
or mass dry weight to turbidity.
— Growth curve can be generated over time.
—
Drawbacks: Limited to suspended/free-living cells
—
Bacteria that produce pigments or grow as
biofilms.
Figure 4.7. Turbidity
measurements of microbial
growth.
IV. Environmental Effects on Microbial Growth
Page 162-173
Temperature
 Each organism has its own cardinal temperatures
(minimum, optimum and maximum (Fig. 4.20; pp 162)
 Temperature classes (Fig. 4.21; pp 162):
— Psychrophiles – low temperature optima
— Mesophiles – midrange temperature optima
— Thermophiles – high temperature optima
— Hyperthermophiles – very high temperature optima
Temperature
 Temperature classes
• Psychrophiles
— Unusually cold (e.g., glaciers) habitats.
— Cold-active proteins (e.g., enzymes and cold shock proteins) and membranes
(psychrophiles) (properties/functions?).
—
Produce cryoprotectants; antifreeze proteins and solutes (glycerol/other
sugars).
— Help prevent the formation of ice crystals.
Temperature
 Temperature classes
• Thermophiles:
•
Unusually warm (e.g., boiling hot springs) habitats.
•
Enzymes and CM can withstand these conditions.
•
Enzymes:
—
Ionic bonding between basic and acidic amino acids.
—
Highly hydrophobic regions.
— Commercial uses: PCR
•
Membrane:
— Heat-stable.
—
Higher content of long-chain and saturated fatty acids.
—
Lipid monolayer rather than a lipid bilayer.
pH, Osmolarity, and Oxygen
 Effects of pH:
•
Acidity or alkalinity of a solution:
•
pH values <7 are acidic and those >7 are alkaline (Fig. 4.26; pp 168).
•
Microbes show a well-defined pH optimum.
•
—
Highly acidic habitats (e.g., acid mine drainage) or alkaline habitats (e.g., soda lakes).
—
Acidophiles (pH 5.5) and Alkaliphiles (pH 8 or higher).
Cytoplasmic pH and Buffers:
—
Internal pH of a cell must remain relatively close to neutral.
—
Internal pH neutrality is important.
—
—
DNA and RNA are acid and alkaline labile, respectively.
Buffers are commonly added to culture media to prevent major pH changes.
pH, Osmolarity, and Oxygen
 Osmolarity and microbial growth
• Water availability affects the growth of microorganisms.
― A function of dissolve solutes (salts, sugars, or other substances)
― Diffuses from regions of high water concentration to regions of lower water
concentration.
― A cell must have strategies to prevent water loss or negative effects of
osmosis.
•
Halophiles and related organisms?
― Halophiles, halotolerant, extremehalophiles, osmophiles, and xerophiles.
― What are compatible solutes?
― Page 169-170; Fig. 4.27.
pH, Osmolarity, and Oxygen
 O2 and microbial growth.
• O2 is an essential nutrient for many microbes.
• Other microbes cannot grow in the presence of O2.
• Oxygen classes of microorganisms:
— Aerobes, microarophiles, facultative aerobes, and anaerobes
(aerotoelrant and obligate).
— pp 172; Fig. 4.28.
pH, Osmolarity, and Oxygen
 Why Is O2 Toxic?
• O2 can be converted to toxic oxygen by-products
•
Superoxide anion (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (OH·)
•
By-products of cellular respiration (Fig. 4.30; pp 172).
—
Oxidizing agents that cause damage to DNA, proteins, and lipids.
— Negative effect on the structure and activity.
—
•
Damage cell components  cell death.
How do microbes keep toxic oxygen molecules under control?
•
pp 173
V. Controlling Microbial Growth
Page 173-206
Microbial Growth Control
 Sterilization:
•
The killing or removal of all microorganisms
•
Physical methods:
•
Heat, radiation, and filter sterilization
•
Heat sterilization commonly used by microbiologists.
― Dry heat, incineration, and autoclave sterilization.
―
Effectiveness of heat as a sterilant is quantified by decimal
reduction time (D).
―
Time or doze at a given temperature to reduce viable bacteria
by 90%.
Figure 4.33. The effect of temperature
on the heat killing of microorganisms.
Microbial Growth Control
 Sterilization:
• Chemical methods::
•
Antiseptics, disinfectants, and sterilants.
•
Antimicrobial agents
— Bactericidal agents kill bacteria.
— Bacteriostatic agents only inhibit bacterial growth.
— Bacteriolytic agents lyse cells.
— Viable and Turbidimetric growth assays (Fig. 4.39; pp 178).
Microbial Growth Control
 Assaying antimicrobial activity:
•
Minimum inhibitory concentration (MIC):
—
The lowest concentration of an antimicrobial that will inhibit visible growth of a
microorganism.
—
•
Varies with the organism used, inoculum size, temperature, pH, etc.
Dilution method (liquid media; Fig. 4.4):
— Series of tubes inoculated with test culture and different concentrations of the
chemical agents.
•
Disc diffusion assay (solid media; Fig. 4.41):
—
Known amount of antimicrobial agent added to filter paper disc
—
Disc arranged on surface of medium on which a lawn of test culture has been
evenly spread.
Recap
i.
Cell Division and Population Growth
ii.
Culturing Microbes and Measuring Their Growth
iii.
Environmental Effects on Microbial Growth
iv.
Controlling Microbial Growth
Homework/self study:
1) Halophiles and related organisms.
•
pp 169-170; Fig. 4.27
2) Oxygen classes of microorganisms.
•
pp 172; Fig. 4.28.
3) How do microbes keep toxic oxygen molecules under control?
•
pp 173.
4) Method for testing a microbial culture for the presence of catalase.
•
pp 173; Fig. 4.32.
Microbial Metabolism: Energetics,
Enzymes, and Redox
(Chapter 3: pp. 112-121)
01 March 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Energetics, Enzymes, and Redox
Learning outcomes
I.
Energetics, Enzymes, and Redox
i.
Understand what free energy is.
ii.
Principles of bioenergetics: energy conservation.
iii.
Energy classes of microorganisms.
Learning outcomes
Check Your Understanding
• Use MINIQUIZZes to test yourself.
• Very important to enhance your understanding of
the concepts.
I. Energetics, Enzymes, and Redox
Page 112-121
Principles of bioenergetics

Energy, the ability to do work is measured in kJ.

Cellular chemical reactions accompanied by changes in energy.
—
Energy being either required (endergonic reactions) or released (exergonic reactions)
as a reaction proceeds.
—


Exergonic reactions release, and endergonic reactions require free energy
Free energy: energy released that is available to do work (G)
—
Can be conserved by cells in the form of ATP.
—
Change in free energy during a reaction expressed as ΔG0‘.
—
Standard conditions: pH7, 25 oC, 1 atm pressure and [reactants and products] at 1M.
Free energy of formation (Gfo)
—
Energy released or required from elements during formation of a given molecule.
Electron donors and acceptors

Cells conserve energy released from exergonic reactions.
•
Coupling the reaction to the biosynthesis of energy-rich compounds.
•
Oxidation–reduction biochemistry (redox reactions; occur in pairs).
—
Oxidation – removal of electrons from a substance.
—
Reduction – addition of electrons to a substance.
H2 is oxidized – e– donor
O2 is reduced – e– acceptor
Figure 3.2. Example of an
oxidation–reduction reaction
(pp 113)
•
For every oxidation reaction, there must be a subsequent reduction reaction.
—
•
Every redox reaction has an electron donor and an electron acceptor.
Reduction potentials are expressed for half reactions written as reductions (E0’ - Volts (pH 7))
—
Reduction potential: tendency to donate or accept electrons.
—
Half reactions: either the oxidation or reduction reaction component of a redox reaction.
Energy Classes of Microorganisms
 Energy yielding reactions are part of
metabolism called catabolism.
 Catabolic energy classes:
•
Chemotrophs – conserve energy from
chemicals.
―
Chemoorganotrophs – conserve energy from
oxidation of organic chemicals.
―
Chemolithotrophs – conserve energy from
oxidation of inorganic chemicals.
•
Phototrophs – contain chlorophylls and other
pigments that convert light energy into ATP.
―
Do not require chemicals as a source of energy.
―
Oxygenic (yield O2) and Anoxygenic (-O2).
Figure 3.3. Classification of
metabolic types based on
energy sources.
Energy Classes of Microorganisms
 Heterotrophs and Autotrophs:
• Heterotrophs – organisms that obtain carbon from
organic chemical compounds.
• Autotrophs – organisms that use CO2 as carbon
source.
Energy Classes of Microorganisms
Life is carbon-based and it needs energy to grow and develop.
Organic
sources
Heterotrophs
Carbon
Sunlight
Environ.
CO2
Autotrophs
Energy
Inorganic
chemicals
Phototrophs
Photoautotrophs
(Cyanobacteria)
Photoheterotrophs
(Purple & green nonsulfur bacteria)
Chemoheterotrophs
(E. coli)
Chemoautotrophs
(Extremophiles)
Chemotrophs
Electron donors and acceptors
 Redox couples are arranged in the
Most -tive
E0’
redox tower
•
From strongest reductants (top of tower) to
strongest oxidants (bottom of tower).
•
Farther the “drop” of an electron between redox
couples on the tower, the more energy is released.
•
The difference in reduction potential between
redox couple (ΔE0′) is proportional to ΔG0′
Figure 3.4. The redox
tower (pp 114)
Most +tive
E0’
Electron Carriers

Electron carriers act as intermediates between:
•
Primary electron donor and terminal electron acceptor
•
Two classes:
—
Cytoplasmic membrane bound (prosthetic groups): function in membrane
associated electron transport chain.
—
•
Freely diffusible (Coenzymes): e.g. NAD+, NADP+
Are recycled and therefore are required in small amounts.
Electron Carriers

Redox reactions
•
Nicotinamide adenine dinucleotide (NAD+):
Oxidized
Fig. 3.6: NAD+/NADH cycling.
Reduced
Energy-Rich Compounds

Energy released from redox reactions fuels energy-requiring cell
functions.

Free energy released (exergonic reactions) trapped by the cell and
conserved.
•
Biosynthesis of compounds containing energy-rich phosphate or sulfur
bonds.
—
Cell needs >-30 kJ/mol as energy currency
—
Examples:
—
ATP – two bonds are high energy phosphoanhydride bonds.
—
Coenzyme-A – derivatives (e.g., Acetyl-CoA) contains thioester
bonds.
—
Hydrolysis releases energy to drive endergonic reactions.
Fig. 3.8: Energy-rich bonds in
compounds that conserve energy in
microbial metabolism.
Mechanisms of Energy Conservation

Substrate-level phosphorylation:
•
A phosphate group of a substrate is transferred to ADP in order to make
ATP.
—
Investment phase of glycolysis: 1,3 bisphosphoglycerate and
Phisphoenolpyruvate

Oxidative phosphorylation:
•
ATP synthesis coupled to the movement of electrons through electron transport
chain and the associated consumption of oxygen to form water.
—
Redox reactions in the electron transport chain generate a proton motive
force (PMF) that is used to drive ATP synthesis.

Photophosphorylation:
•
Light energy is used to form the proton motive force that powers ATP synthesis.
Fig. 3.8: Energy-rich bonds in
compounds that conserve energy in
microbial metabolism.
Energy storage
 Long-term energy storage:
•
ATP is continuously broken down (anabolic redox reactions)
and resynthesized (catabolic redox reactions).
•
Insoluble polymers:
— Polyglucose (starch, glycogen)
— Polyhydroxyalkanoates (PHB)
— Elemental sulfur
•
Deposited as granules visible under a microscope.
•
Later catabolized for the production of ATP.
Enzymes and catalysis

Enzymes are biological catalysts.
—
Catalyst: a substance that helps a chemical reaction to occur.
—
Mostly proteins (few RNA) catalyzing specific reactions.
—
Specificity determined by the three dimensional structure of the enzyme molecule.
—
Enzyme combine with the substrate to form an enzyme-substrate complex.
Image modified from "Enzymes: Figure 2," by OpenStax College, Biology, CC BY 3.0.
Enzymes and catalysis
 Enzyme catalysis:
—
Enzyme must bind substrate.
— Position it relative to the active amino acid in the active site.
—
Net result: reduction of activation energy required for release of
product from substrate.
—
—
Minimum energy required to begin a reaction.
Endergonic reactions: coupling of reaction to an energy yielding
reaction such as ATP hydrolysis.

Enzyme structure and Nomenclature:
—
pp 120; prosthetic groups and coenzymes.
Figure 3.9. Activation energy
and catalysis.
Enzymes and catalysis

Inhibiting enzyme activity:
Allosteric inhibition
Inhibition by a binding event at a
site different from the active site,
which induces a conformational
change and reduces the affinity of
the enzyme for its substrate
Competitive inhibition
Type of inhibition in which the
inhibitor competes with the substrate
molecule by binding to the active site
of the enzyme
Recap
I.
Energetics, Enzymes, and Redox
i.
Understand what free energy is.
ii.
Principles of bioenergetics: energy conservation.
iii.
Energy classes of microorganisms.
Homework/self study:
•
Enzymes:
pp 120-121
Microbial Metabolism: Aerobic respiration
Glycolysis, Glyoxylate Cycle, and Citric Acid Cycle
(Chapter 3: pp. 121-130)
07 March 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Glycolysis
II.
Pyruvate Decarboxylation
III.
The Citric Acid Cycle
IV.
The Glyoxylate Cycle
I. Glycolysis
Page 121-122
Glycolysis
 First step in breaking down glucose to obtain energy through
cellular respiration.
 Consists of 10 steps:
― Stage I (1st 5-steps/energy investment)
― Stage II (2nd 5-steps/pay-off)
Investment Phase
1
 Step 1: Phosphorylation
• Glucose phosphorylated to glucose-6-phosphate.
Hexokinase
• Costs 1 ATP; needed for the phosphate group.
Glucose
ATP
• Hexokinase
ADP
Glucose-6phosphate
 Step 2: Isomerization
2
• Glucose-6-phosphate isomerizes to fructorse-6-phosphate.
• Phosphoglucoisomerase
 Step 3:
2nd
Phosphorylation
• Fructorse-6-phosphate phosphorylated to fructose-1,6bisphosphate.
• Costs 1 ATP; needed for the phosphate group.
• Phosphofructokinase 1
Phosphofructokinase
Fructose-6phosphate
ADP
ATP
3
Fructose-1,6-bisphosphate
Investment Phase
 Step 4: Cleavage
4
• Fructose-1,6-bisphosphate spit into
Glyceraldehyde-3-phosphate
Fructose-bisphosphate
aldolase
glyceraldehyde-3-phosphate (G3P) and
dihydroxyacetone phosphate (DHAP).
Fructose-1,6-bisphosphate
• Fructose-bisphosphate aldolase
Dihydroxyacetone phosphat
 Step 5: Conversion of DHAP into G3P
5
Triosephosphate
isomerase
• DHAP converted into G3P by triosephosphate
isomerase.
Glyceraldehyde-3-phosphate
• Reaction results in 2 molecules of G3P.
Pay-off Phase
Glyceraldehyde-3-phosphate
NAD+
 Step 6: Oxidation
• G3P is oxidized and
Dehydrogenase
NAD+
6
NADH
is reduced to NADH.
• Exergonic: energy used to phosphorylate G3P to 1,3bisphosphoglycerate using inorganic phosphate (Pi).
• Glyceraldehyde phosphate dehydrogenase.
1,3-bisphosphoglycerate
Phosphoglycerate
kinase
 Step 7: 1st Substrate level phosphorylation
ATP
• 1,3-bisphosphoglycerate donates one of its phosphate
ADP
groups to ADP.
• Turning into 3-phosphoglycerate and making 1 ATP.
• Phosphoglycerate kinase
7
3-phosphoglycerate
Pay-off Phase
8
 Step 8: Phosphate transfer
3-phosphoglycerate
• Phosphoglycerate mutase transfers the remaining
phosphate from the OH to the next making 22-phosphoglycerate
phosphoglycerate.
 Step 9: Dehydration
9
• Enolase catalyzes a dehydration reaction, producing
phosphoenolpyruvate.
10
 Step 10:
2nd
Substrate level phosphorylation
Phosphoenolpyruvate
ATP
• Pyruvate kinase transfers the remaining phosphate group
to ADP, generating ATP and pyruvate.
ADP
Pyruvate
Glycolysis

Ten steps:
Energy investment phase (1st 5 steps)
• 1 Glucose + 2ATPs  2G3P
Payoff phase (last 5 steps)
• 2G3P  2Pyruvate + 4ATP
Net = 2ATP per glucose (2NADH and 2Pyruvate)
II. Pyruvate Decarboxylation
Page 123
Pyruvate decarboxylation
 Pyruvate is decarboxylated:
• CO2, NADH, and acetyl-CoA.
• Acetyl-CoA enters the CAC.
― 2CO2, 3NADH, and 1 FADH2 formed per oxidized pyruvate.
― Oxaloacetate is regenerated as the next acetyl acceptor, thus completing the
cycle.
The Citric Acid Cycle
 Pyruvate oxidation:
• Links glycolysis to the rest of cellular respiration.
• Pyruvate—a three-carbon molecule:
― Converted to acetyl-CoA—a two-carbon molecule attached
to Coenzyme A.
― Producing an NADH.
― Releasing one CO2 in the process.
― Acetyl-CoA carry the acetyl group to the citric acid cycle.
― Pyruvate dehydrogenase complex.
― 2CO2 and 2NADH.
III. The Citric Acid Cycle
Page 122-124
The Citric Acid Cycle
 Sources of Acetyl-CoA:
• Derived from decarboxylation of the end
product of glycolysis, pyruvate.
• Also produced from the oxidation of FAs and
degradation of some aa.
• Collectively, feed the CAC cycle and
contribute to ATP synthesis via oxidative
phosphorylation.
The Citric Acid Cycle
 Citric acid cycle (CAC):
• 8-step pathway
• Takes acetyl-CoA—produced by the oxidation of
pyruvate.
• Harvests its bond energy in the form of NADH,
FADH2, and ATP molecules.
― Pass their electrons into the electron transport
chain.
― Produce ATP through oxidative phosphorylation.
The Citric Acid Cycle
 Step 1: Transfer of acetyl group
• Citrate synthase transfers an acetyl group from acetyl-CoA
and attaches it to oxaloacetate.
• Releasing the CoA group and forming a 6-carbon
molecule, citrate.
 Step 2: Isomerization
• Aconitase converts citrate into its isomer, isocitrate.
― Removal and then the addition of a water molecule.
 Step 3: Oxidation/decarboxylation
• Isocitrate dehydrogenase oxidizes isocitrate and releases
CO2.
• Leaving behind a 5-carbon molecule—α-ketoglutarate.
• NAD+ is reduced to form NADH.
1
2
3
The Citric Acid Cycle
 Step 4: Oxidation and rejoining with CoA
• α-ketoglutarate dehydrogenase oxidizes α-ketoglutarate.
1
2
• NAD+ reduced to NADH and CO2 is released.
• The remaining 4-carbon molecule picks up Coenzyme A.
• Forming succinyl CoA.
3
 Step 5: ATP/GTP formation
• CoA of succinyl CoA is replaced by a phosphate group.
4
• Transferred to ADP to make GTP/ATP.
• This reaction results in the formation of succinate.
• Succinyl-CoA synthetase.
5
The Citric Acid Cycle
 Step 6: Oxidation by FAD (Flavin adenine dinucleotide)
• Succinate dehydrogenase transfers two hydrogen atoms—
with their electrons— to FAD.
• Producing FADH2, oxidizing succinate to fumarate.
8
1
2
 Step 7: Hydration
3
• Fumerase adds water to fumarate.
• Converting it into another 4-carbon molecule, malate.
7
 Step 8: Final oxidation
• Oxaloacetate is regenerated by oxidation of malate.
• Malate dehydrogenase.
• NAD+ reduced to NADH in the process.
4
6
5
The Citric Acid Cycle

Eight steps:
For every Acetyl-CoA that enters CAC:
• 3NADH (steps 3, 4, 8), 1FADH2 (step 6), 1ATP (step 4)
• Since 1 glucose = 2Pyruvate (Glycolysis).
Net = 6NADH + 2FADH + 2ATP per glucose
The Citric Acid Cycle
 Important roles of the CAC:
1) To produce reduced electron donors.
― NADH and FADH2.
2) To produce CO2
― Steps 3 and 4.
3) To produce precursors for the FA and aa
biosynthesis.
IV. The Glyoxylate Cycle
Page 124
The Glyoxylate Cycle
 Related to CAC and bypasses of
some CAC reactions.
 Processing of Acetyl-CoA for
gluconeogenesis.
The Glyoxylate Cycle
 1st 2 reactions identical to 1st 2 reactions of
CAC.
 3rd step:
• Isocitrate split into a 4-carbon molecule, Succinate
and 2-carbon molecule, glyoxylate.
• Isocitrate lyase – unique to organisms having the
glyoxylate cycle.
The Glyoxylate Cycle
 Succinate converted to oxaloacetate via CAC.
 Malate synthase transfers acetyl group from
acetyl-CoA to glyoxylate to form malate.
 Malate converted to oxaloacetate via CAC.
 In this way excess oxaloacetate produced.
The Glyoxylate Cycle
 Excess oxaloacetate:
• Decarboxylated into PEP.
• In turn converted to glucose
via gluconeogenesis.
• Organisms with an active
glyoxylate cycle synthesize
glucose from acetyl-CoA.
Next lecture
I.
Electron Carriers
II.
Proton Motive Force
III.
Oxidative Phosphorylation
Microbial Metabolism: Aerobic respiration
Oxidative Phosphorylation
(Chapter 3: pp. 121-130)
08 March 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Electron Carriers
II.
Electron Transport Chain
III.
Oxidative Phosphorylation
In today’s lecture
I.
Electron Carriers
II.
Electron Transport Chain
III.
Oxidative Phosphorylation
ATP
Overview
Complex I
Complex II
Complex III
CoQ
Oxidative
Phosphorylation
Complex IV
ATPase
CytC
Electron Transport Chain
(Shuffle electrons and generates PMF)
Chemiosmosis
(Pumps the proton gradient to generate ATP)
I. Electron Carriers
Page 125-127
Electron carriers
 Coenzymes
• Glycolysis and CAC
― Glucose  Pyruvate  ATP, NADH and FADH2 are formed.
― NADH and FADH2 move on to OP.
• Must be reoxidized back to NAD+ and FAD.
• Generates by far the most ATPs.
Electron carriers
 NADH dehydrogenase, flavoproteins, iron–sulfur
proteins, cytochromes, quinones.
•
Oxidation–reduction enzymes participate in electron transport.
• Arranged in the membrane in order of increasingly more positive
reduction potential.
― NADH dehydrogenase first and the cytochromes last.
Electron carriers
 NADH Dehydrogenase:
• Contain an active site that binds NADH.
• Transfers 2 e– and H+ from NADH, and one H+ from cytoplasm to a
flavoproteins.
• This regenerates NAD+.
― Glycolysis, CAC or other processes.
Electron carriers
 Flavoproteins:
• Contain a derivative of the vitamin riboflavin.
• Flavin portion is bound to its protein as a prosthetic group.
― Accepts 2 e– + 2 H+, donate only electrons.
― Protons are ultimately released to the cytoplasm.
― Flavin mononucleotide (FMN) and flavin adenine dinucleotide
(FAD); Fig. 3.15, pp 126.
― FMN2 and FADH2 are reduced forms.
Electron carriers
 Cytochromes:
• Contain heme prosthetic groups.
― Iron atoms (exist as either Fe2+ or Fe3+) act as redox sites.
• Form into complexes with other cytochromes or with
iron–sulfur proteins.
• cytochrome bc1 (complex III)
• Carry electrons only.
• Fig. 3.6; pp 126
Electron carriers
 Iron–sulfur proteins:
• Nonheme iron proteins called ferredoxins.
― Contain prosthetic groups made up of clusters of iron and sulfur atoms.
― Fe2S2 and Fe4S4 being the most common.
― Carry electrons only.
― Fig. 3.17; pp 127.
Electron carriers

Ubiquinones (Coenzyme Qs):
• Small hydrophobic redox molecules that lack a protein component.
• Due to size and hydrophobicity, they can move about within the
membrane.
• Accept 2 e– and 2H+ from the cytoplasm, forming ubiquinol (QH2).
• Donate electrons to other carriers and lose 2H +, contributing to pmf.
• Ubiquinol interacts with Complex I, II, and III.
• Fig 3.18; pp 127
I. Electron Transport Chain
Page 125-124
Electron Transport Chain

Complex I:
• Also called NADH:quinone oxidoreductase or NADH dehydrogenase.
• Oxidizes NADH to NAD+, resulting in 2 electrons (e–).
• e– transferred to FMN and Fe/S, then to ubiquinone (Q)
• Q accepts 2 e– and 2 H+ from the cytoplasm to become ubiquinol (QH2).
• Transport of 4 H+ across the membrane.
Electron Transport Chain

Complex II:
• Also called the succinate dehydrogenase complex.
― Oxidation of succinate to fumarate in the citric acid cycle reduces
FAD to FADH2.
• 2 e– from FADH2 are transferred through Complex II to Q.
• Q accepts 2 e– and 2 H+ from the cytoplasm to become QH2.
Electron Transport Chain

Complex III:
• Consists of the cytochrome bc1 complex.
― Cytochrome b (L and H subunits) and cytochrome c1.
• Supplies e– to cytochrome c (not part of Complex III), a water
soluble component.
• Also contains Fe/S center.
• Two binding sites:
1) Q0 – binds QH2.
2) Qi – binds Q.
Electron Transport Chain

Complex III:
• Flow of e– through Complex III is called the Q-cycle.
• 2 e– from QH2 enter at the Qo site.
― Donated to cytochrome c, then 2 H+ released, contributing to pmf.
― QH2 carries 2 e– and cytochrome c carries 1 e–.
― 2 e– from QH2 sent in different directions (electron bifurcation):
• 1st e– goes to Cytochrome c via Ferrodoxin (Fe/S) and
cytochorme c1
• 2nd e– goes to Cytochrome b (subunits L and H), then to Q at the
Qi site, forming semi-quinone radical (Qe–).
― The net result of the Q-cycle is that 4 H+.
Electron Transport Chain
 Complex IV:
• Functions as the terminal oxidase, containing
cytochromes a and a3.
• Receives 2 e– from cytochrome c.
• Reduces O2 to 2H2O.
• Requires 4 H+.
• Pumps out 2 H+ from the cytoplasm.
Electron Transport Chain
•
For every 2 e– transported from NADH to O2, a total of 10 H+ are transferred.
•
For every 2 e– transported from FADH2 to O2, a total of 6 H+ are transferred.
•
In both cases, 2 H+ are consumed in the cytoplasm during the formation of water.
II. Chemiosmosis
Page 127-128
Complex V (ATPase)
 ATP Synthase:
https://www.youtube.com/watch?v=kXpzp4RDGJI
• Two components:
1) F1:
• Multiprotein complex that sticks into the cytoplasm and catalyzes
ATP synthesis.
• Catalyzes a reversible reaction between ATP and ADP + Pi.
2) F0:
• Membrane-integrated multiprotein complex that carries out
proton translocation across the membrane.
For every full rotation of the c ring within the Fo subunit, three
ATP are formed by the F1 subunit.
Proton Motive Force
 PMF is the driving force for ATP synthesis by ATP synthase (ATPase) or Complex V.
 Protons flow through a channel in the enzyme.
―
The movement spins the protein, much like wind drives a turbine.
 The mechanical movement of this rotor provides the energy to add an inorganic
phosphate group to ADP to form ATP.
 ATPase can run backward within the cell;
―
ATP hydrolysis can be the driving force to cause the enzyme to eject protons out of the
cytoplasm, resulting in the generation of PMF.
 Some bacterial and archaeal ATPases are linked to a sodium (Na+) rather than a
proton (H+) gradient.
How many ATP do we get per glucose in
cellular respiration?
 ATP = 3.3 H+:
Stage
Direct products (net)
Ultimate ATP yield (net)
Glycolysis
2 ATP
2 ATP
2 NADH (= 20 H+)
6 ATP
Pyruvate oxidation
2 NADH (= 20 H+)
6 ATP
Citric acid cycle
2 ATP/GTP
2 ATP
6 NADH (= 60 H+)
18 ATP
2FADH2 (= 12 H+)
3.6 ATP
Total
Fig. 3.21; pp 130 (Aerobic respiration).
37.6 ATP (~38 ATP)
OP generates
most ATPs.
Homework/self study:
•
Anaerobic respiration: Fermentation
1) What is the role of substrate-level phosphorylation?
2) How is NADH recycled during fermentation?
pp 124-125; Lactic acid fermentation (Fig. 3.21 pp 130).
Microbial Metabolism: Biosynthesis
(Chapter 3: pp. 134-141)
14 March 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Carbon and Nitrogen Fixation
II.
Sugars and Polysaccharides
III.
Amino Acids and Nucleotides
IV.
Fatty Acids and Lipids
Intro: Biosynthetic Reactions
 Biosynthetic reactions:
• Aspect of metabolism called anabolism.
• Require a source of carbon (C) and nitrogen (N).
― Present in the atmosphere as inorganic carbon (CO2) and nitrogen (N2).
― In this form, they are highly oxidized and require large amounts of ATP and
reducing power.
• CO2 and N2 fixation
I. Carbon and Nitrogen Fixation
Page 134-136
CO2 Fixation
 The calvin cycle:
• Used by autotrophs to turn CO2 from the air into sugar.
• CO2 is incorporated into organic molecules and used to build three-carbon sugars.
• Requires:
― CO2, a CO2-acceptor, ATP, and NADPH.
― Enzymes: ribulose bisphosphate carboxylase-oxygenase (RuBisCo) and
phosphoribulokinase (PRK).
• Four main steps: carbon fixation, reduction phase, carbohydrate formation, and
regeneration phase.
CO2 Fixation
 Carbon fixation:
• CO2 combines with 5C Ribulose-1,5bisphosphate (RuBP)
― With help from RuBisCo.
― Makes 6C molecule
― Splits into two 3-phosphoglyceric acid
(3-PGA), 3C
CO2 Fixation
 Reduction phase:
• ATP and NADPH are used to
convert the 3-PGA into G3P.
― NADPH donates electrons to, or reduces 3PGA
1) Each molecule of 3-PGA receives a
phosphate group from ATP – 1,3-bisPG.
2) 1,3-bisPG reduced by NADPH – G3P.
CO2 Fixation
 Regeneration phase:
• Some G3P molecules go to make glucose.
• Others recycled to regenerate the RuBP acceptor.
• Requires ATP.
 Glucose (C6H12O6):
• It takes six turns of the Calvin cycle to make one glucose
molecule.
N2 Fixation
 Fixed as ammonia (NH3) by soil
microorganisms.
• Important for synthesis of protein and nucleic
acids.
• Enormous ecological and agricultural importance.
 Catalyzed by the enzyme complex
nitrogenase.
N2 Fixation
 Nitrogenase complex.
• Dinitrogenase and dinitrogenase reductase.
Nitrogenase
• Both contain iron
• Dinitrogenase contains molybdenum as well.
• Iron and molybdenum in dinotrgenase
• part of iron—molybdenum cofactor (FeMo-co).
• FeMo-co site of N2 fixation.
N2ase
(FeMo-co protein)
N2ase
Reductase
(Fe protein)
N2 Fixation
 Nitrogenase activity:
• N2 requires 16 ATPs to be converted to NH3.
― Energetically costly reaction (~900 kJ/mol).
• Requires 8 e–.
• 2e– transferred from pyruvate to Flavodoxin
and to N2ase reductase.
• e– transferred from N2ase reductase to
N2ase one at a time.
• Each cycle of reduction requires two ATP.
• Net ATP = 16ATPs.
N2 Fixation
 Nitrogenase is inhibited by oxygen:
• Many nitrogen-fixing bacteria are obligate aerobes.
• N2ase inactivation by prevented by:
1) Rapid removal of O2 by respiration.
2) Production of O2-retarding slime layers on the outer cell.
3) N2ase localization in a hetrocyst (cyanobacteria).
― Conditions are anoxic – no O2 production in heterocyst.
II. Sugars and Polysaccharides
Page 127-138
Polysaccharide Biosyntheses
 Synthesized by adding activated glucose to a
preexisting polymer fragment.
 Activated glucose:
• Uridine diphosphoglucose (UDPG)
― Precursor for glucose derivatives (e.g., N-acetylglucosamine and N-
acetylmuramic acid).
• Adenosine diphosphoglucose (ADPG).
• Play a role in glycogen biosythssis.
Gluconeogenesis
 Precursors:
• Lactate – Lactic acid fermentation
• Pyruvate – Glycolysis
• Glycerol-3-phosphate – Fatty acid breakdown
• Amino acids – Protein breakdown
― Converted into citric acid cycle intermediates.
― Then to malate then to glucose.
― Fig. 3.30b, pp 137
Pentose Phosphate Pathway
 Pentoses:
• 5C sugars formed by the removal of 1C atom from a hexose.
• Needed for nucleic acid synthesis, ribose (in RNA) and
deoxyribose (in DNA).
• Ribonucleotide reductase converts ribose into deoxyribose.
• Reduction of –OH group on the 2nd carbon of a 5C pentose ring.
• Fate of ribonucleotides:
• Reduced to deoxyribonucleotides for use as precursors of DNA.
Pentose Phosphate Pathway
 Pentose biosynthesis:
• Glucose oxidized to CO2, NADPH, and ribulose-5-phosphate.
• From ribulose-5-phosphate, several pentose derivatives formed.
Pentose Phosphate Pathway
 Importance of pentose biosynthesis:
• Production of 4-7C sugars – eventually converted to hexoses.
• Generation of NADPH – reductant of deoxyribonucleotides.
III. Amino Acids and Nucleotides
Page 138-139
Amino Acids
 Monomers of proteins.
 Can be synthesized from glucose or other carbon sources.
 Glycolysis or citric acid intermediates provides skeleton.
 Amino group (–NH) derived from inorganic nitrogen sources (e.g., NH3).
Amino Acids
 Incorporation of NH3:
• During glutamate (glutamate
dehydrogenase) or glutamine (glutamine
synthetase).
• Glutamate dehydrogenase – NH3 is present
at high levels.
• Glutamine synthetase – NH3 is present at
low levels.
 NH3 shuffled into various C skeletons for
further biosynthetic reactions.
Nucleotides
 Monomers of nucleic acids.
 Purines:
• Synthesized from carbon and nitrogen
sources.
• Nucleotide skeleton is inosinic acid,
• A precursor for adenine and guanine.
• Synthesized (triphosphate form) and attached to
ribose.
• Then incorporated into DNA or RNA
Nucleotides
 Pyrimidines:
• Also constructed from several
sources.
• Pyrimidine nucleotide skeleton
is uridylate.
• Thymine, cytosine, and uracil can
are derived.
IV. Fatty Acids and Lipids
Page 127-128
Fatty Acid Biosynthesis
 Lipids: polymers of fatty acids (FAs).
 FAs: long chains of hydrocarbons with a carboxylic acid
group at one of the ends.
• Only in bacteria.
― Synthesized 2C atoms at a time.
― Acyl carrier protein (ACP).
― Each C2 (acetyl) originates from malonate (3C), attached to
ACP to form malonyl-ACP.
― Malonyl-ACP condenses with acetyl-ACP to form
acetoacetyl-ACP.
― ACP adds carbon subunits to a growing fatty acid chain
Homework/self study:
•
Difference between saturated and unsaturated FAs.
•
Lipid biosynthesis
pp 140
Metabolic Diversity
Energy and Redox, and Autotrophic Pathways
(Chapter 14: pp. 461-466)
14 March 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Principles of Metabolic Diversity
II.
Autotrophic Pathways
I. Principles of Metabolic Diversity
Page 461-464
Metabolic Diversity: Energy and Redox
 Metabolic diversity: different metabolic strategies that
organisms have evolved to obtain energy.
 Metabolic pathways are modular and often reversible.
• Facilitates metabolic diversity through horizontal gene transfer.
• Adaptation to new ecological niches.
Conjugation
Transformation
Transduction
Horizontal Gene Transfer (HGT)
Metabolic Diversity: Energy and Redox
 Bacteria are metabolically diverse:
(Chapter 3: pp. 112-121)
• Classified according to their energy requirements.
― Phototrophs – energy comes from solar radiation.
― Chemotrophs – energy source is inorganic (Chemolithotroph) or organic
(Chemoorganotroph) reduced compounds.
• Classified according to the mechanism used:
― Photosynthetic (oxygenic and anoxygenic),
― Respirers (aerobic and anaerobic), and
― Fermentors (alcoholic and lactic acid)
Metabolic Diversity: Energy and Redox
 Components of electron transport allow a diversity of respirations
(Figure 14.1; pp 461).
 All organisms need to conserve energy:
• By converting chemical or light energy into ATP.
• Utilizing a source of reducing power.
― Ferredoxin (Fdox) and NADH.
• By achieving redox balance via regeneration of oxidized electron carriers
― NAD+, FAD
Reducing Power
 Reducing power often in the form of low-potential electron carriers such
as NAD(P)H and Fdox.
 Chemoorganotrophs:
• Reducing power readily generated during the oxidation of reduced organic
molecules.
• They need a way to export electrons from the cell in order to regenerate the
oxidized NAD(P)+ or Fdox they need for catabolic reactions.
•
Achieve redox balance by donating electrons to external electron acceptors or
metabolic intermediates.
Reducing Power
 Chemolithotrophs and phototrophs:
• Lack reducing power.
• Their electron donors (e.g., S0, H2S, and NH3) are unable to reduce
NAD(P)+ or Fdox needed for catabolic and biosynthetic reactions.
• Must generate reducing power by coupling the endergonic
reduction of these electron carriers to some other exergonic
reaction.
Reducing Power
 Mechanisms of energy coupling:
1) Coupling a reaction directly to ATP.
• Requires a substantial energy expenditure.
• Used primarily for highly exergonic catabolic reactions.
2) Reverse electron transport.
• Endergonic reduction of NAD(P)+ or Fdox driven by dissipation of PMF.
• NADH:quinone oxidoreductase transfers e– from QH2 to NAD+.
― An endergonic reaction driven by the pmf.
Reducing Power
 Mechanisms of energy coupling:
3) Flavin-based electron bifurcation
• Very low energy yield microbes: fermenters,
sulfate reducers, acetogens, and methanogens.
• Endergonic reduction of a low-potential electron
acceptor (Fdox) driven by exergonic reduction of
a higher-potential electron acceptor (NAD+).
• Allows the generation of reduced Fdred from
NADH.
• Then Fdred can perform difficult reactions such
as CO2 fixation.
Reducing Power
 Mechanisms of energy coupling:
• Flavin-based electron-bifurcating enzyme functions.
1) Allow the cell to make a highly electronegative intermediate (such as
Fdred ) that can drive difficult endergonic reductions and can also be used
to conserve energy through PMF formation.
2) Can increase fermentative energy yields by allowing the cell to oxidize
NADH to NAD+ through H2 production from Fdred.
Assimilative and Dissimilative Processes
 Assimilative processes:
• Used to assimilate inorganic nutrients into cell material.
― Chemotrophs and phototrophs assimilate nutrients by reducing
inorganic molecules (e.g., N2, NO3-, SO42-, and CO2).
• Energy is consumed as nutrients are assimilated.
• Only performed to acquire nutrients needed for
biosynthetic reactions.
• CO2 fixation is the most important assimilative process in
the biosphere.
Assimilative and Dissimilative Processes
 Dissimilative processes:
• Are those processes used to conserve energy.
• A large amount of e– acceptors must be reduced and then excreted
from the cell.
• Dissimilative reduction reactions are the result of anaerobic
respiration.
II. Autotrophic Pathways
Page 464-466
Autotrophs and CO2 fixation
 Autotrophs are organism that can assimilate CO2 into cell material.
 CO2 fixation:
• Most CO2 fixation pathways contain enzymes inhibited by O2.
• Shares some enzymes with catabolic pathways.
• Creates opportunities for some organisms to grow as mixotrophs.
― Organisms that conserve energy from the oxidation of inorganic compounds but
requires organic compounds serve as a carbon source.
Autotrophs and CO2 fixation
 The calvin cycle:
• Bacteria that perform the Calvin cycle have carboxysomes.
― Proteinaceous compartments acting as the site for RuBisCO activity.
― RuBisCO has very low affinity for CO2 and is inhibited by O2.
• O2 competes with CO2 for access to the RuBisCO active site.
• Reducing power is wasted and autotrophic efficiency is reduced.
― Carboxysomes improve the efficiency of RuBisCO
• Concentrate CO2 and exclude O2 at the site of RuBisCO activity.
The Reverse Citric Acid Cycle
 Not all phototrophic organisms rely on the Calvin cycle for CO2 fixation.
 The reverse citric acid cycle (rCAC):
• A pathway of CO2 fixation used by green sulfur bacteria such as Chlorobium
and many microaerophilic and anaerobic chemolithotrophs.
• CO2 is reduced by a reversal of steps in the citric acid cycle.
• More energy efficient than the Calvin cycle.
—Requiring 24 H (that come from 4 NADH, 2 NADPH, 2 FADH, and 4 Fdred) but only
10 ATP to fix 6 CO2 into one molecule of fructose-1,6-bisphosphate.
The Reverse Citric Acid Cycle
 Takes CO2 and water to make carbon compounds.
 Two ferredoxin-linked reductions:
• Carboxylation of succinyl-CoA to α-ketoglutarate.
• Carboxylation of acetyl-CoA to pyruvate.
 Key enzymes:
• ATP citrate lyase (replaces citrate synthase.
• Fumarate reductase (replaces succinate
dehydrogenase).
Other Pathways of CO2 Fixation

Other pathways of CO2 fixation are
known:
• Hydroxypropionate bi-cycle
• 3-hydroxypropionate/4-hydroxybutyrate cycle
• Reductive acetyl-CoA pathway.
Homework/self study:
•
Diversity of respiration reactions
pp 461; Fig. 14.1
• Other Pathways of CO2 Fixation
pp 465
Metabolic Diversity: Phototrophy
Photosynthesis and Chlorophylls, Carotenoids and Phycobilins;
An/oxygenic Photosynthesis
(Chapter 14: pp. 466-475)
14 March 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Photosynthesis and Chlorophylls
II.
Carotenoids and Phycobilins
III.
Anoxygenic Photosynthesis
IV.
Oxygenic Photosynthesis
I. Photosynthesis and Chlorophylls
Page 466-470
Phototrophy
 Phototrophy:
• Organisms trap light energy (photons) and store it as chemical energy in the form
of ATP and/or reducing power in NADPH.
• The use of light energy to drive biosynthesis.
• Phototrophs:
― Organisms that convert light energy into chemical energy.
― Photosynthetic organisms are phototrophs that are also autotrophs.
― Not all phototrophs are autotrophic.
― Phototrophs use organic carbon as their carbon source are called Photoheterotrophs.
― Acidobacteria, Chlorobi, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes,
and Proteobacteria
Patterns of Photosynthesis
 Reactions that support phototrophic growth:
• Light reactions:
— Reactions that convert light energy into chemical energy in the form of the proton motive force and
ATP.
• Light-independent reactions:
― Use ATP and reducing power, NADPH, to “fix” carbon from CO2 into molecules that can be used to
build glucose.
• H2O is the electron donor for photosynthesis in cyanobacteria, algae, and green
plants.
• Oxygenic photosynthesis:– consume H2O and produce O2 as a waste product.
• Anoxygenic photosynthesis:– photosynthetic organisms that do not produce O2.
Photosynthesis
Chlorophyll and Bacteriochlorophyll
 Photosynthesis requires light-sensitive pigments.
• Chlorophylls:– Present in plants, algae, and
cyanobacteria.
• Bacteriochlorophylls:– Present in anoxygenic
phototrophs.
• Related to the parent structure of cytochromes:
― Chlorophylls contain magnesium instead of iron at the center
of the ring.
― Hydrophobic alcohol that helps anchor the chlorophyll into
photosynthetic membranes.
― Figure 14.6; pp 467.
Chlorophyll and Bacteriochlorophyll
 Chlorophylls:
• Chlorophyll a is green because it absorbs red and blue light and transmits green light.
• Chlorophyll d in cyanobacteria and chlorophyll a and b in prochlorophytes.
 Bacteriochlorophyll
• Present in anoxygenic phototrophs.
• Bchl a of purple bacteria absorbs between 800 and 925 nm.
• Other bacteriochlorophylls absorb in regions of visible and near infrared spectra.
What is the significance of different chlorophylls and bacteriochlorophylls?
pp 467.
Reaction Centers and Antenna Pigments
 Photosynthetic pigments (50-300) attached to proteins and are
housed within membranes to form photocomplexes.
 Small number of pigments present within Reaction Centers.
• Macromolecular structures that participate directly in the
reactions that lead to energy conservation.
• Surrounded by larger numbers of light harvesting pigments
called Antenna pigments.
― They funnel light energy to reaction centers.
― Arrangement allows the RC to receive light even at low light
intensities.
Photosynthetic membranes, Chloroplasts,
Chlorosomes
 Chlorophyll pigments and components of the light-
gathering apparatus exist within membranes in the cell.
 Eukaryotic phototrophs photosynthesis takes place in
intracellular organelles – chloroplasts
 Chloroplasts:
• Thylakoids – sheet like photosynthetic membrane systems.
• Grana – stacks of thylakoids within the chloroplast.
• Lumen – the inner space within the thylakoid array.
• This arrangement facilitates the generation of a light-driven PMF
used to synthesize ATP.
Photosynthetic membranes, Chloroplasts,
Chlorosomes
 Prokaryotes:
• Pigments integrated into membrane systems that arise
from invagination of CM.
― Purple bacteria: These can be chromatophores or
membrane stacks called lamellae.
― Cyanobacteria: Lamellar membranes called
thylakoids due to their resemblance to algal
thylakoids.
Photosynthetic membranes, Chloroplasts,
Chlorosomes
 Prokaryotes: pigments integrated into membrane
systems that arise from invagination of CM
• Anoxygenic phototrophs (Green sulfur bacteria, green
nonsulfur bacteria and Acidobacteria) have chlorosomes
(giant antenna pigments not attached to proteins)
– Capture energy and transfer it to RC through a small
protein called FMO protein.
– They allow sulphur bacteria to absorb light of very low
intensities in deep water lakes, inland seas, biofilms in
hotsprings and highly saline environments.
II. Carotenoids and Phycobilins
Page 470-471
Carotenoids and Phycobilins
 Carotenoids
•
•
•
•
Always found in phototrophic organisms.
Typically yellow, red, brown, or green.
Energy absorbed by carotenoids transferred to a reaction center.
Prevent photo-oxidative damage to cells.
 Phycobiliproteins and Phycobilisomes
• They are main light harvesting systems of cyanobacteria and red algae
• They consist of red and blue-green tetrapyroles called billins, bound to proteins
• Different ones absorb light of different wavelengths
― Phycoerythrin (red): 550nm
― Phycocyanin (blue): 620nm
― Allophycocyanin: 650nm
• Phycobiliproteins form aggregates called phycobilisomes
• Facilitate energy transfer to RC of cyanobacteria
III. Anoxygenic Photosynthesis
Page 471-474
Electron Flow in Anoxygenic Bacteria
 Found in at least four phyla of Bacteria: proteobacteria, green sulfur
bacteria, green nonsulfur bacteria and Gram positive bacteria.
 Electron transport reactions occur in the reaction center of anoxygenic
phototrophs.
 Apparatus contained in intracytoplamic membrane vesicles called
chromatophores or membrane stacks called lamellae.
 Three polypeptides (L, M and H) traversing the membranes are found in
reaction centers.
Electron Flow in Anoxygenic Bacteria
 Function: Bind pigments in reaction centre photocomplex.
 Reaction centre photocomplex: have pairs of different complex that function
together to facilitate fast electron transfer reaction resulting in ATP
formation.
• Bacteriochlorophyl a (special pair)
• Bacteriochorophyll a (function unknown)
• Bacteriopheophytin a
• Quinones
• Carotenoid pigment
Electron Flow in Anoxygenic Bacteria
 Energy from the photons is transferred to the reaction center.
 This energy strikes and excites the special pair of
bacteriochlorophyll a.
 The electrons then reduce a molecule of bacteriopheophytin a.
 Reduced bacteriopheophytin a reduces several quinone
molecules.
 Electrons are then transported through a series of Fe-S proteins
and cytochromes (bc1 and c2), and then back to the reaction
centre.
IV. Oxygenic Photosynthesis
Page 474-475
Oxygenic Photosynthesis
 Oxygenic phototrophs have FeS-type and Q-type
photosynthetic reaction centers.
 Electrons flow through two distinct photosystems:
• Photosystem I (PSI, or P700).
― FeS-type reaction center.
• Photosystem II (PSII, or P680).
―
Q-type reaction center.
• PSI and PSII interact in the “Z scheme” of
photosynthesis.
―
Resembles the letter Z turned on its side.
Electron Flow and ATP synthesis
 Photosystem II (PSII) is activated
by photons, causing H2O to be
oxidized on the Mn4Ca cluster of
the water-oxidizing complex.
 Electrons are transferred from
PSII to the plastoquinone pool
(PQ/PQH2).
Electron Flow and ATP synthesis
 Protons are exchanged across the membrane when
plastoquinone is oxidized by cytochrome b6f.
 Per two molecules of water oxidized to one O2, a total of 12
protons are released to the lumen to fuel ATP synthase.
 Electrons are then transferred to plastocyanin (PC), which
carries them to photosystem I (PSI).
 Upon activation by light, PSI reduces ferredoxin (Fd), with
sequential reduction of ferredoxin:NADP oxidoreductase
(FNR), and then NADP+.
 The ATP and NADPH produced by the light reactions are used in
CO2 fixation by the Calvin cycle.
Electron Flow and ATP synthesis
 Cyclic photophosphorylation occurs when FNR
donates electrons to cytochrome b6f instead of
to NADP+.
 During cyclic photophosphorylation, more ATP
and less NADPH are produced than during
noncyclic photophosphorylation.
Electron Flow and ATP synthesis
 Two protons are generated for each water molecule that is split by PSII.
 Four protons are translocated across the membrane for every two
electrons transferred through the electron transport chain.
 Resulting in a total of 12 protons translocated for every molecule of O 2
produced.
 This proton motive force is then used by ATP synthase to produce ATP
(Noncyclic phosphorylation)
 When cells require less NADPH, cyclic phosphorylation occurs
• Electrons from PS I recycled back to PS II.
• Recycled electrons used to generate PMF which supports additional ATP.
Respiratory Processes Defined by donor
acceptor
(Chapter 14: pp. 476-481; 482-488)
28 March 2022
mby_251_biology_of_microorganisms | up | BGM | ThabisoMotaung
In today’s lecture
I.
Oxidation of Sulfur Compounds
II.
Iron (Fe2+) Oxidation
III.
Nitrification
IV.
Anaerobic Ammonia Oxidation
V.
Nitrate Reduction and Denitrification
VI.
Sulfate and Sulfur Reduction
Respiratory Processes defined
by electron donor
Respiratory Processes defined
by electron acceptor
Respiratory Processes
defined by electron
donor
I. Oxidation of Sulfur Compounds
Page 125-127
Chemolithotrophy
 Organisms that obtain energy from the oxidation of
inorganic compounds.
 Most use CO2 as a carbon source and are therefore
autotrophs.
 Some chemolithotrophs require an organic
compound as a carbon source and are called
Mixotrophs.
Chemolithotrophy
 Sources of reduced molecules (inorganic electron donors) exist in the
environment.
• Geological: volcanic activity source of H2S.
• Agriculture and mining; Nitrogen and Iron compounds.
• Biological sources especially production of H2S, H2 and NH3.
 The E0’ of a number of inorganic compounds when oxidized with O 2 as
an electron acceptor, can provide sufficient energy for ATP synthesis.
 The oxidation of different reduced compounds yields varying amounts
of energy.
Energetics of Sulfur Oxidation
 Sulfur oxidation:
• An essential component of the earth's sulfur cycle.
• Hydrogen sulfide (H2S), elemental sulfur (S0), thiosulfate (S2O32−) and sulfite (SO32−).
• In most cases, the final oxidation product is sulfate (SO 42−).
• First oxidation step yields S0:
― Deposited inside the cell as energy (electron) reserve (Beggiatoa).
― External S0 is insoluble – bacterial cell must attach itself to the sulfur particle.
― Additional energy can be conserved from the oxidation of sulfur to SO42−.
• Protons are one product of reduced sulfur.
― Sulfur chemolithotrophy acidifies the environment.
― Many sulfur bacteria have evolved to be acid-tolerant or even acidophilic.
The Sox System
 Sox system (Paracoccus pantotrophus):
• Four periplasmic proteins:
1.
SoxXA, attaches a reduced sulfur compound to the carrier protein SoxYZ.
2.
SoxYZ, carrier protein.
3.
SoxB, releases the sulfur compound as SO42− from SoxYZ.
4.
SoxCD, sulfur dehydrogenase that catalyzes removal of 6 e- from the bound sulfur
atom to ETC.
• Electrons go to ETC while protons acidify the environment (NB: Not for PMF
generation).
• Electrons transferred from the donor enter at cyt c.
Alternative pathway of sulphur
oxidation
 Sulfur atom bound to carrier Sox YZ added to growing sulphur granule in periplasm.
 The sulphur granule becomes reductively activated and transported to cytoplasm.
•
Oxidized to SO32- by reverse activity of DsrAB (dissimilatory sulfite reductase).
 SO32- then oxidised to SO42− by one of two ways:
• Reverse activity of sulphite reductase:- oxidizes SO42− and transfer electrons to ETC.
• Reverse activity of adenosine phosphosulfate reductase. The process yields 1 ATP by substrate
level phosphorylation. Electrons go to ETC.
 Electrons enter ETC at flavoproteins or cyt c, eventually to O2 establishing PMF.
 Reducing power:
•
Electrons for CO2 fixation formed by reverse electron transport to yield NADH
• Autotrophy occurs by Calvin cycle or some other pathway
1.
SoxXA, attaches a reduced sulfur compound to
the carrier protein SoxYZ.
2.
SoxYZ, carrier protein.
3.
SoxB, releases the sulfur compound as SO42−
from SoxYZ.
4.
SoxCD, sulfur dehydrogenase that catalyzes
removal of 6 e- from the bound sulfur atom to
ETC.
II. Iron (Fe2+) Oxidation
Page 478-479
Iron-Oxidizing Bacteria
 Bacteria (Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans) that
use ferrous iron as electron donor are common in acid-polluted environments
(pH = 1).
 Ferroplasma (Archaea) is an extremely acidophilic iron oxidizer (pH < 0).
 Neutral pH:
• Fe2+ spontaneously oxidizes to Fe3+.
• Iron bacteria are restricted to locations where Fe2+ transitions to Fe3+.
• Iron bacteria oxidize Fe2+ to Fe3+ before it oxidizes spontaneously.
• Gallionella ferruginea, Sphaerotilus natans, and Leptothrix discophora.
Energy from Iron Oxidation
 The ETC has cyt c and cyt aa3 and cytoplasmic protein called rusticyanin.
 Cyt c oxidizes Fe2+ to Fe3+, releasing one electron and transmits it to
rusticyanin.
 Rusticyanin then reduces periplasmic cyt c, which transfers electrons to cyt
aa3, which interacts with O2 to form H2O.
 ATP is synthesized from proton-translocating ATPase in the cytoplasmic
membrane.
 Autotrophy is driven by the Calvin cycle and energy is consumed in reverse
electron flow reactions to obtain reducing power (NADH for CO2 fixation).
Energy from Iron Oxidation
 Ferrous oxidation under anoxic conditions
• Some anoxygenic phototrophs can oxidize Fe2+ anaerobically using
Fe2+ as an electron donor and/or reductant for CO2 fixation.
• Fe2+ reduce cyt c to initiate ETC.
• Chemolithotrophs use nitrate as acceptor, producing nitrite or
dinitrogen.
• Phototrophs use either Fe2+ or iron sulfide as donor.
Energy from Iron Oxidation
• Rusticyanin receives electrons from Fe2+
oxidized by a c-type cytochrome located in
the outer membrane.
• Electrons travel through a short electron
transport chain, resulting in the reduction of
O2 to H2O.
III. Nitrification
Page 479-481
Nitrification
 Oxidation of inorganic nitrogen compounds (NH3 and NO2–)
by nitrifying bacteria.
 Two sets of reactions work in concert to fully oxidize.
ammonia to nitrate:
• Oxidation of NH3 to nitrite by ammonia oxidizers (Nitrosomonas) .
• Oxidation of nitrite to nitrate by nitrite oxidizers.
NB: Only Nitrospira can catalyze both reactions, oxidizing NH3 completely to
nitrate.
Nitrification
 Complete oxidation of NH3 to nitrate involves transfer of 8e–.
 Process involves several enzymes:
• Ammonia monooxygenase (membrane protein): Oxidizes NH3 to
hydroxylamine (NH2OH) and H2O.
• Hydroxylamine oxidoreductase (periplasmic): oxidizes NH2OH to
NO2–. Removes 4e–.
Nitrification
 Electrons originate from oxidation of NH2OH and are supplied to AMO
from NH2OH reductase via cyt c and ubiquinone.
 Only 2 of the 4 electron reach cyt aa3 to form water.
 In nitrite oxidizing bacteria using nitrite reductase, electrons travel from
NO3/NO2– to cyt aa3. Activity of cyt aa3 generate PMF.
 NADH to drive the Calvin cycle is formed by reverse electron flow.
 Aerobic nitrifying bacteria employ the Calvin cycle for CO2 fixation.
Nitrification
 Ammonia oxidizing bacteria are either obligate chemolithotrophs or
mixotrophs.
 Ammonia oxidizing Archaea fix CO2 using a variation of
hydroxypropionate cycle.
 Ecological role of nitrifiers:
• They convert NH3 to nitrate, a key plant nutrient.
• In wastewater treatment, they remove toxic amines and ammonia from water,
producing less toxic nitrogen compounds.
• They decompose nitrogen compound to nitrite, a usable nitrogen source for algae
and cyanobacteria.
Nitrification
NH3-oxidizing bacteria
Oxidation of NO2− to NO3− by nitrifying bacteria
IV. Anaerobic Ammonia Oxidation
Page 481-482
Anammox
• Obligate anaerobic bacteria oxidize ammonia under anoxic conditions by
process of annamox.
• NH3 is oxidized with nitrite as the electron acceptor to yield nitrogen gas.
• NO2- reduced to nitric oxide (NO) by nitrite reductase.
• NO reacts with NH4+ to form hydrazine (N2H4) by activity of N2H4 synthase.
• Hydrazine dehydrogenase oxidizes N2H4 to N2 plus electrons.
• Electrons channeled to ETC; ATP formed by ATPase in annamoxosome
membrane.
Anammox
 Anammoxosome is compartment where anammox reactions occur.
• Has strong diffusion resistant membrane to protects cell from reactions occurring
during anammox.
• N2H4 a strong reductant, is an intermediate of anammox.
• It is toxic and is enclosed in the annamoxosome until it is degraded to harmless
N2.
 Reducing power derived from ETC (independent set of electrons).
 Annamox bacteria fix CO2 using reductive acetyl-coA pathway.
 Anammox is very beneficial in the treatment of sewage and wastewater
(removes NH3 and amines).
Anammox
Respiratory Processes
defined by electron
acceptor
III. Nitrate Reduction and Denitrification
Page 482-483
Nitrate Reduction and Denitrification
• Inorganic nitrogen compounds are the most common
electron acceptors in anaerobic respiration.
• All products of nitrate reduction (denitrification) are gaseous
(e.g. N2O, NO, N2).
• Denitrification is the main biological source of gaseous N2.
• The first enzyme, nitrate reductase requires absence of
oxygen and presence of nitrate to be fully expressed.
Nitrate Reduction and Denitrification
• It catalyses reduction of nitrate to nitrite (NO2-).
• In E. coli: Nitrate reductase accepts electrons from b-type cytochrome.
• In Paracoccus denitrificans and Pseudomonas stutzeri: Nitrogen oxide
formed from nitrite by enzymes Nitrite reductase, NO reductase and
Nitrous oxide reductase.
• NO and N2O are gaseous and they are lost from the cell.
• During electron transport the PMF is established and ATP is
synthesized.
Nitrate Reduction and Denitrification
Nitrate Reduction and Denitrification
 Ecological importance of denitrification:
• BAD:
― In agriculture it removes nitrate from soil (Makes nitrogen less available to
plants)
― N2O is a greenhouse gas (contributes to climate change)
― N2O can also be converted to NO by sunlight. NO consumes ozone layer to
form acid rain.
• GOOD:
― In sewage treatment, it removes fixed nitrogen and thereby
reduce algal growth (i.e., Reduce eutrophication).
IV. Sulfate and Sulfur Reduction
Page 483-486
Sulfate and Sulfur Reduction
• Inorganic sulphur compounds used as electron acceptors.
• Sulfate reduced by sulfate reducing bacteria to form H2S.
• Assimilative sulfate metabolism: sulfate incorporated for biosynthesis purposes
(cysteine, methionine, etc).
• Dissimilative sulfate metabolism: sulfate used as electron acceptor for energy
conservation.
• Sulfate reduction less favourable electron acceptor than O2 and NO3-.
• Hydrogen used as donor, but other alternative donors can be used by few organisms.
• Reduction of SO42- to H2S requires 8 electrons.
Sulfate and Sulfur Reduction
• The reduction of SO42- to H2S proceeds through several intermediates
and requires activation of sulfate by ATP.
• The enzyme ATP sulfurylase activates sulfate forming adenosine
phosphosulfate (APS).
• In dissimilative metabolism APS is reduced directly to sulfite by the
enzyme APS reductase.
• In assimilative reduction another phosphate is added to APS, forming
PAPS, which is then reduced to sulfite.
Sulfate and Sulfur Reduction
• In both cases sulfite is converted to sulfide by sulfite reductase.
• Dissimilative reduction: ETC generates PMF that drives ATP synthesis.
• Major electron carrier is cyt c3 which accepts electrons from periplasmic
hydrogenase.
• Cytochrome c3 transfers electrons to APS reductase to form sulfite or to
sulfite reductase to form sulfide.
• Hydrogenase player major role to establish PMF.
Sulfate and Sulfur Reduction
Homework/self study:
Fig. 14.5: pp 467
Theme 5
Prof Moleleki
Chapter 10 pg 328- - 359 16th Edition
Chapter 9 pg 277 – 307 15th Edition
Learning outcomes
• Understand the terminology – not memorise
• What is ‘Omics? [Systems Biology]
• Intergrates
genomics,
transcriptomics,
proteomics and metabolomics
• Understand the different ‘omics (DNA, RNA,
Protein, Metabolome)
• Technologies used in each ‘omic
• What type of information to obtain
• What problems can be solved the ‘omics
• Application: How and where do we use it? (which
‘omics)
Omics Terminology:
•
•
•
•
•
Genome (comparative and functional)
Metagenome
Epigenome
Methylome
Mobilome
•
•
Transcriptome
Metatranscriptome
•
Proteome
(Metaproteome)
Translatome
Interactome
Secretome
Metabolome
Glycome
•
•
•
•
•
Introduction to Genomics
• What is Genomics?
• What can we learn from genomics?
• How do we obtain genomes (tools or technologies)?
• First generation
• Second generation
• Third generation
• Functional and comparative genomics
• New gene functions
Utility of Microbial Genome Sequences
Introduction to Genomics
• Genomics: (functional and comparative)
• Discipline of sequencing, analyzing/annotation,
and comparing genomes
• Gene knock outs/mutations (mutagenesis using
Tn-Seq, CRISPR)
Sequencing Technologies
• Sequencing: determining the precise order of
nucleotides in a DNA or RNA molecule
• First generation sequencing: Sanger dideoxy
method
• Invented by Nobel Prize winner Fred Sanger
• Second-generation DNA sequencing
• Generates data 100x faster than Sanger method
• Massively parallel methods
• Large number of samples sequenced side by side
• Uses increased computer power and miniaturization
• 454 Life Sciences pyrosequencing
• Illumina/Solexa sequencing
• SOLID/Applied Biosystems method
Sequencing Technologies
• Third-generation DNA sequencing
• Sequencing of single molecules of DNA
• HeliScope Single Molecule Sequencer
• Pacific Biosciences SMRT
• Single Molecule Real Time sequencing
• Reactions carried out in nanocontainers (zero mode wave guides)
Sequencing Technologies
• Fourth-generation DNA sequencing
• Oxford Nanopore Technologies system
• Optical detection no longer used
• Ion torrent semiconductor sequencing
DNA Sequencing methods/Technologies
Generation
Method
Features
First generation
Sanger dideoxy method (radioactivity or
fluorescence; DNA amplification)
Read length: 700–900 bases
Used for the Human Genome Project
Second
generation
454 Pyrosequencing
(fluorescence; DNA amplification; massively
parallel)
Illumina/Solexa method
(fluorescence; DNA amplification; massively
parallel)
Read length: 400–700 bases
Used to sequence genome of James Watson
(completed 2007)
Read length: 50–150 bases
Giant panda genome (2009; Beijing Genome
Institute); Denisovan genome (2010)
Read length 50–100 bases
SOLiD method
(fluorescence; DNA amplification; massively
parallel)
Ion torrent
(electronic—pH; DNA amplification)
Third generation
Pacific Biosciences SMRT
(fluorescence; single molecule; zero mode
waveguide)
Oxford nanopore
(electronic—current; single molecule; real time)
Read length: 200–400 bases
Used to sequence genome of Intel cofounder
Gordon Moore
Read length: 2500–3000 bases
Read length: 9000 bases
Portable MinION unit is approximately the size of a
flash drive
Introduction to Genomics
• Genome
• Entire complement of genetic information
• Includes genes, regulatory sequences, and noncoding
DNA
Non coding intergenic region
Transcription
regulator
What can genomics tell us?
• Size
• infer lifestyle
• Prediction of metabolic pathways
• Prediction of pathogenicity and
virulence factors
Genome Sizes of Microbial Cells and Higher Organisms
Kb = thousand bases
Mb = million bases
Gb = billion bases
Genome Size and Content
• Correlation between genome size and ORFs
• On average, a prokaryotic gene is 1,000 bp long
• ~1,000 genes per megabase
(1 Mbp = 1,000,000 bp) Mbp – million base pairs
• As genome size increases, gene content proportionally increases
• How many genes would you expect from a bacterium with a genome of
4Mbp?
• Match the following genome sizes in relation with the correct organism.
Organism
Genome size
Sunflower
5 000 000 bp
Escherichia coli
50 Kbp
Bacteriophage
5 Gbp
Genome Sizes: Bacteria
Small (100 500kbp)
Nanoarchaeum
equitans
Mycoplasmas
• Based on their genome sizes,
what can you infer about the
life styles of these two
bacteria – N. equitans and
Mycoplasma and why?
Standard (4 – 5
Mbp)
Escherichia spp
Bacillus spp
Large genomes
(12 Mbp)
Streptomyces
Myxobacteria
• Smallest belong to parasitic or endosymbiotic
prokaryotes
• totally dependent on host for nutrients; provide
host with essential amino acids and other
nutrients the host cannot synthesize
• Most are unable to grow in the absence of the
host
• Based on their genome sizes,
what can you infer about the
life styles of Streptomyces
and myxobacteria – Theme
• Hint structural complexity
Functional and Metabolic Predictions for Vampirovibrio
chlorellavorus Based on Genomic Annotation
Genome Size and Content
• Many genes can be identified by sequence similarity to genes
found in other organisms (comparative genomics/analysis)
• Functional and Comparative genomics
• Predictions of metabolic pathways and
• Transport systems
• Virulence factors
Functional Genomics
• Selection for sequencing often results from
interesting phenotype
• e.g., Paenibacillus strain LC231 (Figure
10.14)
• Cultured from underground cave
ecosystem isolated from surface for
over 4 million years
• Displays resistance to 14 classes of
antibiotics – how?
• 10 ORFs encoding resistance to 7
different antibiotic types
• Heterologous expression (expressing
a gene in a different host) in
Escherichia coli
• Found five new antibiotic resistance
genes
Metagenomics
• Metagenome
• The total gene content of the organisms present in an environment
• Several environments have been surveyed by large-scale metagenome
projects
• Examples: acid mine runoff waters, deep-sea sediments, fertile soils, humans
particularly the gut, plant roots or leaf surfaces
• Metagenomics: total gene content of microbial community
• Metatranscriptomics - gene expression profiles within a microbial community
• Metaproteomics explore protein expression patterns within a microbial
communities
Metagenomics
Metagenomics: Human microbiome
Google human gut microbiome
and health benefits
Omics Terminology:
•
•
•
•
•
Genome (comparative and functional)
Metagenome
Epigenome
Methylome
Mobilome
•
•
Transcriptome
Metatranscriptome
•
Proteome
(Metaproteome)
Translatome
Interactome
Secretome
Metabolome
Glycome
•
•
•
•
•
Transcriptome
• Transcriptome
• The entire complement of RNA produced
under a given set of conditions
• Hybridization to measure gene expression
• Microarrays
• Small solid-state supports to which genes or
portions of genes are fixed and arrayed
spatially in a known pattern
Chip Design and Application
Transcriptome
• What can be learned from microarray experiments?
• Global gene expression
• Expression of specific groups of genes under different
conditions
• Expression of genes with unknown function; can yield
clues to possible roles – help with genome annotation
• Comparison of gene content in closely related
organisms
• Identification of specific organisms
Transcriptome
• RNA sequence (RNA_Seq) analysis
• All RNA molecules from a cell are sequenced
• Measures:
• Amount of expressed mRNA
• Amount of noncoding RNA (advantage over microarrays)
• Used with the DNA sequence, you can determine:
• Which genes are transcribed
• How many copies of each RNA are made
Omics Terminology:
•
•
•
•
•
Genome (comparative and functional)
Metagenome
Epigenome
Methylome
Mobilome
•
•
Transcriptome
Metatranscriptome
•
Proteome
(Metaproteome)
Translatome
Interactome
Secretome
Metabolome
Glycome
•
•
•
•
•
Proteomics and the Interactome
• Proteomics
• Genome-wide study of the structure, function, and regulation of an
organism's proteins
• Proteome: – all proteins encoded in the genome
• Translatome: – proteins translated under specific conditions
• Secretome: - secreted proteins (Theme 6)
• Which is larger and why, the proteome or the translatome?
Proteomics and the Interactome
• Proteomics: genome-wide study of structure, function, and activity of an organism’s
proteins
• Proteome: all proteins encoded or only those present at a given time (translatome:
under specific conditions)
• Methods in Proteomics
• Mass spectrometry allows unambiguous determination of molecular formula, and
can be used to identify peptides (Figure 10.23)
• High-pressure liquid chromatography (HPLC) is used to separate proteins by
differences in chemical properties (e.g., size, charge, hydrophobicity)
• Proteins collected after HPLC, digested by proteases, resulting peptides identified
by mass spectrometry and compared with translated genome
• Matrix-assisted laser desorption ionization (MALDI): advanced mass spectrometry
method where sample is fixed to matrix, ionized, vaporized, and molecular formula
is determined (MALDI-TOF) (Figure 10.24)
Proteomics
Proteomics and the Interactome
• Interactome
• Complete set of interactions among molecules
• Data expressed in the form of network diagrams
• Network diagrams can also be use RNA (Transcription Regulators)
Omics Terminology:
•
•
•
•
•
Genome (comparative and functional)
Metagenome
Epigenome
Methylome
Mobilome
•
•
Transcriptome
Metatranscriptome
•
Proteome
(Metaproteome)
Translatome
Interactome
Secretome
Metabolome
Glycome
•
•
•
•
•
Metabolomics
• Metabolome
• The complete set of metabolic intermediates and other small molecules
produced in an organism
• Mass spectrometry is one of the primary techniques for
monitoring metabolites
•
•
•
•
NMR Nuclear Magnetic Resonance
MALDI: Matrix assisted laser desorption ionization
TOF: Time of flight
NIMS: Nanostructure Initiator Mass Spectroscopy
Metabolomics
• Metabolome: complete set of metabolic intermediates and
small molecules produced in an organism
• Reflects enzymatic pathways
• Confirms reactions occurred
• Advances in Metabolomic Techniques: NIMS
• Technically challenging due to immense chemical diversity
• Nanostructure-initiator mass spectrometry (NIMS) can
directly analyze samples without special preparation
(Figure 10.27)
Metabolomics
• Utility of Metabolomics
• Plant biochemistry: plants produce several thousand
metabolites
• Secondary metabolites: scents, flavors, alkaloids, pigments
• Used to study microbial cultures and communities (e.g.,
biofilms)
• Characterize human microbiome (Figure 10.28) and
diverse metabolites produced by humans and microbes
Theme 6:
Human and Plant-Bacterial
Interactions
05/04/2022
Chapter 25 pages 850 – 868 16th Edition
Chapter 25 pages 794 – 808 15th Edition
Human– Plant Bacterial Interactions
• Terminology
• Infection and disease
• Pathogenicity, virulence, attenuation
• Virulence Factors: Factors that bacteria use to cause disease
• Adherence
• Adhesins, fimbrae, pili and capsule
• Enzymes
• Coagulase
• Streptokinase
• hyauronidase
• Toxins
• AB Toxins Exotoxin
•
•
Exotoxin – botulinun, tetanus and diphtheria toxin
Enterotoxins – Cholerae toxin
• Cytolytic Exotoxin
• Endotoxins
• T3SS Effectors
Human– Plant Bacterial Interactions
• Pathogens
• An organism that grows in or on a
host and causes disease
• Pathogenicity
• The ability of a pathogen to cause
disease
• Virulence
• Measure of pathogenicity
• Virulence factors:
• Factors or strategies used by
bacteria to cause disease
• Compromised host:
• a host with low resistance – eg HIV
or cancer patient
• Opportunistic pathogen
• Causes disease only in the absence
of normal host resistance
• Attenuation
• The decrease or loss of virulence
• Toxicity
• Organism causes disease by means
of a toxin that inhibits host cell
function or kills host cells
• Toxins can travel to sites within host
not inhabited by pathogen
• Effectors
• Secretion and translocated virulence
factors that interfere with host
defenses
Human and Plant-Bacterial Interactions
• Human Entry Points
Microbial infection of an animal cell
• Natural openings
• nose, urogenital, mouth
• Mucous membranes
• Wounds
• Insect bites
• Plants Entry Points
• Natural openings
•
Stomata, hydathodes, lenticels
• Wounds
• Insect bites
• Compatible host/tissue
• the pathogen is able to cause disease
• Incompatible host/tissue
• The pathogen is NOT able to cause
disease
Microbial infection of a plant cell
Entry of the Pathogen into the Host –
Adherence
• Bacterial adherence can be facilitated by
• Extracellular macromolecules that are not covalently
attached to the bacterial cell surface
• Examples: slime layer, capsule
• Adhesins
• Fimbriae and pili
• Flagella
Human and Plant-Bacterial Interactions
• Human Entry Points
Microbial infection of an animal cell
• Natural openings
• Nose, urogenital, mouth
• Mucous mebranes
• Wounds
• Insect bites
• Plants Entry Points
• Natural openings
•
Stomata, hydathodes, lenticels
• Wounds
• Insect bites
• Compatible host/tissue
• the pathogen is able to cause disease
• Incompatible host/tissue
• The pathogen is NOT able to cause
disease
Microbial infection of a plant cell
How do bacteria virulence transported into
host cell?
Human and Plant-Bacterial Interactions
• Virulence Factors: Plant Pathogen
Virulence Factors: Tissue damaging Enzymes
• Coagulase
•
•
•
•
Produced by pus forming Staphylococcus aureus
induces fibrin clotting
localized
protects pathogen from host immunity
• Hyaluronidase
•
•
•
•
Produced by pus forming Streptococcus pyogenes
dissolves hyaluronic acid in connective tissue
allows bacteria to spread
Systemic
Virulence factors: Plant Cell Wall Degrading
Enzymes
• Plant Cell Wall Degrading Enzymes
• Type II Secretion System
• Examples
• Pectinases
• Cellulases
• Proteases
Virulence Factors: Exotoxins
• Toxicity is the ability of an organism to cause disease
by means of a toxin that inhibits host cell function or
kills host cells
• Exotoxins (Table 25.2)
• proteins released from the pathogen cell as it grows
• three categories
• Cytolytic toxins
• AB toxins
• Superantigen toxins
Virulence Factors: Exotoxins
• AB toxins
• Consist of two subunits, A and B
• Work by binding to host cell receptor (B subunit) and
transferring damaging agent (A subunit) across the cell
membrane
• Examples: diphtheria toxin, tetanus toxin, botulinum toxin
• Table 25.2
© 2019 Pearson Education Ltd.
© 2019 Pearson Education Ltd.
© 2019 Pearson Education Ltd.
Virulence Factors: Exotoxins
• Exotoxins - Enterotoxins
• AB Type toxin
• Produced by Vibrio cholerae
• Cholera toxin, Shiga toxin
Virulence Factors: Exotoxins
• Cytolytic Exotoxins
• Work by degrading cytoplasmic membrane integrity,
causing cell lysis and death
• Toxins that lyse red blood cells are called hemolysins
• Clearing zone of Streptococcus pyogenes
Endotoxins
• Endotoxin
• The lipopolysaccharide portion of the cell envelope of
certain gram-negative Bacteria, which is a toxin when
solubilized
• Generally less toxic than exotoxins
• The presence of endotoxin can be detected by the Limulus
amoebocyte lysate (LAL) assay
Endotoxins
• Endotoxins are the toxic lipopolysaccharides found in the cell walls of most gramnegative Bacteria
• Endotoxins are not proteins but are structural components of the gram-negative
outer membrane
• They are the secreted products of living cells
• Endotoxins are cell bound and released in toxic amounts only when the cells lyse
• The basic properties of exotoxins and endotoxins are compared in Table 25.3
Properties of Exotoxins and Endotoxins
Property
Exotoxins
Endotoxins
Chemistry
Proteins, secreted by certain gram-positive or gramnegative Bacteria; generally heat-labile
Lipopolysaccharide–lipoprotein complexes,
released on cell lysis as part of the outer
membrane of gram-negative Bacteria;
extremely heat-stable
Mode of action;
symptoms
Specific; usually bind to specific cell receptors or
structures; either cytotoxin, enterotoxin, or
neurotoxin with defined, specific action on cells or
tissues
General; fever, diarrhea, vomiting
Toxicity
Often highly toxic in picogram to microgram
quantities, sometimes fatal
Moderately toxic in tens to hundreds of
microgram amounts, rarely fatal
Immune response
Highly immunogenic; stimulate the production of
neutralizing antibody (antitoxin)
Relatively poor immunogens; immune
response not sufficient to neutralize toxin
Toxoid potential a
Heat or chemical treatment may destroy toxicity, but
treated toxin (toxoid) remains immunogenic
None
Fever potential
Nonpyrogenic; do not produce fever in the host
Pyrogenic; often induce fever in the host
Genetic origin
Often encoded on extrachromosomal elements or
lysogenic bacteriophages
Encoded by chromosomal genes
aA
toxoid is a modified toxin that is no longer toxic but can still elicit an immune response against the toxin (►Section 28.3).
Review these properties of exotons compared to endotoxins
Virulence factors: T3SS Effectors
1. Work through homework exercises
2. Theme 6 Class Test will be on 11th April
3. BlackBoard Collaborate Session (11th April)
to discuss long questions (Homework)
Theme 7 Ecological/Functional
Diversity
Ecological Diversity of Phototrophs
Lecture 1 of 3
BBOM 16th Edition pg 514 – 528
BBOM 15th Edition pg489 – 494
Important Terminology - simplified
•
•
•
•
•
•
•
•
•
•
•
Lineage – descent or ancestry
Phylum – a major lineage in one of the three domains of life
Phylogeny – evolutionary history of an organism (tree)
Ecology – relationship of an organism with its environment
Physiology – functions and processes allowing an organism to grow and multiply
Adaptation/adaptation strategies – processes/strategies allowing an organism to
become better suited for living in its environment
Anaerobic respiration – a form of respiration in which the electron acceptor is not
oxygen
Aerobic respiration – respiration that requires oxygen
Oxygenic photosysthesis – photosynthesis in which oxygen produced
Anoxygenic photosynthesis – photosynthesis which does not produce oxygen
Enrichment media – media that favor growth of certain bacteria
Microbial Diversity
• Metabolic diversity: Defined in terms of cellular processes
supporting growth
• Nitrogen fixing bacteria, iron oxidizer etc
• Phylogenetic diversity: Defined by evolutionary
relationships between organisms
• diversity of phyla, genera, species
• genetic and genomic diversity of evolutionary lineages
• defined by 16s ribosomal RNA phylogeny or
housekeeping genes (refer to MBY 251 practical)
• Ecological diversity: Defined in terms of microbial
interactions between organisms and their environments
• Useful but incorrect to consider ecological diversity only
in terms of metabolism
• Domain – Bacteria
• Phylum - Proteobacteria
• Class –
Gammaproteobacteria
• Order – Enterobacteriales
• Family –
Enterobacteriaceae
• Genus – Escherichia
• Species - coli
Functional Traits Mapped Across Major Phyla of
Bacteria and Archaea
Factors Driving Microbial Diversity
• At least three reasons metabolic and ecological traits are shared
by divergent organisms
• Gene loss: A trait is present in a common ancestor is lost
during divergence over time
• Convergent evolution: A trait has evolved independently in
two or more lineages and is not encoded by homologous
genes
• Horizontal gene transfer: Genes that code for a trait are
homologous and have been exchanged between distantly
related lineages
Ecological Diversity of Prokaryotes
• Phototrophs:
• Oxygenic and anoxygenic
• Oxygenic phototrophic bacteria
• Cyanobacteria
• Ecology, physiology, Phyla
• Anoxygenic phototrophs
• Ecology, physiology, phyla
• Purple sulfur and purple non sulfur
• Green sulfur and green non sulfur
• Other
Overview of Phototrophic Bacteria
• Ability to conserve energy from light
evolved early on when Earth was
anoxic
• Photosynthesis originated within
Bacteria
• First phototrophs were anoxygenic
phototrophs that do not generate O2
as a product
• instead of water, used H2, Fe+2, or
H2S as electron donor
• Extensive diversity among anoxygenic
phototrophs
Overview of Phototrophic Bacteria (Recap)
• Several common features
• Use chlorophyll-like and accessory pigments to harvest energy from
light and transfer to membrane-bound reaction center to drive
electron transfer that produces ATP
• Two types of reaction centers
• FeS in photosystem I of oxygenic phototrophs
• Q-type found in photosystem II of oxygenic phototrophs
• Both found in cyanobacteria (see Figure 14.16)
• Pigments often found in intracellular membrane systems
that allow phototrophic bacteria to better use light of low
intensities
• Many but not all fix carbon
Phototrophic bacteria: Cyanobacteria
• Oxygenic phototrophic bacteria
• Cyanobacteria
• Ecology, physiology, Phylogeny
• Anoxygenec phototrophs
• Ecology, physiology, phylogeny
• Purple sulfur and purple non sulfur
• Green sulfur and green non sulfur
• Other
Cyanobacteria
• Key genera: Prochlorococcus, Crocosphaera,
Synechococcus, Trichodesmium, Oscillatoria, Anabaena
• First oxygen-evolving phototrophs
• Large, morphologically, ecologically heterogeneous group of
oxygenic, phototrophic Bacteria
• range from 0.5 to 100 μm in diameter cf E. coli 2 μM
• Five morphological groups
• unicellular or filamentous
• Cyanobacteria are distantly related to Gram positive
bacteria
Cyanobacteria: general information
• Chroococcales: unicellular, divide by binary fission
• Includes prochlorophytes (unique, unicellular)
• Pleurocapsales: unicellular, dividing by multiple fission
(colonial)
• Oscillatoriales: filamentous nonheterocystous
• Nostocales: filamentous, divide on single axis, can
differentiate
• Stigonematales: filamentous, divide in multiple planes,
forming branching filaments
Cyanobacteria
• Physiology and Photosynthetic Membranes
• Oxygenic phototrophs with both FeS and Q-type photosystems
• All fix CO2 by the Calvin cycle
• Many fix N2
• Most synthesize own vitamins
• Harvest energy from light and fix CO2 during day
• Generate energy by fermentation or aerobic respiration of carbon storage products (e.g., glycogen)
at night
Cyanobacteria
• Physiology and Photosynthetic
Membranes
• Thylakoids: specialized membrane
systems that increase ability to
harvest light energy typically
arranged in concentric circles
around cytoplasm periphery
• Produce pigments (chlorophyll a
and phycobilins: accessory
pigments)
Thylakoids in Cyanobacteria
Cyanobacteria
• Physiology: germination and vegetative growth
• Some form akinetes (resting structures with
thickened outer walls that protect the organism from
darkness, desiccation, or cold)
• Physiology and motility
• Cyanobacteria have motility mechanisms but no flagella
• Many cyanobacteria display gliding motility when in
contact with solid surface, another cell, or filament
• Some form hormogonia (short, motile filaments that break
off to facilitate dispersal under stress
• Most show phototaxis (toward light); chemotaxis may
occur
• Gas vesicles (regulating buoyancy) important in positioning cells
in water column where light intensity is optimal
Cyanobacteria
•
Nitrogen Fixation
• Many form cyanophycin
• nitrogen storage product
• Nitrogen rich reserve polymer
• Nitrogenase is sensitive to oxygen, so fixation cannot occur
along with oxygenic photosynthesis
• THREE regulatory mechanisms separate nitrogenase from
photosynthesis
1. Many unicellular Cyanobacteria fix nitrogen only at night
2. Some transiently suppress photosynthetic activity within
filaments
3. Many filamentous Cyanobacteria form Heterocysts
• Arise from differentiation of vegetative cells
• Surrounded by thickened cell wall that slows diffusion
of O2 and provides an anoxic environment
Cyanobacteria
• Ecology of Cyanobacteria
• Important for productivity of oceans
• Cyanobacteria are most abundant ocean phototrophs,
contributing 80 percent of marine photosynthesis and 35
percent of total photosynthesis
• Cyanobacterial nitrogen fixation is dominant input of new
nitrogen in oceans
• Also widely distributed in terrestrial and freshwater
environments
• more tolerant of extremes than eukaryotic algae (e.g., hot
springs, saline lakes, desert soils)
• develop freshwater lake blooms
• can be symbionts
Cyanobacteria form symbiotic relationship
with Lichens, ferns and some cycads
• Important metabolic products (e.g., potent neurotoxins and
toxic blooms, geosmin in water)
Death of elephants in Botswana could be due to
toxic blooms caused by gesomins
Homework Question: Check Your
Understanding
• Explain the importance of the following in the physiology and ecology of
Cyanobacteria. In your answer explain how these organelles enable
cyanobacteria to adapt to their environments and perform different
metabolic activities.
• Heterocysts
• Akinetes
• Gas vesicles
Homework Question: Challenge Yourself
• You are looking at a sample from the top green layer of a colorful
bacterial mat. Using phase contrast and fluorescence microscopy, you
see evenly spaced cells in the middle of a filament that are slightly
smaller than the others. The smaller cells are not fluorescent,
indicating they lack photosystem II, but the rest of the filament is
fluorescent. What type of filamentous bacteria are you most likely
looking at?
Homework Question: Challenge Yourself
• You are looking at a sample from the top green layer of a colorful
bacterial mat. Using phase contrast and fluorescence microscopy, you
see evenly spaced cells in the middle of a filament that are slightly
smaller than the others. The smaller cells are not fluorescent,
indicating they lack photosystem II, but the rest of the filament is
fluorescent. What is the most likely function of the smaller cells in the
filament?
Ecological Diversity of Prokaryotes
• Phototrophs:
• Oxygenic and anoxgenic
• Oxygenic phototrophic bacteria
• Cyanobacteria
• Ecology, physiology, Phyla
• Anoxygenec phototrophs
• Ecology, physiology, phyla
• Purple sulfur and purple non sulfur
• Green sulfur and green non sulfur
• Other
Overview of Phototrophic Bacteria
• Ability to conserve energy from light
evolved early on when Earth was anoxic
• First phototrophs were anoxygenic
phototrophs that do not generate O2 as
a product
• instead of water, use H2, Fe+2, or H2S
as electron donor
• Extensive diversity among anoxygenic
phototrophs
• Purple sulfur and purple non
sulfur
• Green sulfur and green non
sulfur
• Other
Purple Sulfur Bacteria
• Key genera: Chromatium, Thermochromatium, Ectothiorhodospira
• Purple sulfur bacteria: Anoxygenic phototrophs that use hydrogen
sulfide (H2S) as an electron donor for photosynthesis
• Found in illuminated anoxic zones where H2S present (lakes, marine
sediments, sulfur springs)
• Have members in Gammaproteobacteria
• Purple color comes from carotenoids (accessory pigments for light
harvesting)
• Use Q-type photosystem, contain bacteriochlorophyll, and fix carbon
with Calvin cycle
Purple Sulfur Bacteria
• During autotrophic growth, H2S oxidized to elemental sulfur (S0) deposited as granules. (Figure 15.10)
• Sulfur can be oxidized to sulfate (SO42−) as an electron donor for photosynthesis
• Some store S0 granules inside (in periplasm) and have vesicular intracellular photosynthetic membrane
systems (Figure 15.11b)
• Some deposit S0 outside cells and have lamellar intracellular photosynthetic membrane systems
• Many can use other reduced sulfur compounds, e.g., thiosulfate (S2O32−)
Sulfur granules inside periplasm
Sulfur granules outside cells indicated
by arrow
Purple Nonsulfur Bacteria and Aerobic Anoxygenic Phototrophs (
• Purple Nonsulfur Bacteria
•
key genera: Rhodospirillum, Rhodoferax, Rhodopseudomonas,
Rhodobacter
• Rhodo - rose
•
most metabolically versatile microbes
• use a wide range of carbon sources and electron donors
for photosynthesis (e.g., organic acids, amino acids,
alcohols, sugars, aromatic compounds) synthesize an array
of carotenoids giving them colors (purple, red, orange)
(Figure 15.12)
• Use very low levels of H2S
•
typically photoheterotrophs (light is energy source, organic
compound is carbon source)
•
use Q-type photosystem and bacteriochlorophyll
•
morphologically and phylogenetically diverse
Alphaproteobacteria or Betaproteobacteria (Figure 15.13)
• Aerobic Anoxygenic Phototrophs (AAP) (related to purple non
sulphur bacteria)
• key genera: Roseobacter, Erythrobacter
Morphologic diversity of Purple nonsulfur bacteria
Green Sulfur Bacteria
• Key genera: Chlorobium, Chlorobaculum, Prosthecochloris, “Chlorochromatium”
• Phylogenetically coherent anoxygenic phototrophs forming the phylum Chlorobi
• Chloro – green
• contain bacteriochlorophyll in chlorosomes in cell periphery
• Chlorosomes are light harvesting organelles
• allow these bacteria to grow at much lower light intensities
• green- and brown-colored species (Figure 15.16)
• Little metabolic versatility; typically nonmotile, strictly anaerobic anoxygenic
phototrophs
• Morphologically restricted, short to long rods (Figure 15.14)
• Oxidize H2S to S0 and SO42− for autotrophy;
• S0 produced form oxidation is deposited only outside cell
Green Sulfur Bacteria
• Green Sulfur Bacteria Consortia
• consortium: intimate twomembered association
• involves the green sulfur
bacterium (epibiont) attached
to a chemoorganotrophic
bacterium (Figure 15.17)
• examples: Chlorochromatium
aggregatum (mixed green
culture) and Pelochromatium
roseum (brown)
Green Nonsulfur Bacteria)
• Key genera: Chloroflexus, Heliothrix, Roseiflexus
• Filamentous anoxygenic phototrophs
• Phylum Chloroflexi
• Class: Chloroflexi are green nonsulfur bacteria.
• Also includes other metabolically diverse organisms
• aerobic and anaerobic chemoorganotrophs
• Most species are uncultivated and uncharacterized
Other Phototrophic Bacteria
• Key genera: Heliobacterium, Heliorestis
• Heliobacteria are phototrophic gram-positive bacteria within phylum Firmicutes
• anoxygenic phototrophs with FeS-type photosystem
• produce unique pigment bacteriochlorophyll g
• grow photoheterotrophically using pyruvate, lactate, acetate, butyrate
• five genera: Heliobacterium, Heliophilum, Heliorestis, Heliomonas, Heliobacillus
• all rod-shaped or filamentous cells (Figure 15.20)
• Heliophilum forms bundles of cells that move as a unit
• strict anaerobes that can ferment pyruvate
• produce endospores
• reside in soils or in highly alkaline environments (e.g., soda lakes and alkaline
soils)
Other Phototrophic Bacteria
• Phototrophic Acidobacteria
• Key genus: Chloracidobacterium
• Found in thermal spring photosynthetic microbial mats of
Yellowstone National Park (USA)
• Chloracidobacterium thermophilum (Figure 15.21)
• thermophilic oxygen-tolerant anoxygenic phototroph
• similar to green sulfur bacteria, producing chlorosomes,
bacteriochlorophyll, and using FeS-type photosystem
• can grow aerobically
• photoheterotroph that uses short-chain fatty acids as
carbon sources
• not autotrophic
Homework: REALLY Challenge Yourself
• Based on the FUNCTIONAL/METABOLIC characteristics of the Cyanobacteria
and Proteobacteria phyla, which phylum do you think is more deeply rooted
in the tree of life (i.e., which phylum branches off closer to the last universal
common ancestor)? Use at least two pieces of evidence to support your
answer. How could you test your hypothesis? (See Figure 15.1 (BBOM 16)
Proteobacteria include Beta, Gamma, Alpha, Zeta and Epsilon)
Theme 7
Ecological Microbial diversity – Chemotrophs and Morphology
BBOM 16th Edition pgs 510 – 552
© 2015 Pearson Education, Inc.
Three
Diversity of Bacteria chemotrophs and morphology
Nitrogen cycle BBOM16 528 – 532 or BBOM15 506 - 510
Diversity of Nitrogen Fixers (Diazotrophs)
freeliving vs symbionts
Diversity of Nitrifiers and Denitrifiers
Sulfur cycle - NOT INCLUDED!
Iron cycle BBOM16 538 – 540 or BBOM15
Dissimilative Iron-Reducers
Dissimilative Iron-Oxidizers
Methanotrophs and Methylotrophs
Morphologically distinct bacteria
© 2015 Pearson Education, Inc.
Nitrogen Cycle
© 2015 Pearson Education, Inc.
Nitrogen fixation
• Importance of nitrogen fixation
• N is an important component of DNA and proteins
• Nitrogen is inert, does not react easily with other
compounds
• Fixation frees N atoms to react with either oxygen or
hydrogen to form nitrates or ammonia
• Why is this important
• N essential for DNA, RNA, protein synthesis
• Plays important role in agriculture
© 2015 Pearson Education, Inc.
Nitrogen Cycle
Objectives
• Know examples of bacteria involved in the following steps
of the nitrogen cycle
• Nitrogen fixing bacteria
• Including mechanisms they use to protect
nitrogenases
• Nitrifying bacteria and archaea that are:
• ammonia oxidizers and their habitats
• nitrite oxidizers and their habitats
Denitrifying and their habitats
© 2015 Pearson Education, Inc.
1. Diversity of Nitrogen-Fixing Bacteria
• Diazotrophs are organisms that can fix N2 gas
into ammonia (NH3)
• Free living diazotrophs
• Key genera: Azotobacter and Azospirullum
• Symbiotic diazotrophs
• Key genera: All rhizobia such as Mesorhizobium,
Bradyrhizobium
© 2015 Pearson Education, Inc.
Diversity of Nitrogen Fixers
• Symbiotic Diazotrophs
• several relationships with plants, animals, and fungi
• Microbial symbiont provides fixed nitrogen to host
• symbiosis between rhizobia and leguminous plants
• Alphaproteobacteria, Betaproteobacteria,
Actinobacteria
• other symbioses
• shipworms and Teredinibacter
• termite guts and Treponema
• Endomycorrhizal fungi and Glomeribacter
• fungi, algae, and plants with Cyanobacteria
© 2015 Pearson Education, Inc.
Rhizobia nitrogenases
• Nitrogen-fixing bacteria need O2 to generate
energy for N2 fixation, but nitrogenases are
inactivated by O2
• In the nodule, O2 levels are controlled by the
O2-binding protein leghemoglobin
© 2015 Pearson Education, Inc.
1. Nitrogen-Fixing Bacteria – Free living
Diazotrophs
• Major genera capable of fixing N2 nonsymbiotically
are Azotobacter and Azospirillum
• All genera produce extensive capsules or slime layers
• Believed to be important in protecting nitrogenase from O2
•
© 2015 Pearson Education, Inc.
Strategies for protection of nitrogenases
• Free-Living Diazotrophs
•
Need to protect nitrogenase from oxygen
•
Strategies for protection of nitrogenase include the following:
1. Simplest solution: Grow anoxically
2. Fix N2 only when oxygen absent or low concentration
• Example: facultative aerobes fix only when anaerobic (Klebsiella)
• Example: microaerophiles (typically less than 2 percent oxygen)
3. Compartmentalise nitrogen fixation in area of low oxygen
• Example Cyanobacteria fix nitrogen within heterocysts
4. Protect with capsule or slime layer
• Example: Azotobacter produce extensive capsules or slime layers and
respire fast to help protect nitrogenase;
5. Oxygen binding proteins
• Example: Rhizobia produce Leghemoglobins to bind oxygen
6. Use alternative nitrogenases
• Examples Azotobacter chroococcum was the first nitrogen-fixer shown
to grow on N2 without molybdenum
• forms alternative, less efficient nitrogenases containing either vanadium
(V) or Fe in place of Mo
• Example Cyanobacteria and Archaea produce backup nitrogenases
© 2015 Pearson Education, Inc.
Diversity of Nitrifying Bacteria and Archaea
• Nitrifying bacteria
• Nitrification (oxidation of ammonia to nitrate) occurs as
two separate steps by different groups of bacteria and
archaea
• Ammonia oxidizers (nitrosifiers; e.g., Nitrosococcus)
• Nitrite oxidizer (e.g., Nitrobacter)
• The two steps were thought to require the two distinct
groups of prokayotes i.e ammonia oxidisers as well as
nitrite oxidizer.
© 2015 Pearson Education, Inc.
Diversity of Nitrifying Bacteria and Archaea
• Nitrifying bacteria
• Many species have internal membrane systems that
house key enzymes in nitrification
• Ammonia monooxygenase: oxidizes NH3 to NH2OH
• Nitrite oxidase: oxidizes NO2− to NO3−
• Widespread in soil and water
© 2015 Pearson Education, Inc.
Diversity Denitrifying Bacteria
• Denitrifying bacteria
• Key genera: Paracoccus, Pseudomonas
• NO3 − and NO2 −  NO, N2O and N2
• Growth by anaerobic respiration of nitrate or nitrite
• Important in agricultural soils
© 2015 Pearson Education, Inc.
Diversity in Other Distinctive Chemotrophic
Bacteria
A. Dissimilative Iron-Reducing Bacteria
B. Dissimilative Iron-Oxidizing Bacteria
C. Methanotrophic and Methylotrophic Bacteria
D. Predatory Bacteria
E. Morphologically Diverse bacteria
© 2015 Pearson Education, Inc.
What you need to know
• A. Dissimilative Iron-Reducing Bacteria
• Give examples - two key genera and their habitats
• B. Dissimilative Iron-Oxidizing Bacteria
• Give examples of two key genera and their habitats
• Describe a distintive physiological feature of Gallionella
© 2015 Pearson Education, Inc.
What you need to know
• C. Methanotrophic Bacteria
• Differentiate between type I and II methanotrophs
• D. Predatory Bacteria
• Describe the lifestyles of Bdellovibrio compared to
Myxococcus
© 2015 Pearson Education, Inc.
What you need to know
• Spirochaetes
• Study Table 15.2: Important genera of spirochaetes
and diseases
• focus on Treponema, Borellia, and Leptospira and
know diseases they cause
• Prosthecate/stalked/budding bacteria
• Describe prosthecate and stalked bacteria.
• You must know benefits of appendages to bacteria
© 2015 Pearson Education, Inc.
What you need to know
• Sheathed bacteria
• How do sheathed bacteria grow under favourable vs
unfavourable condition?
• Magnetic Bacteria
• What are the benefits of magnetosomes to bacteria
© 2015 Pearson Education, Inc.
A. Dissimilative Iron-Reducing Bacteria
• Key genera: Geobacter, Shewanella
• Phylogenetically diverse
• Most species use iron oxide or manganese oxides as
electron acceptors
• Electron donors are organic compounds
• Common in anoxic freshwater and marine
sediments
© 2015 Pearson Education, Inc.
B. Dissimilative Iron-Oxidizing Bacteria
• Key genera: Acidithiobacillus, Gallionella
• Couple oxidation of Fe2+ to cellular growth
• Diversity based on pH and O2
• Iron oxidizers are divided into four functional
groups
• Acidophilic aerobic iron oxidizers iron pyrite
• Neutrophilic aerobic iron oxidizers
• Anaerobic chemotrophic iron oxidizers
• Anaerobic phototrophic iron oxidizers
© 2015 Pearson Education, Inc.
B. Dissimilative Iron-Oxidizing Bacteria
• Acidophilic aerobic iron oxidizers
• Key genera: Acidithiobacillus,
• Ferroplasma – Archaea
• Found in acid mine drainage
© 2015 Pearson Education, Inc.
B. Dissimilative Iron-Oxidizing Bacteria
• Gallionella - nuetrophilic
• Chemolithotrophic iron-oxidizing bacteria
• Possess twisted, stalk-like structure composed of
ferric hydroxide
• Common in waters draining bogs, iron springs, and
other environments rich in Fe2+
© 2015 Pearson Education, Inc.
C. Methanotrophic and Methylotrophic Bacteria
• Methylotrophs
• Organisms that can grow using carbon compounds that
lack C–C bonds
• CH4 – methane, CH3OH – methanol, HCOO- formate
• Most are also methanotrophs
• Methanotrophs
• Organisms that use methane for growth
© 2015 Pearson Education, Inc.
C. Methanotrophic and Methylotrophic Bacteria
• Aerobic methanotrophs
• Key genera: Methylomonas, Methylosinus
• Classification of methanotrophs
• Two major groups:
• Type I
• Type II
• Contain extensive internal membrane systems for
methane oxidation
© 2015 Pearson Education, Inc.
C. Methanotrophic and Methylotrophic Bacteria
• Type I methanotrophs
• Assimilate C1 compounds via the ribulose
monophosphate pathway
• Gammaproteobacteria
• Membranes arranged as bundles of disc-shaped
vesicles
• Most DO NOT fix nitrogen
© 2015 Pearson Education, Inc.
C. Methanotrophic and Methylotrophic Bacteria
• Type II methanotrophs
• Assimilate C1 compounds via the serine pathway
• Alphaproteobacteria
• Paired membranes that run along periphery of cell
• Fix Nitrogen
© 2015 Pearson Education, Inc.
Some Characteristics of Methanotrophic Bacteria
Gammaproteobacteria
Organism
Internal membranes a
Carbon assimilation pathwayb
Methylomonas
One
I
Ribulose monophosphate
I
I
Ribulose monophosphate
I
Ribulose monophosphate and
Calvin cycle
Methylomicrobium
Methylobacter
Methylococcus
One
One
One
Ribulose monophosphate
Alphaproteobacteria
Organism
Internal membranes a
Carbon assimilation pathwayb
Methylosinus
Two
II
II
Serine
II
Serine
Methylocystis
Methylocellac
© 2015 Pearson Education, Inc.
Two
Two
Serine
C. Methanotrophic and Methylotrophic Bacteria
• Ecology and isolation of methanotrophs
• Widespread in aquatic and terrestrial environments
• Certain marine mussels have symbiotic relationships
with methanotrophs
• Reduce global warming
• Agricultural importance as N2 fixers
© 2015 Pearson Education, Inc.
D. Predatory Bacteria
• Key genera: Bdellovibrio, Myxococcus
• Members of Proteobacteria and Bacteroidetes and
Cyanobacteria
• Bdellovibrio
• Prey on other bacteria
• Two stages of penetration
• Obligate aerobes
• Widespread in soil and water, including marine
environments
© 2015 Pearson Education, Inc.
D. Predatory Bacteria
• Myxobacteria
• Group of gliding bacteria that form multicellular
structures (fruiting bodies) and show complex
developmental life cycles
• Lifestyle includes consumption of dead organic (plant)
matter or other bacterial cells
© 2015 Pearson Education, Inc.
D. Predatory Bacteria
• Fruiting myxobacteria exhibit complex behavioral
patterns and life cycles
• lyse other bacteria
• Life cycle consists of :
• Vegetative cells  fruiting bodies  myxospores
© 2015 Pearson Education, Inc.
E. Morphologically Diverse bacteria
© 2015 Pearson Education, Inc.
Spirochetes: tightly coiled bacteria
• Treponema
• Anaerobic host-associated spirochetes that are
commensal or parasites of humans
• Causes syphilis
• Borrelia
• Majority are human or animal pathogens
• Borrelia burgdorferi is the causative agent of Lyme
disease
• B. burgdorferi has a linear chromosome
© 2015 Pearson Education, Inc.
• Syphilis : Caused by Treponema pallidum
• Often transmitted at the same time as gonorrhea
• T. pallidum can be transmitted from an infected woman
to the fetus during pregnancy (congenital syphilis)
• Penicillin highly effective for primary and secondary
stages
© 2015 Pearson Education, Inc.
Prosthecate/Stalked Bacteria
• Prosthecate and stalked Bacteria
• Appendaged bacteria that attach to particulate matter,
plant material, and other microbes in aquatic
environments
• Appendages increase surface-to-volume ratio of the
cells
© 2015 Pearson Education, Inc.
Prosthecate/Stalked Bacteria
• Caulobacter – stalked bacteria
• Produces a cytoplasm-filled stalk
• Often seen on surfaces in aquatic environments with
stalks of several cells attached to form rosettes
• Holdfast structure present on the end of the stalk used
for attachment
© 2015 Pearson Education, Inc.
Sheathed Bacteria
• Key genera: Sphaerotilus, Leptothrix
• flagellated swarmer cells form within a long tube or
sheath
• swarmer cells to in search of nutrients
© 2015 Pearson Education, Inc.
Magnetic Bacteria
• Key genera: Magnetospirillum
• Are magnetotactic, demonstrating directed movement in
a magnetic field
• Magnetospirillum
© 2015 Pearson Education, Inc.
Diversity of Bacteria
Theme 8 Proteobacteria
BBOM 16th pg 556 – 567
Chapters 16 and 23
Taxonomic hierarchy
• Domain – Bacteria
• Phylum - Proteobacteria
• Class –
• Order –
• Family –
• Genus –
• Species -
Deferribacter
Cytophaga
Flavobacteria
Spirochetes
Planctomyces/
Pirellula
Verrucomicrobiaceae
Green sulfur
bacteria
Deinococci
Green nonsulfur
bacteria
Chlamydia
Cyanobacteria
Thermotoga
Actinobacteria
Firmicutes and Mollicutes
Gram-positive
bacteria
Thermodesulfobacterium
Nitrospira

Aquifex




See Figure 17.2
Proteobacteria

See Figure 16.1 in the text book
© 2012 Pearson Education, Inc.
Diversity of Bacteria
• Phylogenetic overview of Bacteria
• More than 90% of characterized genera and species come from four
phyla
•
•
•
•
Proteobacteria
Actinobacteria
Firmicutes
Bacteroidetes
Proteobacteria • Alphaproteobacteria
• Betaproteobacteria
• Gammaproteobacteria – Enterobacteriales
• Gammaproteobacteria – Pseudomonadales and Vibrionales
• Deltaproteobacteria and Epsilonproteobacteria
• Zetaproteobacteria: Mariprofundus ferrooxydans
Alphaproteobacteria:
Chapter 16 pg 557 – 561
Chapter 23 pg 785 -790; 793 - 795
• What you need to know
• Describe steps in the development of root nodules in plants
• What are the benefits of root nodulation to agriculture
• How are nitrogenases protected by root nodulating bacteria
• Agrobacterium and T-DNA transfer (describe the role of individual vir genes in T-DNA
transfer)
• Compare recognition of rhizobia and Agrobacterium, what are the similarities and
differences?
• Name two key genera in the family Rickettsiales and diseases that these cause
Alphaproteobacteria:
Chapter 16 pg 557 – 561
Chapter 23 pg 785 -790; 793 - 795
• Rhizobiales
• Key genera: Bartonella, Methylobacterium, Pelagibacter, Rhizobium, Agrobacterium,
• Largest and most metabolically diverse order
• Contains phototrophs, chemolithotrophs, symbionts, nitrogen-fixing bacteria, pathogens,
and chemoorganotrophs
The Legume–Root Nodule Symbiosis pg 785 -790
• Infection of legume roots by nitrogenfixing bacteria leads to the formation
of root nodules that fix nitrogen
• Leads to significant increases in combined
nitrogen in soil
The Legume–Root Nodule Symbiosis
• Nitrogen-fixing bacteria need O2 to
generate energy for N2 fixation, but
nitrogenases are inactivated by O2
• In the nodule, O2 levels are controlled by
the O2-binding protein leghemoglobin
The Legume–Root Nodule Symbiosis
• Critical steps in root nodule formation
• Step 1: Recognition and attachment of bacterium to root
hairs
• Step 2: Excretion of Nod factors by the bacterium
• Step 3: Bacterial invasion of the root hair
• Step 4: Travel to the main root via the infection thread
• Step 5: Formation of bacteroid state within plant cells
• Step 6: Continued plant and bacterial division, forming the
mature root nodule
The Legume–Root Nodule Symbiosis
Rhizobial cell
Invaded plant cells
and those nearby are
stimulated to divide
Root hair
Infection
thread
Root hair
Soil
1. Recognition
and attachment
(rhicadhesinmediated).
2. Bacterium
secretes Nod factors
causing root hair
curling.
3. Invasion. Rhizobia
penetrate root hair
and multiply within
an "infection thread."
4. Bacteria in
infection
thread grow
toward root
cell.
5. Formation of
bacteroid state
within plant
root cells.
6. Continued plant
and bacterial
cell division
leads to nodules.
Nodules
Effect of root nodulation on plant growth: unnodulated vs nodulated soybean plants in nitrogen poor soils
Agrobacterium and Crown Gall Disease 793 - 795
Agrobacterium tumefaciens causes tumor
formation on plants (Black arrow)
Ti plasmid (tumor inducing plasmid) is required
For tumor formation
T-DNA (transferred DNA) is part of the Ti plasmid
plasmid which gets transferred and integrated into
the host genome
T-DNA carries genes for synthesis of opines required
by Agrobacterium for growth
Agrobacterium and Crown Gall Disease
Transmissibility
genes
Oncogenes
Opine
catabolism
genes
C
vir
genes
(encode
virulence
factors)
Opine
synthesis
Ti Plasmid – tumour inducing
T-DNA – transferred DNA
Agrobacterium and Crown Gall Disease
• To initiate tumor formation, A. tumefaciens cells must attach to the
wound site on the plant
• Attached cells synthesize cellulose microfibrils and transfer a portion
of the Ti plasmid to plant cells
•
• DNA transfer is mediated by vir-encoded proteins
Phenolics from
plant wound
Transcription of
other vir genes
ADP
VirG
P
Nicking
by VirD
Transfer
to plant
E
E
VirA
Vir
B
E
ATP
T-DNA
VirG
VirD
Agrobacterium
cell
Plant
cell
The Ti plasmid has been used in the genetic engineering of plants
Mobilized
region
Foreign DNA
Kanamycin
resistance
Chromosomes
Grow transgenic
plants from
plant cells
D-Ti
Transfer to
E. coli cells
Origin
A. tumefaciens
Spectinomycin
Origin E. coli
resistance
Cloning vector
Transfer to
plant cells
“Disarmed”
Ti plasmid
Transfer by
conjugation
E. coli
Nucleus
A. tumefaciens
Plant cell
Take note as a benefit derived from understanding Agrobacterium-plant infection,
You do not need to learn this process.
Genetic Engineering of Plants – what are
your ideas
Alphaproteobacteria
• Rickettsiales
• Key genera: Rickettsia, Wolbachia
• Rickettsia
• Small, coccoid or rod-shaped cells
• Most are obligate intracellular parasites
• Causative agent of several human diseases
• Typhus – spread by body lice
• Rocky Mountain spotted fever
• Wolbachia
• Rod-shaped
• Intracellular parasites of arthropod insects
• Affect the reproductive fitness of hosts
Beta and Gammaproteobacteria
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Betaproteobacteria (pg 560 – 562)
• Burkholderiales,
• Niesseriales,
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Betaproteobacteria
• Burkholderiales
• Key genus: Burkholderia
• Wide range of metabolic and ecological characteristics
• Fix nitrogen
• B. cepacia
• Opportunistic human pathogens
• Soil pathogens
• Plant pathogen
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Betaproteobacteria
• Neisseriales
• Key genera: Neisseria,
• Neisseria
• Always cocci
• Can be isolated from animals
• Some species and NOT pathogenic
• Two species important human
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Meningitis and Meningococcemia pg 994-995
• Neisseria meningitidis (miningococcus)
• Gram-negative, obligate aerobes
• encapsulated diplococcus
• airborne
• Meningitis
• Inflammation of the meninges
• Membranes that line the central nervous system,
especially the spinal cord and brain
• Can be caused by viral, bacterial, fungal, or protist
infections
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Gonorrhea pg 1007- 1008
• N. gonorrhoeae colonises mucosal surfaces of
urethra, uterine cervix, anal canal
• Transmitted via sexual contact
• Die-off in environment,
• No natural immunity
• Can be treated with antibiotics
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Gonorrhea
• Females
•
•
•
•
Often undiagnosed / Reservoir
Aggravated by contraceptives
Vaginitis similar to mild vaginal infections
Develops into pelvic inflamatory diseases (PID) –
leading to sterility
• Male
• Infection of urethral canal
• Newborns
• Eye infections – during birth
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Gammaproteobacteria – 562 - 566
Enterobacteriales
Pseudomonadales
Vibrionales
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Gammaproteobacteria – Enterobacteriales
• Key genera: Enterobacter, Escherichia, Klebsiella,
Proteus, Salmonella, Serratia, Shigella
• Enteric bacteria
• Phylogenetic group within the Gammaproteobacteria
• Motile (petritrichous flagella) or nonmotile, nonsporulating
rods
• Ferment sugars to a variety of end products
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Gammaproteobacteria – Enterobacteriales
• Enteric bacteria can be separated into two broad
groups by the type and proportion of fermentation
products generated by anaerobic fermentation of
glucose (Figure 15.12)
• Mixed-acid fermenters
• 2,3-butanediol fermenters
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Glucose
Glycolysis
Pyruvate
Lactate
CO2
Uninoculated tube
Succinate
Ethanol
Acetyl~CoA
Acid + gas
reaction
(H2 + CO2)
Acetate
+
CO2
Formate
H2
Gas collection tube
Mixed-acid fermentation (for example, Escherichia coli)
Glucose
Glycolysis
Pyruvate
2,3-Butanediol + CO2
Ethanol
Lactate
Uninoculated
Succinate
Acetate
CO2 + H2
Butanediol color reaction
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Butanediol fermentation (for example, Enterobacter aerogenes)
Mixed acid vs butanediol fermenters
Mixed acid fermenters
Butanediol fermenters
• Escherichia spp
• Enterobacter spp
• NO butanediol
• Butanediol produced
• Three acids formed in
• Smaller amounts of acids
significant amounts
• Equal amounts of CO2
and H2
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• More CO2 than H2
• Mixed-acid fermenters
• Escherichia
• Universal inhabitants of intestinal tract of humans and
warm-blooded animals
• Synthesize vitamins (K) for host
• Some strains are pathogenic
• enterohemorrhagic E. coli (EHEC, i.e., O157:H7), produces
verotoxin and shiga toxins
• Raw/under cooked meat
• Toxin secreted
• Toxin destroys intestinal lining  bloody diarrhea
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• Salmonella and Shigella
• Closely related to Escherichia
• Usually pathogenic – gastroenteritis, bacillary dysentery
and typhoid fever
• Salmonella is characterized immunologically by surface
antigens
• Salmonellosis is a gastrointestinal illness
• Ingested in food or water
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Gammaproteobacteria – Enterobacteriales
• Butanediol fermenters
• Closely related group of organisms
• Enterobacter
• Found in water, sewage, and intestinal tract of
warm-blooded animals
• May cause urinary tract infection
• Klebsiella
• Found in soil and water
• Most strains fix nitrogen
• Klebsiella pneumoniae occasionally causes pneumonia
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Gammaproteobacteria – Pseudomonadales and
Vibrionales
• Key genera: Aliivibrio, Pseudomonas, Vibrio
• Pseudomonadales
• Can cause plant and animal diseases
• Pseudomonas aeruginosa – cystic fibrosis
• Pseudomonas syringae – many plants
• Vibrionales
• Vibrio cholerae human pathogen
• V. parahaemolyticus fish pathogenic
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Deltaproteobacteria and Epsilonproteobacteria
• Deltaproteobacteria
• Key genera: Bdellovibrio, Myxococcus,
• Epsilonproteobacteria
• Key Genera: Campylobacter and Helicobacter
• Motile spirilla, microaerophilic
• Mostly pathogenic to humans
• Campylobacter - acute gastroenteritis  bloody diarrhea
• Helicobacter – peptic ulcers
• Zetaproteobacteria
• Iron oxidizer – Mariprofundus ferrooxydans
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Firmicutes, Tenericutes, and Actinobacteria
Gram positive and related
•
Firmicutes – Lactobacillales
•
Firmicutes – Sporulating Bacillales and Clostridiales
•
Firmicutes – Non Sporulating Bacillales and Clostridiales
•
Tenericutes: The Mycoplasmas aka Mollucutes
•
Actinobacteria: Coryneform and Propionic Acid
Bacteria
•
Actinobacteria: Mycobacterium
•
Filamentous Actinobacteria: Streptomyces and
Relatives
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Firmicutes – Lactobacillales (pg 567 – 572)
• Key genera: Lactobacillus, Streptococcus
• Fermentative bacteria that produce lactic acid
• Homofermentative: lactic acid only
• Heterofermentative: lactic acid, ethanol and CO2
• Lactobacillus
• Rod-shaped and grow in chains
• Common in dairy products
• Resistant to acidic conditions
• Grow in pH as low as 4
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Firmicutes – Lactobacillales
• Streptococcus
• Coccus-shaped and grow in chains
• Homofermentative
• Some species are pathogenic
• Streptococcus pyogenes – strep throat
• S. mutans – dental caries
• Virulence factors
• Streptolysin O and S – beta hemolysis
• Blood agar diagnosis of pyogenes and viridans
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Streptococcal Diseases
• Streptococcus pyogenes
• Also called group A streptococci
• Found in upper respiratory tract of healthy individuals
• streptococcal pharyngitis or strep throat
• Streptolysin virulence factor
• beta-hemolysis
• Can also cause these infections:
• Mastatis – mammary gland
• Otitis media – middle ear
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• Impetigo – skin
Streptococcal Diseases (see 31.2 pg 988 – 990)
• Streptococcus pyogenes
• Occasionally cause sudden and severe systemic
infections
• Scarlet fever – streptococcal toxic shock syndrome
• Cellulitis
• Subcutaneous skin infection
• Necrotizing fasciitis
• Flesh-eating bacteria
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Firmicutes – Nonsporulating Bacillales and
Clostridiales
• Key genera: Listeria, Staphylococcus
• Listeria
• Gram-positive, coccobacillus, are aerobic
chemoorganotrophs
• Forms chains 3 to 5 cells long
• Obligate aerobe
• Listeria monocytogenes causes
• listeriosis – affects pregnant women and minigitis
• Outbreak in South Africa (2017 – 2018)
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Firmicutes – Nonsporulating Bacillales and
Clostridiales
• Staphylococcus
• Facultative aerobe, Gram-positive coccus
• Dessication tolerant
• High salt tolerance – select on 7.5% NaCl medium
• S. epidermidis non pigmented skin flora
• Many species are pigmented
• Staphylococcus aureus
• Yellow pigment, causes boils, pneumonia, pimples,
arthritis
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Firmicutes – Sporulating Bacillales and
Clostridiales
• Key genera: Bacillus, Clostridium,
• Distinguished on the basis of cell morphology and on
the shape and cellular position of endospore
• Generally found in soils
• Endospores are advantageous for soil microorganisms
• Selected for by heating samples to 80 ◦C
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Firmicutes – Sporulating Bacillales and
Clostridiales
• Clostridium
• Lacks a respiratory chain; anaerobic
• Saccharolytic – ferment sugars and produce butyric acid
• Cellulolytic - ferment cellulose forming acids and alcohols
• Proteolytic - fermentation of amino acids aka Stickland
reactions – foul smell
• Mainly found in anaerobic pockets in the soil
• Also live in mammalian intestinal tract
• Some species are pathogenic; diseases include
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botulism, tetanus, and gangrene
Firmicutes – Sporulating Bacillales and
Clostridiales
• Bacillus and Paenibacillus
• Many produce extracellular hydrolytic enzymes that
break down polymers
• Many bacilli produce antibiotics
• Paenibacillus popilliae and Bacillus thuringiensis
produce insect larvicides
• Used to genetically engineer insect resistant plants e.g.
Bt-corn
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Tenericutes: The Mycoplasmas (pg 572 – 574)
• Key genera: Mycoplasma, Spiroplasma
• Lack cell walls
• Some of the smallest organisms capable of autonomous
growth
• Parasites that inhabit animal and plant hosts
• Key components of peptidoglycan are missing
• Sterols and lipoglycans required to stabilise cytoplasmic
membranes
• Mycoplasma cells are pleomorphic
• Cells may be cocci or filaments of various lengths
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Mycoplasma mycoides: showing pleomorphic cells
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Tenericutes: The Mycoplasmas
• Growth of mycoplasmas
• Media for the culture of mycoplasmas are typically quite
complex (e.g Yeast extract, peptone, beef heart
infusion)
• Mycoplasma colonies show a characteristic "fried-egg"
appearance
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Fried egg appearance of Mycoplasma colonies
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Firmicutes: Summary
• Firmicutes:
• Lactobacillales
• Lactobacillus and Streptococcus
• Non spore forming Bacillales and Clostridiales
• Listeria and Staphylococcus
• Sporulating Bacillales and Clostridiales
• Bacillus and Paenibacillus GMO and Biological control
agent
• Clostridium – ferment sugars, amino acids, cellulose
• Tenericutes aka Mollicutes (Mycoplasma)
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Firmicutes, Tenericutes, and Actinobacteria
Gram positive and related
•
Firmicutes – Lactobacillales
•
Firmicutes – Sporulating Bacillales and Clostridiales
•
Firmicutes – NonSporulating Bacillales and Clostridiales
•
Tenericutes: The Mycoplasmas aka Mollucutes
•
Actinobacteria: Coryneform and Propionic Acid
Bacteria
•
Actinobacteria: Mycobacterium
•
Filamentous Actinobacteria: Streptomyces and
Relatives
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Actinobacteria: Coryneform and Propionic Acid
Bacteria
• Key genera: Corynebacterium, Arthrobacter,
Propionibacterium
• Gram positive
• Actinobacteria form their own phylum
• Over 30 taxonomic families
• Rod-shaped to filamentous, aerobic, non-motile
• Mostly harmless commensals (Mycobacterium is an
exception)
• Valuable for antibiotics and certain fermented dairy
products
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Actinobacteria: Coryneform and Propionic Acid
Bacteria
• Corynebacterium
• Gram-positive, aerobic, nonmotile, rod-shaped
• Form club-shaped, irregularly shaped, or V-shaped cell
arrangements – snapping division
• Extremely diverse
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Actinobacteria: Coryneform and Propionic Acid
Bacteria
• Propionic acid bacteria
• First discovered in Swiss cheese
• Gram-positive anaerobes
• Have metabolic strategy called secondary fermentation
• Obtain energy from fermentation products (e.g. lacic acid)
produced by other bacteria
• Fermentation produces CO2  produces holes on Swiss
cheese
• Propionic acid  responsible for flavor of the cheese
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Swiss cheese
Starter cultures e,g homofermentative
streptococci or lactobacillus carry out initial
fermentation of lactose forming curd
Secondary fermentation: Propionic
bacteria develop after curd drainage  formation of
CO2 and Propionic Acid as well as Acetic acid
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Actinobacteria: Mycobacterium
• Mycobacterium
• Rod-shaped organisms, exhibit acid-fastness
• Presence of mycolic acids
• Cells are somewhat pleomorphic
• Separated into two groups:
• slow and fast growers
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Actinobacteria: Mycobacterium
• Mycobacteria form tight, compact, wrinkled
colonies
• Virulence of Mycobacterium tuberculosis
correlated to cordlike structure
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Mycobacterium: Tuberculosis, and Hansen’s
Disease
• Tuberculosis
• Worldwide infectious disease of humans
• Leading cause of death in South Africa
• Highly contagious
• M. tuberculosis transmitted by airborne droplets
• Treatable with antibiotics
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© 2012 Pearson Education, Inc.
Mycobacterium: Tuberculosis,
• Hansen’s disease (leprosy)
• M. leprae is the causative agent
• armadillo is the only experimental host
• Lepromatous:
• Most serious form is characterized by folded,
bulblike lesions on the body
• Tuberculoid:
• Less pronounced lesions
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Filamentous Actinobacteria:
Streptomyces & Relatives
• Key genera: Streptomyces, Actinomyces
• Filamentous, gram-positive bacteria
• Produce mycelium
• Over 500 species
• Produce spores are called conidia
• responsible for earthy odor of soil (geosmins)
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Filamentous Actinobacteria: Streptomyces and
Relatives
• Streptomyces
• Produce colored colonies
• 50% of all isolated Streptomyces produce antibiotics
• Over 500 distinct antibiotics are produced by
Streptomyces
• Some produce more than one antibiotic
• Genomes are typically quite large (8 Mbp and larger)
• Knowledge of the ecology of Streptomyces remains
poor
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Bacteroidetes
• Bacteroidales
• Cytophagales, Flavobacteriales, and
Sphingobacteriales
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Bacteroidales
• Key genera: Bacteroides
• Bacteroides
• Obligately anaerobic
• Numerically dominant bacterium in human intestinal
tract
• Synthesize sphingolipids, which are normally found in
mammalian tissues
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Chlamydiae, Planctomycetes, and
Verrucomicrobia
• Chlamydiae
• Planctomycetes
• Verrucomicrobia
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Chlamydiae
• Key genera: Chlamydia, Chlamydophila,
Parachlamydia
• Obligately parasitic with poor metabolic capacities
• Some of the simplest biochemical capacities of all
known bacteria
• Chlamydia is currently one of the leading sexually
transmitted diseases
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Elementary bodies: dispersal and infectious, non multiplying
Reticulate bodies: non infectious and multiplication
Release of
elementary bodies
Elementary bodies
Conversion to
elementary bodies
Elementary
body
Elementary body
attacks host cell.
Reticulate
body
Multiplication
of reticulate
bodies
Phagocytosis of
elementary body
Conversion to
reticulate body
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Summary: Actinobacteria
• Coryneform
• Corynebacterium: can be pathogenic to animals plants
• Arthrobacter are soil organisms
• Propionic acid bacteria
• Important in Swiss Cheese production
• Filamentous Actinobacteria: Streptomyces
• Production of antibiotics
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Archaea: Expected outcomes


What are the major phyla in Archaea
Eurachaeota

How do haloarchaea tolerate extreme salt
conditions – maintain osmotic balance?
Archaea

Refer to Fig 17.1 Phylogeny of all major
taxonomic orders within domain Archaea
Phylogenetic and Metabolic Diversity of
Archaea


Archaea share many characteristics with
both Bacteria and Eukarya
Archaea are split into five major phyla





Crenarchaeota
Thaumarchaeota
Nanoarchaeota
Korachaeota
Euryarchaeota
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Selected Euryarchaeota






Extremely Halophilic Archaea
Methanogenic Archaea
Thermoplasmatales
Thermococcales and Methanopyrus
Archaeoglobales
Nanoarchaeum and Aciduliprofundum
II. Euryarchaeota
 Euryarchaeota


Physiologically diverse group of Archaea
Many inhabit extreme environments

Examples: high temperature, high salt, high acid
Extremely Halophilic Archaea
 Halophilic
archaea
 Key genera: Halobacterium, Haloferax,
Natronobacterium


Extremely halophilic Archaea
Have a requirement for high salt concentrations


Typically require at least 1.5 M (~9%) NaCl for
growth
Found in artificial saline habitats

e.g., salted foods, solar salt evaporation ponds, and
salt lakes such as Utah USA (Fig 17.2) and Etosha
Namibia
Great salt lake
A lake in Egypt - haloalkalophiles
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Extremely Halophilic Archaea


Hypersaline environments are rare, examples
are salt lakes, or salt pans
Salt lakes can vary in ionic composition,





Great Salt Lakes are similar to concentrated
seawater
They have high concentrations of ions such as Cl-,
Na+
Selected microbes –called halophiles are adapted
to these extreme environments
Halophiles require high concentrations of NaCl for
growth
How do these microbes withstand osmotic
pressure?
Adaptations of Halophilic Archaea
 Water
Balance in Extreme Halophiles
Water balance is essential to withstand osmotic
pressure
 Halophiles maintain osmotic balance by



accumulation or synthesis of compatible solutes
E.g. Halobacterium species instead pump large
amounts of K+ into the cell from the environment

Intracellular K+ concentration exceeds extracellular
Na+ concentration and positive water balance is
maintained
Extremely Halophilic Archaea
 Proteins


of halophiles
Are highly acidic
Contain fewer hydrophobic amino acids and
lysine residues
Methanogenic Archaea
 Methanogens
Key genera: Methanobacterium,
Methanocaldococcus, Methanosarcina



Microbes that produce CH4
Found in many diverse environments
Taxonomy based on phenotypic and phylogenetic
features
 Process
of methanogenesis first demonstrated
over 200 years ago by Alessandro Volta
Methanogenic Archaea
 Substrates


for Methanogens
Obligate anaerobes
11 substrates, divided into 3 classes, can be
converted to CH4 by pure cultures of
methanogens

Other compounds (e.g., glucose) can be
converted to methane, but only in cooperative
reactions between methanogens and other
anaerobic bacteria
Thermoplasmatales
 Thermoplasmatales
 Key
genera: Thermoplasma, Ferroplasma,
Picrophilus



Taxonomic order within the Euryarchaeota
Thermophilic and/or extremely acidophilic
Thermoplasma and Ferroplasma lack cell walls
Thermoplasmatales
 Thermoplasma





Chemoorganotrophs
Facultative aerobes via sulfur respiration
Thermophilic
Acidophilic
Found in self-heating coal piles
A typical self-heating coal refuse pile,
habitat of Thermoplasma.
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Thermoplasmatales
 Thermoplasma

Evolved unique cytoplasmic membrane
structure to maintain positive osmotic pressure
and tolerate high temperatures and low pH
levels


Membrane contains lipopolysaccharide-like
material (lipoglycan) consisting of tetraether lipid
monolayer membrane with mannose and glucose
Membrane contains glycoproteins but not sterols
Thermoplasmatales
 Ferroplasma




Chemolithotrophic
Acidophilic
Oxidizes Fe2+ to Fe3+, generating acid
Grows in mine tailings containing pyrite
(FeS2)
Thermoplasmatales
 Picrophilus

Extreme acidophiles


Grow optimally at pH 0.7
Model microbe for extreme acid tolerance
Nanoarchaeum and Aciduliprofundum
 Nanoarchaeum





equitans
One of the smallest cellular organisms (~0.4 µm)
Obligate symbiont of the crenarchaeote
Ignicoccus
Contains one of the smallest genomes known
Lacks genes for all but core molecular processes
Depends upon host for most of its cellular needs
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Crenarchaeota
 Habitats
and Energy Metabolism
 Crenarchaeota from Terrestrial Volcanic
Habitats
 Crenarchaeota from Submarine Volcanic
Habitats
 Crenarchaeota from Nonthermal Habitats
and Nitrification in Archaea
Habitats and Energy Metabolism
 Crenarchaeota


Inhabit temperature extremes
Most cultured representatives are
hyperthermophiles


Found in extreme heat environments
Other representatives found in extreme cold
environments
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Terrestrial habitats of hyperthermophilic
Archaea: Yellowstone National Park
(Wyoming, USA).
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Habitats and Energy Metabolism
 Hyperthermophilic


Crenarchaeota
Most are obligate anaerobes
Chemoorganotrophs or chemolithotrophs with
diverse electron donors and acceptors
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Crenarchaeota from Terrestrial
Volcanic Habitats
 Sulfolobales

Key genera: Sulfolobus and Acidianus

Sulfolobus



Grows in sulfur-rich acidic hot springs
Aerobic chemolithotrophs that oxidize reduced sulfur
or iron
Acidianus


Also lives in acidic sulfur hot springs
Uses elemental sulfur both aerobically and
anaerobically
Crenarchaeota from Terrestrial
Volcanic Habitats

Thermoproteales


Key genera: Thermoproteus, Thermofilum,
and Pyrobaculum
Inhabit neutral or slightly acidic hot springs or
hydrothermal vents
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Crenarchaeota from Submarine
Volcanic Habitats
 Shallow-water
thermal springs and deepsea hydrothermal vents harbor the most
thermophilic of all known Archaea



Pyrodictium and Pyrolobus
Desulfurococcus and Ignicoccus
Staphylothermus
Crenarchaeota from Submarine
Volcanic Habitats

Pyrodictium and Pyrolobus



Optimum growth temperature above 100C
Pyrolobus fumarii is one of the most thermophilic
Strain 121 can grow up to 121C
© 2012 Pearson Education, Inc.
Crenarchaeota from Submarine
Volcanic Habitats

Desulfurococcus and Ignicoccus



Desulfurococcus is a strictly anaerobic
S0-reducing organism
Ignicoccus grows optimally at 90Cand its
metabolism is H2/S0 based
Ignicoccus contains an outer membrane similar to
that of gram-negative Bacteria
Crenarchaeota from Submarine
Volcanic Habitats

Staphylothermus




Spherical cells
About 1 mm in diameter
Forms aggregates of up to 100 cells
Chemoorganotroph that grows optimally at 92C
Figure 19.21
The hyperthermophile Staphylothermus
marinus.
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Crenarchaeota from Nonthermal
Habitats
 Nonthermophilic
Crenarchaeota have been
identified in cool or cold marine waters and
terrestrial environments by culture-independent
studies


Abundant in deep ocean waters
Appear to be capable of nitrification
Figure 19.22
Cold-dwelling Crenarchaeota.
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Figure 19.23
Nitrosopumilus maritimus, a nitrifying
species of Archaea.
© 2012 Pearson Education, Inc.