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Introduction to Environmental Microbiology
ENV 411
1
Chapter 1
Introduction to
Environmental Microbiology
Chapter Overview
INTRODUCTION TO ENVIRONMENTAL
MICROBIOLOGY & MICROBES
Environmental Microbiology
Significance of Environmental Microbiology
Microbial cell, it structure and function
Microbial growth
Microbial metabolism
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LEARNING OBJECTIVES:
After studying this chapter, you should be able to:
1.1
1.
Define environmental microbiology
2.
Understand the impact of microorganisms on the biosphere
3.
Describe the microbial cell, its structure and function.
4.
Explain differences in cell walls, cytoplasmic membrane of
Bacteria, Archaea & Eucarya
5.
Explain the different types of transport across cytoplasmic
membrane
6.
Elaborate the microbial growth, development and characteristics of
spores.
7.
Differentiate the different types of microbial metabolism.
WHAT
1. IS ENVIRONMENTAL MICROBIOLOGY?
The2.study of how microorganisms affect the earth and its atmosphere is called
3. microbiology or microbial ecology. The study of the relationships that exist
environmental
4.
between microorganisms
and the environment. The study of relationship of microorganisms
with themselves and with their surroundings.
1.2
TRAITS OF MICROORGANISMS AND IMPACT ON BIOSPHERE
Microorganisms’ unique combination of traits and their broad impact on the
biosphere
Traits of microorganisms
Ecological consequences of traits
Small size
Geochemical cycling of elements
Ubiquitous distribution throughout Detoxification of organic pollutants
the earth’s habitats
High specific surface areas
Detoxification of inorganic pollutants
Potentially high rate of metabolic Release of essential limiting nutrients from
activity
the biomass in one generation to the next
Physiological responsiveness
Maintaining the chemical composition of
Genetic malleability
soil, sediment, water and atmosphere
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Potential rapid growth rate
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required by other forms of life
Unrivaled nutritional diversity
Unrivaled enzymatic diversity
1.3
MICROBIAL CELL, ITS STRUCTURE AND FUNCTION
Based on ribosomal RNA sequence comparisions (16S, 23S). 3 basic groups or
domains established (domains are a higher order than kingdoms, ie are superkingdoms).
The 3 domains (i) Bacteria, (ii) Archaea and (iii) Eucarya. 3 domains are related to each
other; progenote = hypothetical ancient universal ancestor of all cells. Natural relationships
amongst cells established (phylogeny).
Microbes have different shapes and is of advantage: Cell wall establishes the shape of a
microbial cell but environmenta conditions can change it

Shapes include: (i) Spheres called cocci (greek = berry) can divide once in one axis
to produce diplococci (Neisseria gonnorrhoeae, N. meningitidis), or more than once
to produce a chain (Streptococcus pyogenes), divides regularly in two planes at right
angles to produce a regular cuboidal packet of cells (xxx) or in two planes at different
angles to produce a cluster of cells (Staphyloccus aureus). (ii) Cylinders called rods
or bacilli (Latin bacillus = walking stick). (iii) Spiral or spirilli (Greek spirillum = little
coil)

Shape offers an advantage to the cell: (i) Cocci: more ressistant to drying than rods
(ii) Rods: More surface area & easily takes in dilute nutrients from the environment.
(iii) Spiral: Corkscrew motion & therefore less ressistant to movement (iv) Square:
Assists in dealing with extreme salinities.
Microbes are small but this feature is crucial : Nutrients and wastes are transported in and
out the cell via the cytoplasmic membrane. The rate of transport determines the metabolic
rates and therefore the growth rates of microbial cells The smaller the size, the larger the
surface area of the cytoplasmic membrane to volume and therefore the faster is it's potential
growth rate. This can be seen as follows:
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radius (r) of cell A =
1um
radius (r) of cell B =
2um
Surface area (SA) of cell =
4pir2
12.6um2
50.3um2
Volume (V) of cell = 4/3pir3
4.2um3
33.5um3
3
1.5
Ratio of SA to V
Features of bacterial, archaeal and eucarya cells
This section deals with the structure and functions of cells. Cells are of three types as
described above (Bacteria, Archaea & Eucarya) and the description below provides
similarities and differences amongst these cell types.
Diagrammatic representation of cells
Cell walls are external structures that shape and protect cells
a. Bacterial Cell Walls:
All the members of domain Bacteria, with the exception of the genera Mycoplasma,
Ureaplasma, Spiroplasma, and Anaeroplasma contain cell walls Cell walls are
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chemically peptidoglycans ie peptides (short amino acids chains) and glycans
(sugars); peptidoglycans are a.k.a. mureins, mucopeptide.
o
Glycans: are modified sugars viz, N-acetyl muramic acid (NAM or M) & Nacetly glucose amine (NAG or G).M and G are linked to each other by a beta
1, 4 glycosidic bond & alternate to form the wall backbone. Lysozyme (an
enzyme produced by organisms that consume bacteria, and normal body
secretions such as tears, saliva, & egg white = protect against would-be
pathogenic bacteria) digests beta 1,4 glycosidic bonds. Lysozyme lyses
growing or non growing cells but cell wall-less microbes are not affected High
osmotic pressure in high solute concentrations prevents lysis of Gram +ve &
Gram -ve cells when treated with lysozyme:
o

spheroplasts = part of cell wall removed (Gram -ve)

protoplasts = complete removal of cell wall (easier for Gram +ve)
Peptides: Short peptides (4 amino acids, tetrapeptides) attached to M. Some
of the amino acids are only found in cell walls & not in other cellular proteins
(D- amino acids, eg D-alanine & diaminopimelic acid, DAP). Tetrapeptides
chains are cross linked (interlinked) by a peptide bridge (the carboxyl group of
one tetrapeptide with an amino group of an adjacent (direct interbridge) or a
different tetrapeptide chain (indirect interbridge). Transpeptidase enzyme
builds peptide bridges in actively dividing cells; penicillin binds to it stoping
cell wall synthesis. Autolysins restructure and reshape cell walls by breaking
specific bonds in the peptidoglycan in actively growing cells. Cell wall
synthesis stops but cell degrading enzymes still function resulting in
weakened cell walss and ultimately death.
Glycans and peptides therefore forms a single, large and strong cross-linked
molecule in a form of a multilayered sheet, (sacculus, Latin = little sac) that surrounds
the entire bacterial cell.
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Differences Between Gram-positive And Gram-negative Bacterial Cell Walls
Gram-positive wall
Gram-negative wall
Peptidoglycan
Thick layer
Thin layer
Peptidoglycan tetrapeptide
Most contain lysine
All contain diaminopimelate
Peptidoglycan cross linkage
Generally via pentapeptide
Direct bonding
Teichoic acid
Present
Absent
Teichuronic acid
Present
Absent
Lipoproteins
Absent
Present
LPS
Absent
Present
Outer Membrane
Absent
Present
Periplasmic Space
Absent
Present
b. Archaeal Cell Walls:
Archaeal cells have more variations in their cell wall chemistries, and some do not
contain cell walls (eg Thermoplasma). Methanobacterium sp. contain glycans
(sugars) and peptides in their cell walls:
o
Glycans: are modified sugars viz, N-acetyl talosaminouronic acid (NAT or T)
& N-acetly glucose amine (NAG or G). T and G are linked to each other by a
beta 1, 3 glycosidic bond & alternate to form the cell wall backbone.
Lysozyme (an enzyme produced by organisms that consume bacteria, and
normal body secretions such as tears, saliva, & egg white = protect against
would-be pathogenic bacteria) cannot digest beta 1,3 glycosidic bonds.
o
Peptides: Short peptides attached to T. The amino acids are only of the Ltype. Penicillin is ineffective in inhibiting the cell wall peptide bridge formation.
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Methanosarcina sp. cell walls contain non-sulfated polysaccharides. Halococcus sp.
contain sulfated polysaccharides similar to Methanosarcina sp. Halobacterium sp.
contain negatively charged acidic amino acids in their cell walls which counteract the
positive charges of the high Na+ environment. Therefore, cells lyse in NACl
concentrations below 15%. Methanomicrobium sp. & Methanococcus sp. cell walls
are exclusively made up of proteins subunits.
c. Eucaryal Cell Walls:
Cell walls of algae have a variety of different cell wall types and include cellulose,
calcium carbonate, silcone dioxide, proteins and even polysaccharides. The cell walls
of fungi are made up of chitin (a nitrogen-containing polysaccharide) and is similar to
that found in the exoskeletons of arthropods & crabs . Protozoa do not have a true
cell wall. In some species, silicon dioxide, calcium carbonate or strontium sulfate are
found but do not provide the cell wall with a protective function.
d. Glycocalayx, Capsules, Slime Layers & S layers:
Various external structures which have different functions surround the bacterial cell
wall, and are collectively called glycocalyx. Glycocalyx varies in different species:

Are thick & rigid structures which exclude stain.

Adhere externally to the to cell walls

Negative stain allows capsules to be observed.

Chemically
polysaccharides.
Found
in
pneumonia
causing pathogens such as Streptococcus pneumoniae,
Haemophilus influenzae & Klebsiella pneomoniae.
Capsules

Chemically D-glutamic acid found in some Bacillus sp.

Capsulated variants of a species are pathogenic whereas
non-capsulated variants of the same species are nonpathogenic. Capsules protect against phagocytosis by
human white blood cells.
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
Similar in composition to capsules but are not as tightly
bound to the cell wall.
Slime layers

Protects cells against dehydration and a loss of nutrients.

Some bacteria have a crystalline protein layer called a S
layer.

S layer
Found outside the cell walls of some species of Gramnegative, Gram-positive Bacteria, and outside the cell
membranes of some Archaea.

Function is unknown.
Cytoplasmic membranes are involved in transport of molecules
(A) Structural & Biochemical Diversity

Thickness 4-5nm

Regulates flow of molecules in and out of the cell but is a differentially permeable
barrier- movement across the membrane is selectively restricted (structure &
chemistry is key to this)

Small, neutrally charged molecules (H2O, O2 & CO2) easily transportable but large
molecules & ions (glucose) or small charged atoms (protons, H+) require specific
transport systems.

Provides increased surface area to volume & is very important to small cells

Bilayered structural backbone are the phospholipids (bacteria & eucaryotes only);
forms a separation barrier with water inside and outside the cell

"Fluid mosaic model": Proteins are integrated into the lipid layer and both "float"
laterally in the membrane ie are in dynamic rather than static state (lipids float more
than proteins)
o
o
Peripheral proteins: confined to the membrane surface
Integral proteins: partially / completely buried & may span the entire
membrane

Distribution & properties of proteins on each side of the layer are different & therefore
the functions of the 2 layers are different

The structure and chemical properties of archaeal, bacterial and eucaryotic
membranes are "phylogenetically" distinct
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Characteristics of Bacterial Eucaryotic Archaeal cytoplasmic membranes
Characteristics
Bacteria
Eucaryotic
Archaea
Protein
content
High
Low
High
Lipid
composition
Phospholipid
Phospholipids
Sulfolipids,
glycolipids,
nonpolar
isoprenoid
lipids,
phospholipids
Lipid structure
Straight chain
Branched
Straight chain
Lipid linkage
Ester linked(1)
Ester linked
Ether linked
(di&
tertaethers)
Sterols
Absent(2)
Present
Absent
(1) Aquifex pyrophilus contains phospholipids & ether linked lipids
(2) Cell wall-less bacteria (Mycoplasma, Ureaplasma, Spiroplasma, Anaeroplasma) contain sterols
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A. Bacterial cytoplasmic membranes:
i.
Phospholipids: (structure, functions & utility)
Made of phospholipids - a phosphate group joined to 2 fatty acids by glycerol
(glycerol diester); oleate, stearate The phosphate group is -vely charged & is therefore
hydrophilic
("water
loving")-
exposed
to
cell
wall
&
cytoplasm
The fatty acid group is nonpolar & therefore hydrophobic ("afraid of water")- exposed
within the internal membrane matrix Electron micrographs of thin sections of bacteria
cells show a pair of electron dense dark railroad track-like appearance (hydrophilic
portion) & electron light middle layer (hydrophobic) Form a bilayer due to hydrophobic /
hydrophilic interactions (spontaneous aggregation)- contributes to flow of molecules.
Phospholipid composition varies with species & environmental conditions. Psychrophiles:
high proportion of unsaturated fatty acids enhance membrane fluidity (saturated fatty
acids pack together more tightly & produce a rigid less-fluid membrane). Bacteria can be
identified on phospholipid composition (computerized databanks available) but cells have
to be grown under standard conditions (Why?)
ii.
Protein
Are in dynamic state and distribution is according to the fluid mosaic model
Function
Location in Membrane
Example
Energy
transformation
Inside membrane
ATPase F1
Transport of
molecules
Inside membrane
HPr
Protein export
Inside membrane
Docking protein
Association of
DNA with
membrane
Inside membrane
DNA binding protein
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Transport of
molecules
Both sides
Permease
Chemotaxis
Both sides
Methylase-accepting chemotaxis
proteins
Electron &
proton transport
Both sides
Flavoproteins
Flagellar
activity
Outside surface
M protein (basal body of flagella)
Penicillinbinding proteins
Outside surface
Cell wall biosynthesis
B.
Archaeal cytoplasmic membranes:
Structure fundamentally different to bacterial & eucaryotic membranes Glycerol
molecules may be linked: (i) to a phosphate group (similar to bacteria & eucaryotes) and / or
(ii) to a sulfate and carbohydrates (unlike bacteria & eucaryotes) & therefore phospholipids
are not the structural lipids. Lipids are hydrocarbons (isoprenoid hydrocarbons) not fatty
acids, are branched (straight chain in bacteria & eucaryotes) and linked to glycerol by ether
bonds (ester linked in bacterial & eucaryotes). Lipids are diverse in structure:
o
Glycerol diether (Glycerol + C20 hydrocarbons)- Bilayered membrane
o
Glycerl tetraether (Glycerol + C40 hydrocarbons)- Monolayered membrane
o
Mixture of di- & tetra- Mono /Bi layered membrane
o
Cyclic tetraethers (Glycerol + > C40)- maintain the 4-5nm membrane thickness
Diversity of membranes is related to the diverse habitats that archaea live in
o
Sulfolobus (90oC, pH 2)- branched chain C40 hydrocarbons. Branched chains increase
membrane fluidity (unbranched & saturated fatty acids limit sliding of fatty acid
molecules past one another)- required for growth at high temperatures (upto 110oC,
hyperthermophies)
o
Halobacterium (saturated salts)-
o
Thermoplasma- high temperature, cell wall-less archaea
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C. Eucaryal cytoplasmic membranes:
Phospholipids similar to bacterial membranes but terols make upto 25% of the lipids
(cholestrol in humans,ergosterol in fungi). Polyene antibiotics (eg nystatin, candicidin)
targets sterols & has more affinity for ergosterol than cholesterol (more effective against
fungi rather than human cells)
Transport Across Cytoplasmic membrane
Membranes must selectively regulate transport of materials and waste ie semipermeable &
several mechanisms are available for this: (i) Pass directly enter thro' the lipid layer or via
proteins , (ii) Altered / modified as it passes thro' , (iii) Process requires cellular energy and
(iv) Solutes are concentrated against a gradient.
Passive Processes: Transport does not require energy & include diffusion, osmosis and
facilated diffusion
Diffusion: Unassisted movement of molecules from a higher concentration to lower
concentration (concentration gradient) until equilibrium is reached is called passive diffusion.
Rate of diffusion depends on membrane permiability & solute concentrations. Some solutes
after moving into the cell binds with some other proteins or are metabolically transformed.
Therefore concentration is not built up in the cell & the diffusion process continues at a faster
rate. Passive diffusion is slow eg glucose and tryptophan have diffusion rates of 1/10,000
that of water, & not enough for cellular growth & reproduction.
Osmosis : Process by which water croses the membrane in response to concentration
gradient of the solute 30 minutes at 70o C . Water moves from a region of low solute
concentration to high solute concentration.
o
isotonic- solute conc. outside the cell = solute conc. inside the cell
o
hypertonic- solute conc. is higher than that inside the cell; water flows out causing the
cell to shrink, plasmolysis
o
hypotonic- reverse of hypertonic; water will flow into the cell & the cell will burst
Usually water moves into the cell as cytoplasm has solutes resulting in increased pressure
on the membrane- osmotic pressure. Cells can lyse due to osmotic shock but have
developed strategies to protect against this (see shock-sensitive proteins later)
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Facilitated Diffusion: Enhanced rate of diffusion found mainly in eucaryotic cells but rarely
in bacteria & archaea (glycerol is the only known substrate that undergoes facilitated
diffusion in some bacteria). Facilitator proteins (membrane proteins) selective increase the
permeability of the membrane for certain solutes. Facilitator proteins are very specific & act
as carriers ie solutes bind to the facilitator protein changing its 3D properties. This change in
shape allows the solute to be carried across the membrane.
Active Energy-linked transport processes
Require energy for transport and the processes include active transport, group translocation,
binding protein transport and cytosis
a. Active Transport:
Active transport requires energy but the molecule is not modified during transport.
Transport occurs against concentration gradients
Permeases are very specific membrane protein transport carriers .Uniporters- carry one
substance at a time .Cotransporters- carry more than one type of substance . SymporterTwo substances carried in the same direction simultaneously [(eg lactose & proton (H+)]
Antiporter- Substances are transported across the membrane in opposite directions (eg Na+
are pumped outside the cell at the same time H+ are transported inside the cell).
Protonmotive
force
(PMF):
Energy
for
active
transport
in
bacteria
(oxidative
phosphorylation) in archaea, algae, mitochondria & chloroplasts generally comes from PMF.
PMF force is essential. Various metabolic activities produce protons (H+) and these are
translocated outside the cell. Higher concentrations & an increase in positive charge
outside the cell favours movement of protons back into the cell but cannot do so on their
own. Uncharged molecules (eg amino acids & sugars) are usually transported into the cell
with protons The various means by which PMF is produced will be discussed later
Sodium-potassium pump: A gradient between Na+ & K+ similar to protonmotive force &
known as sodium-potasium pump . Found in many eucaryotes.Three Na+ are pumped out of
the cell and two K+ are pumped into the cell by Na+-K+ ATPase enzyme; ATP is expanded.
Unequal distribution of positive ion with a higher Na+ conc. outside the cells and a higher
conc. of K+ inside the cells; leads to a powerful electrochemical gradient used for active
transport (eg symport protein binds both Na+ and glucose for transport therebye lowering
Na+ conc gradient across the membrane)
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b. Group translocation- Phosphoenol pyruvate: Phosphotransferase system (PEP:PTS).
Transported substance is chemically altered during passage thro' the membrane by the
addition of phophate . Carbohydrates, fatty acids, some nucleic acid building blocks. In E.
coli, glucose outside the cell is phosphorylated during transport (G6-P) into the cell.
Metabolism almost instantaneous once inside (couples energy resources efficiently thro
transport & initiation of energy-generating metabolism. Concentration gradient of glucose is
prevented (not in the same chemical state). Prokaryotic specific; in anaerobes, facultative
anaerobes but not in aerobes (active transport occurs).
c. Binding protein transport
Specialized transport system associated with the outer membrane of Gram negative
bacteria only. Periplasmic space(periplasm, periplasmic gel) is the space between the outer
membrane & the cytoplasmic membrane. There is interplay between porins, binding
proteins, permeases & transport proteins, eg maltose transport in E. coli . Binding protein
transport is also called shock-sensitive transport (cells that are osmotically shocked loose
the transport proteins of the periplasm).
d. Cytosis- Eucaryotic specific transport
A transport process in which a substance is engulfed by the cell membrane to form a
vesicle. Cytosis requires energy:
o
Endocytosis- movement into the cell
o
Exocytosic- movement out of the cell
o
Phagocytosis- engulfing by a cell of a smaller cell or a particle (protozoa, Amoeba)
o
Pinocytosis- cell engulfs liquid
o
Receptor-mediated endocytosis- receptor binds to a substance and assist in
transport (viruses and host cells)
Sites of cellular energy transformations where ATP is generated. ATP generation &
utilization is a central metabolic activity. The location & structures involved in cellular-energy
generating reactions will be discussed here (i) Some reactions occur in the cytoplasm, (ii)
Some cell membrane structures are a key to generate cellular energy. Two mechanisms for
generating cellular energy; (i) Substrate level phosphorylation and (ii) Chemiosmosis:
Movement of microbial cells : (i) Flagella / Cilia, (ii) Axial Filament, (iii) Gas vacuoles, (iv)
Magnetosomes, (v) Pseudopodia and (vi) Chemotaxis, magnetotaxis and phototaxis.
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Microbes do not die?
There are structures to ensure survival
Ordinary microbes are killed by minor stresses eg chilling, antibiotics, disinfectants but cells
with protecive bodies, namely endospores and cysts ressist such stresses. In most cases,
the cells that produce endospores and cysts are a part of the soil microflora. Soil heats &
dries in summer but is periodically flooded by rain -- harsh fluctuating environment.
a. Endospores:
Historical Developement & Importance
100's of species mainly of the genera Bacillus (aerobic rods, facultative anaerobes), and
Clostridium (anaerobic rods); Few others include Sporosarcina (aerobic cocci),
Desulfotomaculum (anaerobic rods, sulfate-reducers) . Food industries (canning, milk
etc) heat treat products to reduce microbial spoilage & kill pathogens; spore-formers are
a problem (swelling of tins; putrification of meat etc). Mainly found in soils --> vegetables
--> meat where spores germinate to produce toxins (eg veg / meat salad stored
improperly prior to use; wooden choping boards prefered over synthetic) . Mainly found
in soils --> infect wounds (problem with farm associated workers)
Some strains were being developed for biological warfare eg B. anthracis (anthrax).
Some strains produce important biopesticides (biotechnology) eg B. thuringiensis var.
israelensis produces toxic proteins against mosquito & blackfly larvae. Commercial
variants available which produce toxins towards slightly different insect pests eg
Thuricide, Teknar, M-one. Spore which can germinate have been found from structures
7200 year old temples have been found and recently from GI tract of a bee preserved in
amber (1 million years old)
Distribution
Bacteria
Fungi
Present in some genera
Present
Protective & dispersal function
Reproductive
function
Endospores
Endo- or Exo- spores
One per cell but C. disporicum=2; C.
Numerous
polypendens=5
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
Size : Larger (distends the cell) or smaller than the cell

Shape : (i) Cylindrical, (ii) Ellipsoidal (iii) Spherical

Location : (i) Central, (ii) Terminal, (iii) Sub-terminal

Cells with endospores can be identified by spore-staining: (i) B. megaterium,an
aerobe: Small cylindrical sub-terminal spores, (ii) C. tetani, an anaerobe: Large
(distend) spherical terminal spores .

Heat ressistance
Endospore-forming cell

Time required to kill a suspension in
boiling water (100oC)
B. anthracis
1-2 min (not very heat ressistant)
C. botulinum
2-6 hours
C. tetani
1-3 hours
E. coli & S. aureus (nonendospore formers)
30 minutes at 70o C
Spore structure
Spores are formed during unfavourable growth conditions & germinate under
favourable conditions. The spore can be differentiated into 4 distinct parts: Core: Nucleic
acids, ribosome, low levels of enzyme activity, Calcium dipicolonic acid (CDPA) & low
water content. Low level of metabolic activity .Two wall like layers:

Cortex: Surrounds the core, mainly electron light peptidoglycan

Coat: Surrounds the cortex, mainly protein
Exosporium: The outer most thin layer

Mechanism of heat ressistance
Physical (sporecoat): Ressistance to staining demonstrates imperability & therefore
ressistant to dehydration & effects of toxins (multilayered thick peptidoglycan) . Chemical
(core): Low water content (15% instead of 80% found in cells) makes prteins & nucleic
acids more ressistant. CDPA complexes with proteins & other labile components &
makes them more ressistant. Medium lacking calcium or mutant strains that do not form
CPDA produce less "tolerant" spores.
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Cysts
Ressistant to dehydration but not to heat and hence unlike spores. Deposition of layers &
layers of cell wall around the cell rather than within the cell as in case of spores. Azotobacter
(free living nitrogen fixing bacterium found in soil) and Myxbacteria .Involved in nitrogen
fixation and protection.
Cellular storage of genetic information
i.
Bacterial & archaeal chromosome
Usually a single circular chromosome (Streptomyces & Borrelia = linear, Rhodobacter
sphaeroides = 2 separate chromosomes). "Naked DNA" - not membrane bound (nucleoid
region). Negatively supercoiled (highly twisted)- can expand to 1mm in length uncoiled
(length of a "typical" bacterium is a few micrometers and not associated with histone proteins
(histones responsible for eucaryotic DNA coiling) but histone-like proteins found. Genome
size extremely heterogenous, determined in nucleotide base pairs (bp).
Microbe
Characteristics
Size (Mb)
Mycoplasma genitalium No cell wall, bact
0.58
Haemophilus influenzae bact pathogen
1.83
Helicobacter pylori
bact pathogen
Neisseria meningitidis
bact pathogen
Escherichia coli
GI bact
Thermotoga maritima
bact thermo
Sequence information
4.4
Archaeoglobus fulgidus archaea thermo
Pyrodictium occultum
Methanococcus
jannaschii
archaea thermo
archaea therm

G+C content between 28% to 72%

Cell division (binary fission) & DNA duplication are synchronised. DNA duplication is
slower than cell division & therefore new rounds of DNA synthesis are initiated by the
cell even though the previous copy has not fully replicated. A cell can carry one full
copy & several partial copies.
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ii. Plasmids

small, circular, self replicating extrachromosomal genetic elements- >= 1

the genetic information supplements the chromosomal genetic information

o
antibiotic ressistance
o
tolerance to toxic metals
o
production of toxins
o
mating capabilities
genetic information is 1 - 5% of chromosomal DNA information but means 0% or
100% survival eg antibiotic ressistance


classfied on the basis of its function
o
"mating" plasmids - F (fertility) factor
o
antibiotic, metal ressistance- R (ressistance) factor
benefits & hazards
o
multiple drug ressistant pathogens
o
genetic engineering- cloning & expression of useful substances
iii. Nucleus & chromosomes of Eucarya cells

linear chromosomes associated with chromatins; chromatins are histone proteins
(basic proteins) around which DNA coils (~ 200 nucleotides/histone) to form
nucleosomes- "beads on a string" under EM

chromosomes are located in the nucleus
o
nucleus is separated from the cytoplasm by pore containing nuclear
membrane (double layered bilayered membrane)
o
more processing of the DNA is needed before it can be expressed & hence
this type of separation is necessary

usually greater than 1 different sized chromosomes present

Dinoflagellate algae is an evolutionary link between eucaryotes and bacteriao
DNA inside nucleus (similar to eucaryotes)
o
not histone associated ie not coiled like eucaryotic chromosomes
o
DNA arangement is similar to that of bacterial DNA (nucleoid region)
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Information flow in cells: the role of ribosomes
DNA -------> RNA (tRNA, mRNA, rRNA)-------->proteins
~10,000 ribosomes in archaea & bacteria depending on growth rates but many more in
eucarya. Measured in Svedgurg (S) units: rate of sedimentation in an ultracentrifuge
dependent on shape & size. Bacteria & Archaea: (i) 70S (30S = 21 proteins, 16SrRNA
[~1542 nucleotides] + 50S = 34 proteins, 23S rRNA [2900 nucleotides, 5S [120
nucleotides]) -- Phylogeny. (ii) Similar 70S but differences exist in protein composition

archaea are not sensitive to antibiotics that inhibit bacterial protein synthesis
tetracycline, erythromycin, chloramphenicol

diptheria toxin & anisomycin affects ribosomes of archaea but not bacterial
Eucarya:
o
80S (40S = 18SrRNA, 60S = 25 to 28S rRNA, 5.8S rRNA)
o
synthesised in the nucleolus & transported via nuclear pores into cytoplasm
o
Primitive protozoa Giardia contains 70S
o
mitochondria & chloroplast contain 70S; rRNA sequence shows similarity to
noncultured archaea & Rickettsia (proteobacteria) endosymbiotic theory.
Differences in 70S & 80S can be targeted for treatment of animal / plant diseases
o
Streptomycin & Erythromycin bind & alter 70S shape of bacteria not eucaryotes
Storage of materials
1. Inclusion bodies of bacteria
Bacteria store chemicals under certain conditions. eg, increased carbon availability but
not inadequate nitrogen-containing compounds for protein synthesis available. Not
separated by membranes & display differential solubility.
o
Nutrient reserves synthesised by the cell: poly-beta-hydroxybutyrate (PHB)
o
Energy reserves: inorganic polyphosphates (volutin, metachromatic granules) for
ATP synthesis; viewable after staining by light microscopy
o
Metabolic deposits: Sulfur deposited as a result of metabolism (photosynthetic
bacteria)
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2. Membrane bound organelles in Eucarya

Endoplasmic reticulum

Golgi apparatus

Lysosomes

Microbodies

Vacuoles

Cytoskeletal network
Cell surface structures involved in attachment
Glycocalyx: Bind cells togethr forming multicellular aggregates. In some cases the bacterial
cells adhere to solid surfaces using these structures.
o
Some pathogenic bacteria adhere to animal tissues
o
Some aquatic bacteria adhere to rocks
o
Some are involved in plaque formation leading to dentall caries
Fimbrae:

Not all bacteria posses fimbrae -- it is an inherited trait

Arise from the cytoplasmic membrane or just below the membrane

Can be mistaken for flagella but are not involved in motility

Much shorter and more numerous than flagella

Adhesion functions which enables cells to form a pellicle on liquid surfaces
Pili:
Similar to fimbrae but longer and fewer; sometimes only one per cell. Three functional types
of bacterial pili:
o
Act as receptors sites for some attachment of some phages ie phage infection
o
Act as sex pilus for bacterial conjugation processes (F aka Fertility pili of E. coli)
o
Attachment for pathogenic bacteria to human tissues (Neisseria gonorrhoeae)
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MICROBIAL GROWTH
Microbial growth may be described as occurring in different ways under different
circumstances. Increase in both population size and population mass: (i) Microbial
populations tend to increase in number and in cell mass simultaneously. (ii) Increase in cell
number and increase in cell population mass both usually occur in a measurably coordinated
fashion.
Note that, for bacteria, while the cell population and population mass typically increase with
time (with growth), over the course of population growth individual cells actually cycle
through increases and decreases in cell mass (i.e., growth, division, growth, division, growth
. . .). Bias toward cell number:
i. When a microbiologist speaks of microbial growth it is usually increase in cell number
that she is after.
ii. The reason for this bias is that a typical microbiologist is more interested in population
characteristics than in the characteristics of individual cells, or both (since the
characteristics of individual cells tend to be studied, by necessity, within the context of
populations of cells).
iii. Consequently, there is a tendency for microgiologists to follow microbial growth as
populations rather than following the growth of individual cells, and therefore
microbiologists tend to be more interested in population sizes than the size (mass) of any
indvidual cell. Futhermore, the typical measurement of microbial growth will be done over
the span of more than one microbial generation.
a. Increase in cell number :
An increase in cell number is an immediate consequence of cell division. Because most
bacteria grow by binary fission, doubling in cell number usually occurs at the same rate that
individual cells grow and divide.
b. Increase in cell mass
Doubling in size: Individual cells of many species double in size between divisions. Cell
mass thus increases at the same rate as cell number. The implication of this is that while
increase in cell number may be emphasized while considering microbial growth, increases
(and decreases) in individual cell masses are also occurring, though these increases and
decreases ballance each other out such that the average cell size tends to remain constant
under constant conditions.
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Anabolic process: The increase in mass is a consequence of anabolism. For anabolism to
occur a cell must be situated in an environment that supplies all necessary nutrients and
which physically falls into a range in which growth can occur.
c. Binary fission
i.
Procaryotic cell division:
Binary fission is the process by which most procaryotes replicate. Binary fission
generally involves the separation of a single cell into two more or less identical daughter
cells, each containing, among other things, at least one copy of the parental DNA.
ii. Stepwise process:
The first steps of binary fission include cell elongation and DNA replication. The cell
envelope then pinches inward, eventually meeting. A cross wall is formed and ultimately two
distinct cells are present, each essentially identical to the original parent cell.See illustration
below.
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d. Generation time [doubling time]
Procaryotic cell division: A bacterial generation time is also know as its doubling time.
Doubling time is the time it takes a bacterium to do one binary fission starting from
having just divided and ending at the point of having just completed the next division.
Generation times vary with organism and environment and can range from 20 minutes
for a fast growing bacterium under ideal conditions, to hours and days for less than ideal
conditions or for slowly growing bacteria.
e. Standard bacterial growth curve
The standard bacterial growth curve describes various stages of growth a pure culture of
bacteria will go through, beginning with the addition of cells to sterile media and ending
with the death of all of the cells present. The phases of growth typically observed
include: (i) lag phase, (ii) exponential (log, logarithmic) phase, (iii) stationary phase and
(iv) death phase (exponential or logarithmic decline).
In standard bacterial growth curves one keeps track of cell growth by some measure or
estimation of cell number.
Exponential [log or logarithmic] growth (phase)
Back-to-back divisions: Exponential growth is a physiological state marked by back-toback division cycles such that the population doubles in number every generation time.
Note that during exponential growth there is no change in average cell mass, though
individuals cells are constantly changing in mass as they increase in mass, then divide
thus rapidly decreasing in mass (while increasing in number).
The algebra of exponential growth: Note that during exponential growth the number of
cells present at any given time is a multiplicative function of the number of cells present
at a previous time. Under constant conditions the multiplicative increase in cell number
consequently is constant for any given interval of the same duration.
If a log phase culture goes from 2 cells to 4 cells during a 20 minute interval, then the
culture will go from 4 cells to 8 cells during the next 20 minutes. If a log phase culture
goes from 2 cells to 6 cells during a 60 minute interval, then the culture will go from 6
cells to 18 cells during the next 60 minutes. If during exponential phase there are 10 cells
present at time 0, and 100 cells present at time 200, then at time 400 there will be
10,000 (100 * 100) cells present.
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Lag phase
Lag in division: Upon a change in environment (especially from a rich environment to a
poor environment), or when going from stationary phase to exponential phase, there is a
lag before division resumes. For example, stationary phase Escherichia coli placed in an
excess of sterile broth will go through a lag phase during which they increase in cell size
but do not divide. They will divide only once they have reached the size of a cell which is
about to divide during exponential growth under those conditions. During this time a
culture is said to be in lag phase.
Increase in mass: During lag phase cells increase in mass but do not divide. In other
words, there is no change in number, but an increase in mass.
"The length of the lag phase is determined in part by characteristics of the bacterial
species and in part by conditions in the media---both the medium from which the
organisms are taken and the one to which they are transferred. Some species adapt to
the new medium in an hour or two; others take several days. Organisms from old
cultures, adapted to limited nutrients and large accumulated wastes, take longer to
adjust to a new medium than do those transferred from a relatively fresh, nutrient-rich
medium." (p. 138, Black, 1996)
Stationary phase
Stationary phase is classically defined as a physiological point where the rate of cell
division equals the rate of cell death, hence viable cell number remains constant.
No cell division: Note that when cell division = 0 and cell death = 0, then the rate of cell
division = rate of cell death. In other words, when cells stop dividing but have not yet
started dying they are in stationary phase.
A way to distinguish these possibilities is to compare viable count with total count. If both
total counts and viable counts don't change then you know that there is both no cell
division and no cell death. If total count increases while viable counts remain constant,
then you know that you are observing a true balance between ongoing cell division and
cell death.
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Physiological adaptation to cell excess: Stationary phase usually occurs when cell
concentration is so great and that some aspect of the environment is no longer able to
serve the requirements of exponential growth. Stationary phase is a time of significant
physiological change and particularly involves the physiological adaptation of cells to
survival through periods of little growth.
Cell death
In single celled microorganisms cell death is the point at which reinitiation of division is
no longer possible. Qualified definition:
i. Note that the concept of cell death is actually dependent on how one attempt to
reinitiate growth.
ii. Particularly, there are ways to gently revive some microbes from physiological states
that would result in permanent lack of growth in other growth environments.
An analogous situation would be a person with an injury that is inevitably fatal in a thirdworld hospital, but readily treated in a first-world hospital.
Example: seeds: Another analogy is with a plant seed. You can try to sprout it in all kinds
of environments but not all will work out in the seed's favor. You may end up killing the
seed by allowing it to attempt to germinate in the wrong environment. The more
degraded is the seed prior to planting, the greater the likelihood that germination will not
successfully occur unless you take great care to make sure sprouting conditions are as
close to ideal as you can make them.
Death phase [logarithmic decline, exponential decline]
Death phase is a physiological point at which cell deaths exceed cell births. More
specifically, viable count declines. "During the decline phase, many cells undergo
involution---that is, they assume a variety of unusual shapes, which makes them difficult
to identify." (p. 140, Black, 1996
Endospore [spore, sporulation, sporogenesis, activation, germination]. Tough, dormant
state: A very tough, dormant form of certain bacterial cell that is very resistant to
desiccation, heat, and a variety of chemical and radiation treatments that are otherwise
lethal to non-endospore bacterial cells. At least part of the toughness associated with a
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spore is found in its very tough outer layers, called a coat. Only some bacteria produce
endospores. Endospores of some bacteria can last so long under proper conditions that
various endospores found in such things as Egyptian mummies are likely the oldest
living things.
Sporulation and sporogenesis:
Sporulation and sporogenesis refer to the formation of endospores by vegetative (i.e.,
growing) cells. The endospore is actually the intracellular product of sporogenesis. A
spore is an endospore which has been released from a cell, i.e., it exists is a free state.
In bacteria the formation of a spore is not considered to be an act of reproduction.
Indeed, the formation of the endospore is directed by the DNA that will ultimately be
found in the spore, and the sister DNA found in the vegetative part of the cell ultimately is
destroyed.
The first step of germination often requires some kind of coat traumatizing insult such as
high temperature or low pH. The transformation from the endospore state to the
vegetative state. The key thing to worry about with endospores is that they are capable
of germinating despite harsh treatment, and thus can potentially produce actively
replicating cells where there may have been none previously prevent. Of those bacteria
on your list, the following are spore formers (note that all are gram-positives):
1.5
i.
Bacillus anthracis
ii.
Bacillus subtilis
iii.
Clostridium botulinum
iv.
Clostridium perfringens
v.
Clostridium tetani
MICROBIAL METABOLISM
Based on their modes of metabolism, the procaryotes are much more diverse than all
eucaryotes, and the real real explanation for "microbial diversity" rests fundamentally on
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some aspect procaryotic metabolism, especially with regards to energy-generating
metabolism and synthesis of secondary metabolites.
Microbial diversity translates to metabolic diversity. The procaryotes, as a group, conduct
all the same types of basic metabolism as eucaryotes, but, in addition, there are several
types of energy-generating metabolism among the procaryotes that are non existent in
eucaryotic cells or organisms. These include: (i) Unique fermentation pathways that
produce a wide array of end products. (ii) Anaerobic respiration: respiration that uses
substances other than O2 as a final electron acceptor.
 Lithotrophy: use of inorganic substances as sources of energy
 Photoheterotrophy: use of organic compounds as a carbon source during bacterial
photosynthesis
 Anoxygenic photosynthesis: uses special chlorophylls and occurs in the absence of
O2
 Methanogenesis: an ancient type of archaean metabolism that uses H2 as an energy
source and produces methane.
 Light-driven nonphotosynthetic energy production: unique archaean metabolism
that converts light energy into chemical energy; occurs in the archaea (extreme
halophiles).
 Unique mechanisms for autotrophic CO2 fixation, including primary production on
anaerobic habitats
What is metabolism?
The term metabolism refers to the sum of the biochemical reactions required for energy
generation and the use of energy to synthesize cell material from small molecules in the
environment.
Hence,
metabolism
has
an
energy-generating
component,
called
catabolism, and an energy-consuming, biosynthetic component, called anabolism.
Catabolic reactions or pathways produce energy as ATP, which can be utilized in anabolic
reactions to build cell material from nutrients in the environment. The relationship between
catabolism and anabolism is illustrated in Figure 1 below.
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Figure : The relationship between catabolism and anabolism in a cell. During catabolism, energy is
changed from one form to another, and keeping with the laws of thermodynamics, such energy
transformations are never completely efficient, i.e., some energy is lost in the form of heat. The efficiency
of a catabolic sequence of reactions is the amount of energy made available to the cell (for anabolism)
divided by the total amount of energy released during the reactions.
Metabolism is usually visualized as as a series of biochemical reactions mediated by
enzymes, referred to as a metabolic pathway. Catabolic pathways lead to end products,
which are "waste products" and result in the generation of energy which is temporarily
conserved as adenosine triphosphate (ATP). In heterotrophs, the most common catabolic
pathways are the Emden-Meyerhof pathway for degradation of sugars as energy sources
(glycolysis and the tricarboxylic acid cycle (TCA cycle), which can be linked to the further
degradation of almost any organic compound and further leads to the synthesis of ATP.
Model of a catabolic pathway. Each reaction in the pathway is mediated by a specific
enzyme.
s
sugar-------->
X-------->
x
Y-------->
y
Z-------->
Intermediate
z
+
ATP
Anabolic pathways utilize ATP to provide energy for the synthesis of the monomeric
compounds that are required for the manufacture of the small molecules needed in cells,
i.e., carbohydrates, lipids, amino acids, nucleotides, vitamins, etc.
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Model of an anabolic pathway. Each reaction in the pathway is mediated by a specific
enzyme.
a
Intermediate
+
ATP-------->
b
A-------->
c
B-------->
C-------->
d
Final
product
ATP
During catabolism, useful energy is temporarily conserved in the "high energy bond" of ATP
- adenosine triphosphate. No matter what form of energy a cell uses as its primary source,
the energy is ultimately transformed and conserved as ATP. ATP is the universal currency
of energy exchange in biological systems. When energy is required during anabolism, it may
be spent as the high energy bond of ATP which has a value of about 8 kcal per mole.
Hence, the conversion of ADP to ATP requires 8 kcal of energy, and the hydrolysis of ATP to
ADP releases 8 kcal.
The structure of ATP. ATP is derived from the nucleotide adenosine monophosphate (AMP) or
adenylic acid, to which two additional phosphate groups are attached through pyrophosphate
bonds (~P). These two bonds are energy rich in the sense that their hydrolysis yields a great deal
more energy than a corresponding covalent bond. ATP acts as a coenzyme in energetic coupling
reactions wherein one or both of the terminal phosphate groups is removed from the ATP
molecule with the bond energy being used to transfer part of the ATP molecule to another
molecule to activate its role in metabolism. For example, Glucose + ATP -----> Glucose-P + ADP or
Amino Acid + ATP ----->AMP-Amino Acid + PPi.
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NAD
Another coenzyme commonly involved in metabolism, derived from the vitamin niacin, is the
pyridine nucleotide, NAD (Nicotinamide Adenine Dinucleotide). The basis for chemical
transformations of energy usually involves oxidation/reduction reactions. For a biochemical
to become oxidized, electrons must be removed by an oxidizing agent. The oxidizing agent
is an electron acceptor that becomes reduced in the reaction. During the reaction, the
oxidizing agent is converted to a reducing agent that can add its electrons to another
chemical, thereby reducing it, and reoxidizing itself. The molecule that usually functions as
the electron carrier in these types of coupled oxidation-reduction reactions in biological
systems is NAD and its phosphorylated derivative, NADP. NAD or NADP can become
alternately oxidized or reduced by the loss or gain of two electrons. The oxidized form of
NAD is symbolized NAD; the reduced form is symbolized as NADH2. The structure of NAD
is drawn below.
The Structure of NAD. (a) Nicotinamide Adenine Dinucleotide is composed of two nucleotide
molecules: Adenosine monophosphate (adenine plus ribose-phosphate) and nicotinamide ribotide
(nicotinamide plus ribose-phosphate). NADP has an identical structure except that it contains an
additional phosphate group attached to one of the ribose residues. (b) The oxidized and reduced
forms of of the nicotinamide moiety of NAD. Nicotinamide is the active part of the molecule where
the reversible oxidation and reduction takes place. The oxidized form of NAD has one hydrogen
atom less than the reduced form and, in addition, has a positive charge on the nitrogen atom
which allows it to accept a second electron upon reduction. Thus the correct way to symbolize the
reaction is NAD+ + 2H----->NADH + H+. However, for convenience we will hereafter use the symbols
NAD and NADH2.
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ATP Synthesis
The objective of a catabolic pathway is to make ATP, that is to transform either chemical
energy or electromagnetic (light) energy into the chemical energy contained within the highenergy bonds of ATP. Cells fundamentally can produce ATP in two ways: substrate level
phosphorylation and electron transport phosphorylation.
Substrate level phosphorylation (SLP) is the simplest, oldest and least-evolved way to
make ATP. In a substrate level phosphorylation, ATP is made during the conversion of an
organic molecule from one form to another. Energy released during the conversion is
partially conserved during the synthesis of the high energy bond of ATP. SLP occurs during
fermentations and respiration (the TCA cycle), and even during some lithotrophic
transformations of inorganic substrates.
Three examples of substrate level phosphorylation. (a) and (b) are the two substrate level
phosphorylations that occur during the Embden Meyerhof pathway, but they occur in all other
fermentation pathways which have an Embden-Meyerhof component. (c) is a substrate level
phosphorylation found in Clostridium and Bifidobacterium. These are two anaerobic (fermentative)
bacteria who learned how to make one more ATP from glycolysis beyond the formation of
pyruvate.
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Electron Transport Phosphorylation (ETP) is a much more complicated affair that evolved
long after SLP. Electron Transport Phosphorylation takes place during respiration,
photosynthesis, lithotrophy and possibly other types of bacterial metabolism. ETP requires
that electrons removed from substrates be dumped into an electron transport system (ETS)
contained within a membrane. The electrons are transferred through the ETS to some final
electron acceptor in the membrane (like O2 in aerobic respiration) , while their traverse
through the ETS results in the extrusion of protons and the establishment of a proton
motive force (pmf) across the membrane. An essential component of the membrane for
synthesis of ATP is a membrane-bound ATPase (ATP synthetase) enzyme. The ATPase
enzyme transports protons, thereby utilizing the pmf (protons) during the synthesis of ATP.
The idea in electron transport phosphorylation is to drive electrons through an ETS in the
membrane, establish a pmf, and use the pmf to synthesize ATP. Obviously, ETP take a lot
more "gear" than SLP, in the form of membranes, electron transport systems, ATPase
enzymes, etc.
A familiar example of energy-producing and energy-consuming functions of the bacterial
membrane, related to the establishment and use of pmf and the production of ATP, is given
in the following drawing of the plasma membrane of Escherichia coli.
The plasma membrane of Escherichia coli. The membrane in cross-section reveals various transport
systems, the flagellar motor apparatus (S and M rings), the respiratory electron transport system, and the
membrane-bound ATPase enzyme. Reduced NADH + H+ feeds pairs of electrons into the ETS. The ETS is
the sequence of electron carriers in the membrane [FAD --> FeS --> QH2 (Quinone) --> (cytochromes) b -> b --> o] that ultimately reduces O2 to H2O during respiration. At certain points in the electron transport
process, the electrons pass "coupling sites" and this results in the translocation of protons from the
inside to the outside of the membrane, thus establishing the proton motive force (pmf) on the membrane.
The pmf is used in three ways by the bacterium to do work or conserve energy: active transport (e.g.
lactose and proline symport; calcium and sodium antiport); motility (rotation of the bacterial flagellum),
and ATP synthesis (via the ATPase enzyme during the process of oxidative phosphorylation or electron
transport phosphorylation).
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Heterotrophic Types of Metabolism
Heterotrophy (i.e., chemoheterotrophy) is the use of an organic compound as a source of
carbon and energy. It is the complete metabolism package. The cell oxidizes organic
molecules in order to produce energy (catabolism) and then uses the energy to synthesize
cellular material from these the organic molecules (anabolism). We animals are familiar with
heterotrophic metabolism. Fungi and protozoa are all heterotrophs; many bacteria, but just a
few archaea, are heterotrophs, Heterotrophic fungi and bacteria are the masters of
decomposition and biodegradation in the environment. Heterotrophic metabolism is driven
mainly by two metabolic processes: fermentations and respirations.
Fermentation
Fermentation is an ancient mode of metabolism, and it must have evolved with the
appearance of organic material on the planet. Fermentation is metabolism in which energy is
derived from the partial oxidation of an organic compound using organic intermediates
as electron donors and electron acceptors. No outside electron acceptors are involved;
no membrane or electron transport system is required; all ATP is produced by substrate
level phosphorylation.
By definition, fermentation may be as simple as two steps illustrated in the following model.
Indeed, some amino acid fermentations by the clostridia are this simple. But the pathways
of fermentation are a bit more complex, usually involving several preliminary steps to prime
the energy source for oxidation and substrate level phosphorylations.
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Model fermentation. L. The substrate is oxidized to an organic intermediate; the usual oxidizing agent is
NAD. Some of the energy released by the oxidation is conserved during the synthesis of ATP by the
process of substrate level phosphorylation. Finally, the oxidized intermediate is reduced to end products.
Note that NADH2 is the reducing agent, thereby balancing its redox ability to drive the energy-producing
reactions. R. In lactic fermentation by Lactobacillus, the substrate (glucose) is oxidized to pyruvate, and
pyruvate becomes reduced to lactic acid. Redox balance is maintained by coupling oxidations to
reductions within the pathway. For example, in lactic acid fermentation via the EmbdenMeyerhof
pathway, the oxidation of glyceraldehyde phosphate to phosphoglyceric acid is coupled to the reduction
of pyruvic acid to lactic acid.
In biochemistry, for the sake of convenience, fermentation pathways start with glucose. This
is because it is the simplest molecule, requiring the fewest enzymatic ( catalytic) steps, to
enter into a pathway of glycolysis and central metabolism.
In the bacteria there exist three major pathways of glycolysis (the dissimilation of sugars):
the classic Embden-Meyerhof pathway, which is also used by most eucaryotes, including
yeast (Saccharomyces): the heterolactic pathway used by lactic acid bacteria, and the
Entner-Doudoroff pathway used by vibrios and pseudomonads, including Zymomonas.
Although the latter two pathways have some interesting applications in the manufacture of
dairy products and alcoholic beverages, they will not be discussed further in this section..
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The Embden-Meyerhof Pathway
This is the pathway of glycolysis most familiar to biochemists and eucaryotic biologists, as
well as to brewers, breadmakers and cheese makers. The pathway is operated by
Saccharomyces to produce ethanol and CO2. The pathway is used by the lactic acid bacteria
to produce lactic acid, and it is used by many other bacteria to produce a variety of fatty
acids, alcohols and gases. Some end products of Embden-Meyerhof fermentations are
essential components of foods and beverages, and some are useful fuels and industrial
solvents. Diagnostic microbiologists use bacterial fermentation profiles (e.g. testing an
organism's ability to ferment certain sugars, or examining an organism's array of end
products) in order to identify them, down to the genus level.
The Embden Meyerhof pathway for glucose dissimilation. The overall reaction is the oxidation of
glucose to 2 pyruvic acid. The two branches of the pathway after the cleavage are identical.
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The first three steps of the pathway prime (phosphorylated) and rearrange the hexes for
cleavage into 2 triodes (glyceraldehyde phosphate). Fructose 1,6-diphosphate aldolase is
the key (cleavage) enzyme in the E-M pathway. Each triose molecule is oxidized and
phosphorylated followed by two substrate level phosphorylations that yield 4 ATP during the
drive to pyruvate. Lactic acid bacteria reduce the pyruvate to lactic acid; yeast reduce the
pyruvate to alcohol (ethanol) and CO2 as shown in Figure below.
(a) The Embden Meyerhof pathway of lactic acid fermentation in lactic acid bacteria (Lactobacillus)
and (b) the Embden Meyerhof pathway of alcohol fermentation in yeast (Saccharomyces). The
pathways yield two moles of end products and two moles of ATP per mole of glucose fermented.
The steps in the breakdown of glucose to pyruvate are identical. The difference between the
pathways is the manner of reducing pyruvic acid, thereby giving rise to different end products.
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Besides lactic acid, Embden-Meyerhof fermentations in bacteria can lead to a wide array of
end products depending on the pathways taken in the reductive steps after the formation of
pyruvic acid. Usually, these bacterial fermentations are distinguished by their end products
into the following groups.
a. Homolactic Fermentation. Lactic acid is the sole end product. Pathway of the
homolactic acid bacteria (Lactobacillus and most streptococci). The bacteria are used
to ferment milk and milk products in the manufacture of yogurt, buttermilk, sour
cream, cottage cheese, cheddar cheese, and most fermented dairy products.
b. Mixed Acid Fermentations. Mainly the pathway of the Enterobacteriaceae. End
products are a mixture of lactic acid, acetic acid, formic acid, succinate and
ethanol, with the possibility of gas formation (CO2 and H2) if the bacterium
possesses the enzyme formate dehydrogenase, which cleaves formate to the gases.
c.
Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition,
2,3 butanediol from the condensation of 2 pyruvate. The use of the pathway
decreases acid formation (butanediol is neutral) and causes the formation of a
distinctive intermediate, acetoin. Water microbiologists have specific tests to detect
low acid and acetoin in order to distinguish non fecal enteric bacteria (butanediol
formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid
fermenters, such as E. coli, Salmonella and Shigella).
d. Butyric acid fermentations, as well as the butanol-acetone fermentation (below),
are run by the clostridia, the masters of fermentation. In addition to butyric acid, the
clostridia form acetic acid, CO2 and H2 from the fermentation of sugars. Small
amounts of ethanol and isopropanol may also be formed.
e. Butanol-acetone fermentation. Butanol and acetone were discovered as the main
end products of fermentation by Clostridium acetobutylicum during the World War I.
This discovery solved a critical problem of explosives manufacture (acetone is
required in the manufacture gunpowder) and is said to have affected the outcome of
the War. Acetone was distilled from the fermentation liquor of Clostridium
acetobutylicum, which worked out pretty good if you were on our side, because
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organic chemists hadn't figured out how to synthesize it chemically. You can't run a
war without gunpowder, at least you couldn't in those days.
f.
Propionic acid fermentation. This is an unusual fermentation carried out by the
propionic acid bacteria which include corynebacteria, Propionibacterium and
Bifidobacterium. Although sugars can be fermented straight through to propionate,
propionic acid bacteria will ferment lactate (the end product of lactic acid
fermentation) to acetic acid, CO2 and propionic acid. The formation of propionate is a
complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of lactate
are converted to 2 moles of propionate + 1 mole of acetate + 1 mole of CO 2, and 1
mole of ATP is squeezed out in the process. The propionic acid bacteria are used in
the manufacture of Swiss cheese, which is distinguished by the distinct flavor of
propionate and acetate, and holes caused by entrapment of CO2.
The Embden-Meyerhof pathway for glucose dissimilation (Figure 8), as well as the TCA
cycle discussed below (Figure 10), are two pathways that are at the center of metabolism in
nearly all organisms. Not only do these pathways dissimilate organic compounds and
provide energy, they also provide the precursors for biosynthesis of macromolecules that
make up living systems. These are sometimes called amphibolic pathways since the have
both an anabolic and a catabolic function.
Respiration
Compared to fermentation as a means of oxidizing organic compounds, respiration is a lot
more complicated. Respirations result in the complete oxidation of the substrate by an
outside electron acceptor. In addition to a pathway of glycolysis, four essential structural or
metabolic components are needed:
i.
The tricarboxylic acid (TCA) cycle (also known as the citric acid cycle or the Kreb's
cycle): when an organic compound is utilized as a substrate, the TCA cycle is used
for the complete oxidation of the substrate. The end product that always results from
the complete oxidation of an organic compound is CO2.
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ii.
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A membrane and an associated electron transport system (ETS). The ETS is a
sequence of electron carriers in the plasma membrane that transports electrons
taken from the substrate through the chain of carriers to a final electron acceptor.
The electrons enter the ETS at a very low redox potential (E'o) and exit at a relatively
high redox potential. This drop in potential releases energy that can be harvested by
the cells in the process of ATP synthesis by the mechanisms of electron transport
phosphorylation. The operation of the ETS establishes a proton motive force (pmf)
due to the formation of a proton gradient across the membrane.
iii.
An outside electron acceptor ("outside", meaning it is not internal to the pathway,
as is pyruvate in a fermentation). For aerobic respiration the electron acceptor is
O2, of course. Molecular oxygen is reduced to H20 in the last step of the electron
transport system. But in the bacterial processes of anaerobic respiration, the final
electron acceptors may be SO4 or S or NO3 or NO2 or certain other inorganic
compounds, or even an organic compound, such as fumarate.
iv.
A transmembranous ATPase enzyme (ATP synthetase). This enzyme utilizes the
proton motive force established on the membrane (by the operation of the ETS) to
synthesize ATP in the process of electron transport phosphorylation. It is believed
that the transmembranous Fo subunit is a proton transport system that transports
2H+ to the F1 subunit (the actual ATPase) on the inside of the membrane. The 2
protons are required and consumed during the synthesis of ATP from ADP plus Pi.
See Figure 6 -the membrane of E. coli. The reaction catalyzed by the ATPase
enzyme is ADP + Pi + 2 H+ <----------> ATP. (It is important to appreciate the
reversibility of this reaction in order to account for how a fermentative bacterium,
without an ETS, could establish a necessary pmf on the membrane for transport or
flagellar rotation. If such an organism has a transmembranous ATPase, it could
produce ATP by SLP, and subsequently the ATPase could hydrolyze the ATP,
thereby releasing protons to the outside of the membrane.)
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The diagram below of aerobic respiration
integrates these metabolic processes into a
scheme that represents the overall process of respiratory metabolism. A substrate such as
glucose is completely oxidized to to CO2 by the combined pathways of glycolysis and the
TCA cycle. Electrons removed from the glucose by NAD are fed into the ETS in the
membrane. As the electrons traverse the ETS, a pmf becomes established across the
membrane. The electrons eventually reduce an outside electron acceptor, O2, and reduce it
to H20. The pmf on the membrane is used by the ATPase enzyme to synthesize ATP by a
process referred to as "oxidative phosphorylation".
Model of Aerobic respiration.
The overall reaction for the aerobic respiration of glucose is
Glucose + 6 O2 ----------> 6 CO2 + 6 H2O
In a heterotrophic respiration, glucose is dissimilated in a pathway of glycolysis to the
intermediate, pyruvate, and it the pyruvate that is moved into the TCA cycle, eventually
becoming oxidized to 3 CO2. Since 2 pyruvate are formed from one glucose, the cycle must
turn twice for every molecule of glucose oxidized to 6 CO 2. The TCA cycle (including the
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steps leading into it) accounts for the complete oxidation of the substrate and it provides 10
pairs of electrons (from glucose) for transit through the ETS. For every pair of electrons put
into the ETS, 2 or 3 ATP may be produced, so a huge amount of ATP is produced in a
respiration, compared to a fermentation.
The TCA cycle is an important amphibolic pathway, several intermediates of the cycle may
be withdrawn for anabolic (biosynthetic) pathways
The tricarboxylic acid (TCA) or Kreb's cycle. Also called the citric acid cycle because citric
acid is one of the first intermediates formed during the cycle. When an organic compound is
utilized during respiration it is invariably oxidized via the TCA cycle. Combined with the
pathway(s) of glycolysis (e.g. Embden-Meyerhof) TCA is central to the metabolism of all
heterotrophic respiratory organisms.....worth memorizing if you are a biologist.
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Anaerobic Respiration
Respiration in some procaryotes is possible using electron acceptors other than oxygen
(O2). This type of respiration in the absence of oxygen is referred to as anaerobic
respiration. Although anaerobic respiration is more complicated than the foregoing
statement, in its simplest form it represents the substitution or use of some compound
other than O2 as a final electron acceptor in the electron transport chain. Electron
acceptors used by procaryotes for respiration or methanogenesis (an analogous type of
energy generation in archaea) are described in the table below.
Electron acceptors for respiration and methanogenesis in procaryotes
electron
acceptor
reduced end
product
name of process
organism
O2
H2O
aerobic respiration
Escherichia,
Streptomyces
NO3
NO2, NH3 or N2
anaerobic respiration:
denitrification
Bacillus,
Pseudomonas
SO4
S or H2S
anaerobic respiration: sulfate
reduction
Desulfovibrio
fumarate
succinate
anaerobic respiration:
Escherichia
using an organic e- acceptor
CO2
CH4
methanogenesis
Methanococcus
Biological methanogenesis is the source of methane (natural gas) on the planet. Methane
is preserved as a fossil fuel (until we use it all up) because it is produced and stored under
anaerobic conditions, and oxygen is needed to oxidize the CH4 molecule. Methanogenesis is
not really a form of anaerobic respiration, but it is a type of energy-generating metabolism
that requires an outside electron acceptor in the form of CO2.
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Denitrification is an important process in agriculture because it removes NO3 from the soil.
NO3 is a major source of nitrogen fertilizer in agriculture. Almost one-third the cost of some
types of agriculture is in nitrate fertilizers The use of nitrate as a respiratory electron acceptor
is usually an alternative to the use of oxygen. Therefore, soil bacteria such as Pseudomonas
and Bacillus will use O2 as an electron acceptor if it is available, and disregard NO3. This is
the rationale in maintaining well-aerated soils by the agricultural practices of plowing and
tilling. E. coli will utilize NO3 (as well as fumarate) as a respiratory electron acceptor and so it
may be able to continue to respire in the anaerobic intestinal habitat.
Sulfate reduction is not an alternative to the use of O2 as an electron acceptor. It is an
obligatory process that occurs only under anaerobic conditions. Methanogens and sulfate
reducers may share habitat, especially in the anaerobic sediments of eutrophic lakes such
as Lake Mendota, where they crank out methane and hydrogen sulfide at a surprising rate.
Anaerobic respiring bacteria and methanogens play an essential role in the biological cycles
of carbon, nitrogen and sulfur. In general, they convert oxidized forms of the elements to a
more reduced state. The lithotrophic procaryotes metabolize the reduced forms of nitrogen
and sulfur to a more oxidized state in order to produce energy. The methanotrophic bacteria,
which uniquely posses the enzyme methane monooxygenase, can oxidize methane as a
source of energy. Among all these groups of procaryotes there is a minicycle of the elements
in a model ecosystem.
Lithotrophic Types of Metabolism
Lithotrophy is the use of an inorganic compound as a source of energy. Most lithotrophic
bacteria are aerobic respirers that produce energy in the same manner as all aerobic
respiring organisms: they remove electrons from a substrate and put them through an
electron transport system that will produce ATP by electron transport phosphorylation.
Lithotrophs just happen to get those electrons from an inorganic, rather than an organic,
compound.
Some lithotrophs are facultative lithotrophs, meaning they are able to use organic
compounds, as well, as sources of energy. Other lithotrophs do not use organic compounds
as sources of energy; in fact, they won't transport organic compounds. CO 2 is the sole
source of carbon for the methanogens and the nitrifying bacteria and a few other species
scattered about in other groups.
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Most lithotrophs get their carbon from from CO2 and are thus autotrophs and are properly
referred to as lithoautotrophs or chemoautotrophs. The lithotrophs are a very diverse
group of procaryotes, united only by their ability to oxidize an inorganic compound as an
energy source.
Lithotrophy runs through the Bacteria and the Archaea. If one considers methanogen
oxidation of H2 a form of lithotrophy, then probably most of the Archaea are lithotrophs.
Lithotrophs are usually organized into "physiological groups" based on their inorganic
substrate for energy production and growth (see Table 2 below).
Physiological groups of lithotrophs
physiological group energy source oxidized end product organism
hydrogen bacteria
H2
H2O
Alcaligenes, Pseudomonas
methanogens
H2
H2O
Methanobacterium
carboxydobacteria
CO
CO2
Rhodospirillum, Azotobacter
nitrifying bacteria*
NH3
NO2
Nitrosomonas
nitrifying bacteria*
NO2
NO3
Nitrobacter
sulfur oxidizers
H2S or S
SO4
Thiobacillus, Sulfolobus
iron bacteria
Fe ++
Fe+++
Gallionella, Thiobacillus
* The overall process of nitrification, conversion of NH3 to NO3, requires a consortium of
microorganisms.
The hydrogen bacteria oxidize H2 (hydrogen gas) as an energy source. The hydrogen
bacteria are facultative lithotrophs as evidenced by the pseudomonads that fortuitously
possess a hydrogenase enzyme that will oxidize H2 and put the electrons into their
respiratory ETS. They will use H2 if they find it in their environment even though they are
typically heterotrophic. Indeed, most hydrogen bacteria are nutritionally versatile in their
ability to use a wide range of carbon and energy sources. the bacterial electron transport
system.
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The methanogens used to be considered a major group of hydrogen bacteria - until it was
discovered that they are Archaea. The methanogens are able to oxidize H2 as a sole source
of energy while transferring the electrons from H2 to CO2 in its reduction to methane.
Metabolism of the methanogens is absolutely unique, yet methanogens represent the most
prevalent and diverse group of Archaea. Methanogens use H2 and CO2 to produce cell
material and methane. They have unique enzymes and electron transport processes. Their
type of energy generating metabolism is never seen in the Bacteria, and their mechanism of
autotrophic CO2 fixation is very rare, except in methanogens.
The carboxydobacteria are able to oxidize CO (carbon monoxide) to CO2, using an
enzyme CODH (carbon monoxide dehydrogenase). The carboxydobacteria are not
obligate CO users, i.e., some are also hydrogen bacteria, and some are phototrophic
bacteria. Interestingly, the enzyme CODH used by the carboxydobacteria to oxidize CO to
CO2, is used by the methanogens for the reverse reaction - the reduction of CO2 to CO - in
their unique pathway of CO2 fixation.
The nitrifying bacteria are represented by two genera, Nitrosomonas and Nitrobacter.
Together these bacteria can accomplish the oxidation of NH3 to NO3, known as the process
of nitrification. No single organism can carry out the whole oxidative process. Nitrosomonas
oxidizes ammonia to NO2 and Nitrobacter oxidizes NO2 to NO3. Most of the nitrifying bacteria
are obligate lithoautotrophs, the exception being a few strains of Nitrobacter that will utilize
acetate. CO2 fixation utilizes RUBP carboxylase and the Calvin Cycle. Nitrifying bacteria
grow in environments rich in ammonia, where extensive protein decomposition is taking
place. Nitrification in soil and aquatic habitats is an essential part of the nitrogen cycle.
Lithotrophic sulfur oxidizers include both Bacteria (e.g. Thiobacillus) and Archaea (e.g.
Sulfolobus). Sulfur oxidizers oxidize H2S (sulfide) or S (elemental sulfur) as a source of
energy. Similarly, the purple and green sulfur bacteria oxidize H2S or S as an electron donor
for photosynthesis, and use the electrons for CO2 fixation (the dark reaction of
photosynthesis). Obligate autotrophy, which is nearly universal among the nitrifiers, is
variable among the sulfur oxidizers. Lithoautotrophic sulfur oxidizers are found in
environments rich in H2S, such as volcanic hot springs and fumaroles, and deep-sea thermal
vents. Some are found as symbionts and endosymbionts of higher organisms. Since they
can generate energy from an inorganic compound and fix CO2 as autotrophs, they may play
a fundamental role in primary production in environments that lack sunlight. As a result of
their lithotrophic oxidations, these organisms produce sulfuric acid (SO4), and therefore tend
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to acidify their own environments. Some of the sulfur oxidizers are acidophiles that will grow
at a pH of 1 or less. Some are hyperthermophiles that grow at temperatures of 115
degrees C.
Iron bacteria oxidize Fe++ (ferrous iron) to Fe+++ (ferric iron). At least two bacteria probably
oxidize Fe++ as a source of energy and/or electrons and are capable of lithoautotrophic
growth: the stalked bacterium Gallionella, which forms flocculant rust-colored colonies
attached to objects in nature, and Thiobacillus ferrooxidans, which is also a sulfur-oxidizing
lithotroph.
Lithotrophic oxidations. These reactions produce energy for metabolism in the nitrifying and
sulfur oxidizing bacteria.
Phototrophic Metabolism
Phototrophy is the use of light as a source of energy for growth, more specifically the
conversion of light energy into chemical energy in the form of ATP. Procaryotes that can
convert light energy into chemical energy include the photosynthetic cyanobacteria, the
purple and green bacteria, and the "halobacteria" (actually archaea). The cyanobacteria
conduct plant photosynthesis, called oxygenic photosynthesis; the purple and green
bacteria conduct bacterial photosynthesis or anoxygenic photosynthesis; the extreme
halophilic archaea use a type of nonphotosynthetic photophosphorylation mediated by a
pigment, bacteriorhodopsin, to transform light energy into ATP.
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Biosynthesis
The pathways of central metabolism (i.e., glycolysis and the TCA cycle), with a few
modifications, always run in one direction or another in all organisms. The reason - these
pathways provide the precursors for the biosynthesis of cell material. When a pathway, such
as the Embden-Meyerhof pathway or the TCA cycle, functions to provide energy in addition
to chemical intermediates for the synthesis of cell material, the pathway is referred to as an
amphibolic pathway. Pathways of glycolysis and the TCA cycle are amphibolic pathways
because they provide ATP and chemical intermediates to build new cell material. The main
metabolic pathways, and their relationship to biosynthesis of cell material, are shown in
Figure 25 below.
Biosynthesis or intermediary metabolism is a topic of biochemistry, more so than
microbiology. It will not be dealt with in detail here. The fundamental metabolic pathways of
biosynthesis are similar in all organisms, in the same way that protein synthesis or DNA
structure are similar in all organisms. When biosynthesis proceeds from central metabolism
as drawn below, some of the main precursors for synthesis of procaryotic cell structures and
components are as follows.

Polysaccharide capsules or inclusions are polymers of glucose and other
sugars.

Cell wall peptidoglycan (NAG and NAM) is derived from glucose phosphate.

Amino acids for the manufacture of proteins have various sources, the most
important of which are pyruvic acid, alpha ketoglutaric acid and oxalacetic acid.

Nucleotides (DNA and RNA) are synthesized from ribose phosphate. ATP and
NAD are part of purine (nucleotide) metabolism.

Triose-phosphates are precursors of glycerol, and acetyl CoA is a main precursor
of lipids for membranes

Vitamins and coenzymes are synthesized in various pathways that leave central
metabolism. In the example given in Figure 24, heme synthesis proceeds from the
serine pathway, as well as from succinate in the TCA cycle.
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The main pathways of biosynthesis in procaryotic cells
Written and Edited by KennethTodar University of Wisconsin-Madison Department of
Bacteriology. All rights reserved.
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At this point you should be able to:
 Define environmental microbiology
 Understand the impact of microorganisms on the biosphere
 Describe the microbial cell, its structure and function.
 Explain differences in cell walls, cytoplasmic membrane of Bacteria, Archaea &
Eucarya
 Explain the different types of transport across cytoplasmic membrane
 Elaborate the microbial growth, development and characteristics of spores.
 Differentiate the different types of microbial metabolism.
Identify the role of microbes in the following sectors:
 Food
 Water
 Air
 Soil
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PART A: DEFINITION
Please define the following terms:
 Prokaryote
 Eukaryote
 Gram positive/Gram negative
 Binary fission
 Endospore
 Lag phase
 Exponential phase
 Stationary phase
 Death phase
 Catabolism
 Anabolism
 Respiration
 Aerobic respiration
 Anaerobic respirartion
 TCA pathway
 Fermentation
 Emden-Meyerhof pathway
 Lithotrophic Metabolism
 Phototrophic Metabolism
 Metanogens
 Nitrification
 Denitrification
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PART B: SHORT ANSWER
Answer the following questions:
1.
Differentiate structurally between eukaryote and prokaryote.
2.
Differentiate between gram negative and gram-positive bacteria
3.
Explain microbial cell growth.
4.
List the phases of bacterial growth
5.
Name the different microbes involved:
i. Nitrification
ii. Denitrification
iii. Methanogenesis
6.
State the importance of nitrifiers , denitrifiers and methanogens.
7.
Differentiate TCA and Emden-Meyerhof pathways
8.
Identify the energy source in lithotrophic and phototrophic metabolisms.
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STUDY NOTES:
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