Microbial Metabolism
Unit 2: 7 days
February 3rd and 4th:
Microbial Metabolism
• The sum of all chemical reactions in a living
organism is called metabolism
Microbial Metabolism
• Catabolism refers to chemical reactions that
result in the breakdown of more complex
organic molecules into smaller substances
• Catabolic reactions usually release energy
Microbial Metabolism
• Anabolism refers to chemical reactions in
which simpler substances are combined to
form more complex molecules
• These reactions usually require energy
Microbial Metabolism
• The energy of catabolic reactions is used to
drive anabolic reactions
• The energy for chemical reactions is stored in
ATP
Enzymes
• Proteins produced by living cells, that catalyze
chemical reactions by lowering the activation
energy
• Generally globular proteins with characteristic
shapes
Naming Enzymes
• Usually end in – ase
• Six different classes, defined based on the
type of reactions they catalyze
Energy Production
• Oxidation-reduction reaction
– LEO
– GER
• When one substance is oxidized, another is
reduced
• NAD+ is the oxidized form, NADH is the
reduced form
Energy Production
• Glucose is a reduced molecule
• Energy is released during a cell’s oxidation of
glucose
Energy Production
• Energy release can be trapped to form ATP
from ADP and phosphate
• Addition of a phosphate is called
phosphorylation
Energy Production
• A series of enzymatically catalyzed chemical
reactions called metabolic pathways store
energy in and release energy from organic
molecules
Carbohydrate Catabolism
• Most of a cell’s energy is produced from the
oxidation of carbohydrates
• Glucose is the most commonly used carb
• There are two major pathways of glucose
catabolism:
– Respiration
• Completely broken down
– Fermentation
• Partially broken down
Review
Alternatives to Glycolysis
• The pentose phosphate pathway is used to
metabolize 5 carbon sugars
– Operates simultaneously with glycolysis
• The Entner-Doudoroff pathway
– Requires special enzymes
– Found in some gram-negative bacteria
• Both yield one ATP and two NADPH molecules are
produced from one glucose
Cellular Respiration Review
• Organic molecules are oxidized
• Energy is generated from the ETC
• In aerobic respiration, O2 is the final electron
acceptor
• In anaerobic respiration, a different inorganic
molecule is the final electron acceptor
Aerobic Respiration Review
• The Krebs Cycle:
Aerobic Respiration Review
• The Electron Transport Chain:
Aerobic Respiration Review
• The mechanism of ATP synthesis using the ETC
is called chemiosmosis
– Protons being pumped across the membrane
produce force caused by electrons moving along
the chain
– The protons then move back across the
membrane, and ADP is turned into ATP by the
protein ATP synthase
– In eukaryotes the electron carriers are located in
the inner mitochondrial membrane
– In prokaryotes they are in the plasma membrane
Aerobic Respiration Summary
• In aerobic prokaryotes 38 ATP molecules can
be produced from complete oxidation of a
glucose molecule
• In eukaryotes 36 ATP molecules can be
produced from complete oxidation of a
glucose molecule
Anaerobic Respiration Review
• The final electron acceptors can be nitrate,
sulfate, or carbonate
• The total ATP yield is less than aerobic
respiration because only part of the Krebs
cycle is operating
Fermentation Review
• Releases energy from molecules through
oxidation
• Oxygen gas is not required
• Two ATP molecules are produced
• Electrons removed from the substrate reduce
NAD+
• The final electron acceptor is an organic
molecule
Fermentation Review
• In lactic acid fermentation, pyruvic acid is
reduced by NADH to lactic acid
• In alcohol fermentation, acetaldehyde is
reduced by NADH to produce ethanol
• Heterolactic fermenters can use the pentose
pathway to produce lactic acid and ethanol
Photosynthesis Review
• Conversion of light energy from the Sun into
chemical energy
• This chemical energy is then used for carbon
fixation
Photosynthesis Review
Metabolic Diversity
• Photoautotrophs obtain energy through
photophosphorylation and fix carbon from
CO2 using the Calvin cycle to synthesize
organic molecules
• Cyanobacteria are oxygenic phototrophs
• Green and purple sulfur bacteria are
anoxygenic phototrophs
Cyanobacteria
Purple Sulfur Bacteria
Metabolic Diversity
• Photoheterotrophs use light as an energy
source and an organic molecule for their
carbon source or electron donor
• Chemoautotrophs use inorganic compounds
as their energy source and CO2 as their carbon
source
Metabolic Diversity
• Chemoheterotrophs use complex organic
molecules as their carbon and energy sources
February 6th: Microbial Growth
• The growth of a population is an increase in
the number of cells or in mass
• Microbes have both physical and chemical
requirements for growth
Physical Requirements
• Temperature:
– Psychrophiles (cold-loving)
– Mesophiles (moderate-loving)
– Thermophiles (heat-loving)
Psychrophiles at Everest Base Camp
Physical Requirements
• Minimum growth temperature = the lowest
temperature at which a species will grow
• Optimum growth temperature = the
temperature at which a microbe grows the
best
• Maximum growth temperature = the highest
temperature at which growth is possible
Physical Requirements
• Most bacteria grow best at a pH value
between 6.5 and 7.5
• In a hypertonic solution most microbes
undergo plasmolysis
• Halophiles can tolerate high salt
concentrations
Chemical Requirements
• Carbon source
• Nitrogen source
– Needed for nucleic acid and protein synthesis
– Can be obtained:
• From the decomposition of proteins
• From nitrate or ammonium
• Some bacteria are capable of nitrogen fixation (N2)
Chemical Requirements
• Oxygen:
– Obligate aerobes
– Facultative anaerobes
– Obligate anaerobes
– Aerotolerant anaerobes
– Microaerophiles
• Other chemicals:
– S, P, trace elements
Culture Media
• Any material prepared for the growth of
bacteria in a laboratory
• Microbes that grow and multiply in or on a
culture medium are known as a culture
• Agar is a common solidifying agent for a
culture medium
Culture Media
• A chemically defined medium is one in which
the exact chemical composition is known
• A complex medium is one in which the exact
chemical composition is not known
• Selective media allows for growth of only the
desired organism by inhibiting others with
salts, dyes, or other chemicals
Selective Media
Culture Media
• Differential media are used to distinguish
between different organisms
• An enrichment culture is used to encourage
the growth of a particular microbe in a mixed
culture
Culture Media
• The normal reproductive method for bacteria
is binary fission
– One cell splits into two
• Some bacteria can reproduce by budding,
aerial spore formation, or fragmentation
Culture Media
• Generation time is the time required for a cell
to divide
• This is also the time required for a population
to double
Phases of Growth
• During the lag phase the metabolic activity of
cells is high, but there is no change in the
overall number of cells
• During the log phase the bacteria multiply at
the fastest rate allowable by environmental
conditions
Phases of Growth
• During the stationary phase equilibrium
between cell division and death exists
• During the death phase cell death outpaces
cell replication
Phases of Growth
Measuring Growth
• A standard plate count reflects the number of
viable microbes and assumes that each
bacteria grows into a single colony
• This can be done using a pour plate or by a
spread plate
Measuring Growth
• A direct count can be done using a microscope
and specialized slides
• In filtration, bacteria are retained on a
membrane and then transferred to a plate to
grow and be counted
• The most probably number is a statistical
estimation using bacteria growing in a liquid
medium
Indirect Measurements
• A spectrophotometer can be used to measure
turbidity
• Metabolic activity can also be measured by
measuring substance consumption or output
• Measuring dry weight can also be useful for
some organisms (especially fungi)
February 10th: Control of Growth
• Controlling microbial growth is important in
infection prevention and food spoilage
avoidance
• Sterilization is the process of destroying all
microbial life on an object
– Commercial sterilization destroys C. botulinum
with heat
Control of Growth
• Disinfection is the process of limiting or
inhibiting microbial growth on a surface
• Antisepsis is the process of reducing or
limiting microorganisms on a living tissue
• Sepsis is bacterial contamination
• -cide = to kill
• -stat = to inhibit
Control of Growth
• Bacterial population subjected to heat usually
die at a constant rate
• This death curve, when graphed, appears as a
straight logarithmic line
• The time it takes to kill an entire population is
proportional to the number of microbes
Bacterial Death Curve
Control of Growth
• Different species, and different lifecycle
phases, have different susceptibilities to
physical and chemical controls
– e.g. endospores
• Longer exposure to lower heat can produce
the same effect as shorter exposure to high
heat
Actions of Microbial Control Agents
• Alteration of membrane permeability:
– Due to lipid and protein components of the
plasma membrane
– Chemical control agents can damage the
membrane
• Damage to proteins and nucleic acids:
– Some control agents can damage proteins by
breaking hydrogen and covalent bonds
– Other interfere with DNA and RNA synthesis and
replication
Physical Methods of Microbial Control
• Heat:
– Frequently used
– Moist heat denatures enzymes
– Thermal death point – the lowest temperature at
which bacteria in a liquid culture will be killed in 10
minutes
– Thermal death time – the length of time required to
kill bacteria at a given temperature
– Decimal reduction time – length of time in which 90%
of bacteria will be killed at a given temperature
Physical Methods of Microbial Control
• Heat:
– Boiling kills many vegetative cells and viruses within
10 minutes
• Autoclaving (steam under pressure) is the most effective
method of moist heat
– In pasteurization a high temperature is used for a
short time to destroy pathogens without altering the
flavor of food (72°C for 15 seconds)
• Ultra-high-temperature treatment is used to sterilize dairy
products (140°C for 3 seconds)
Physical Methods of Microbial Control
• Heat:
– Methods of dry heat sterilization include direct
flaming, incineration, and hot-air sterilization
– Different methods that produce the same effect
are called equivalent treatments
Physical Methods of Microbial Control
• Filtration:
– The passage of liquid or gas through a filter with
pores small enough to retain microbes
– Microbes can be removed from air with high
efficiency particulate air filters
Physical Methods of Microbial Control
• Low Temperatures:
– The effectiveness of low temperatures depends on
the specific microorganism and the intensity of
the application
– Most microorganisms do not reproduce at
ordinary refrigeration temperatures
– Many microbes can survive, but not grow, at the
subzero temperatures used to store food
Physical Methods of Microbial Control
• Desiccation:
– Absence of water
– Microbe can not grow
– May remain viable
– Viruses and endospores resist desiccation
Physical Methods of Microbial Control
• Osmotic Pressure:
– In high salt and sugar concentrations microbes
undergo plasmolysis
– Molds and yeasts are more resistant
Physical Methods of Microbial Control
• Radiation:
– Effects depend on wavelength, intensity, and duration
– Ionizing radiation has a high degree of penetration
• Reacts with water forming highly reactive hyxdroxyl radicals
– Ultraviolet radiation has low penetration
• Causes cell damage by creating thymine dimers
– Most effective germicidal wavelength is 260nm
– Microwaves cause indirect death due to temperature
increase
Conditions Influencing Control
• The effectiveness of chemical disinfectants
depends on the microorganism and the
physical environment
Conditions Influencing Control
• Gram-positive tend to be more susceptible to
disinfectants than gram-negative
• Pseudomonads can grow in some disinfectants
and antiseptics
• M. tuberculosis is resistant to many
disinfectants
• Endospores and mycobacteria are very
resistant to everything
• Non-enveloped viruses are typically more
resistant than enveloped viruses
Conditions Influencing Control
• Organic matter (such as vomit and feces)
frequently affect the actions of chemical
control agents
• Disinfectant activity is enhanced by warm
temperatures
Chemical Methods
• Types of disinfectants:
– Phenol and phenolics
• Injure plasma membranes, denature proteins, inactivate
enzymes
– Halogens
• Can be used alone or in a molecule
• Form acids and disrupt amino acids
– Alcohols
• Denature proteins and dissolve lipids
Chemical Methods
• Types of disinfectants:
– Heavy metals
• Ag, Hg, Cu, and Zn
• Denature proteins
– Antibiotics
• Often used to preserve food
– Aldehydes
• Inactivate proteins
• Among the most effective chemical disinfectants
February 11th: Microbial Genetics
• Remember that genetics is the study of what
genes are, how they carry information, and
how that information is expressed
• It also looks at how that information is passed
on to subsequent generations
Microbial Genetics
• Hydrogen bonds hold the DNA strands
together
• A gene is a segment of DNA that codes for a
functional product, typically a protein
• Gene expression involves transcription and
translation
Genotype and Phenotype
DNA and Chromosomes
Eukaryotes
Prokaryotes
DNA Replication
Transcription
Translation
Codon Chart
In Prokaryotes…
• Translation can begin before transcription is
complete
• The two processes occur in the same location
Regulation of Bacterial Gene Expression
• Regulating protein synthesis at the gene level
is energy efficient because proteins are
synthesized only as they are needed
• Constitutive enzymes produce products at a
fixed rate
– E.g. genes for the enzymes in glycolysis
Regulation of Bacterial Gene Expression
• Repression controls the synthesis of one or
more enzymes
• When cells are exposed to a specific end
product, the production of that product is
decreased
Regulation of Bacterial Gene Expression
• In the presence of inducers, cells synthesize
more product
• An example of induction is when lactose
causes E. coli to produce the compound that
metabolizes lactose
Lactose Breakdown
Regulation of Bacterial Gene Expression
• The formation of enzymes is determined by
structural genes
• A coordinated group of genes, including the
promoter sequence and the operator sites
that control their transcription, is called an
operon
Mutations
• Types
Mutations
• Mutagens
– Chemicals
– Radiation
• Frequency of mutation
• Identifying mutants
• Identifying carcinogens
Genetic Transfer and Recombination
• Genetic recombination usually involves genes
from different organisms
• Contributes to genetic diversity
• Crossing over helps with this too
• Recombinant cells have donor DNA
incorporated into them
Conjugation
• Donor and recipient cells
Transformation
• The process of transferring genes as ‘naked’
DNA in solution
Transduction
• DNA is transferred with the help of a
bacteriophage
Plasmids and Transposons
• Plasmids – self replicating circular DNA
molecules
• Genes on plasmids are not usually essential
for the cell’s survival
• Many plasmid genes code for toxins and
resistance factors
Plasmids and Transposons
• Transposons – small fragments of DNA that
can move from one area of a chromosome to
another, or to a completely different
chromosome
• Can be simple or complex
Diversity
• Genetic diversity is the prerequisite for
evolution
• Genetic mutation and recombination provide
a diversity of organisms, and natural selection
allows the growth of those best adapted for a
given environment
February 12th and 13th: Recombinant
DNA and Biotechnology
• Closely related organisms can exchange genes
in natural recombination
• Genes can be transferred among unrelated
species through genetic engineering
• Recombinant DNA combines DNA from two
different sources
Overview of Recombination
• A desired gene is inserted into a vector
– Plasmid
– Viral genome
• The vector inserts the DNA into a new cell
• This cell is grown to form a clone
Overview of Recombination
• Large quantities of the gene or the gene
product can then be harvested from the clone
Biotechnology
• Includes all industrial applications of
microorganisms
• Also, industrial uses of genetically engineered
cells
• A DNA molecule used to carry a desired gene
from one organism to another is called a
vector
Biotechnology
• Prepackaged kits are available for many
genetic engineering techniques
Restriction Enzymes
• Recognizes and cuts only one specific
sequence of DNA
• May produce sticky ends
• Fragments can then spontaneously rejoin
Vectors
• Shuttle vectors are plasmids that can exist in
several different species
• A plasmid can be inserted into a cell by
transformation
• A virus containing a new gene can insert the
new gene into the cell
Methods of Inserting DNA
• Chemical treatment can cause cells to take up
naked DNA through transformation
• Electric current can cause electroporation, the
formation of pores which can allow DNA to
enter
• Protoplast fusion involves the joining of cells
whose cell walls have been removed
Protoplast Fusion
Sources of DNA
• Gene libraries can be made by cutting up an
entire genome and inserting the pieces into
plasmids
• Synthetic DNA can be made in vitro with
synthesis machines
Gene Libraries
Selecting a Clone
• Many genes are given markers so that they
can be easily identified later
Making a Gene Product
• E. coli is frequently used to produce proteins
by genetic engineering because it is easily
grown and its genetics are well understood
• However, E. coli does produce an endotoxin,
that must be kept out of end products to be
used in humans
Making a Gene Product
• Yeasts can also be used, and are more likely to
continuously secrete the gene product
• Mammalian cells have been genetically
engineered to produce hormones for medical
use
• Plant cells can be engineered and used to
produce plants with new properties
Applications
• Cloned DNA is used to:
– Produce products
– Study the cloned DNA
– Alter the phenotype of an organism
Applications
• Synthetic genes in E. coli are used to produce
human insulin
• Cells can be engineered to produce a
pathogen’s surface proteins, which can be
used to create a vaccine
Applications
• Recombinant DNA techniques can be used to
increase understanding of DNA for genetic
fingerprinting and gene therapy
• DNA sequencing machines can be used to
determine the exact nucleotide sequence of a
gene
Applications
• Southern blotting can be used to locate a
specific gene in a cell
• Gene therapy can be used to cure diseases by
replacing the defecting gene
• DNA probes can be used to quickly identify a
pathogen in food or body tissues
Applications
• Cells from plants with desirable characteristics
can be identified, isolated, and cloned
• Rhizobium has been genetically modified to
enhance nitrogen fixation
• Pseudomonas has been engineered to
produce toxins against insects
Ethical Issues
• Avoidance of release
• Some are modified and cannot survive outside
of a laboratory
• Organisms used in the environment may
contain ‘suicide genes’