Microbial Metabolism

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


• Catabolism refers to chemical reactions that result in the breakdown of more complex organic molecules into smaller substances

• Catabolic reactions usually release energy


• Anabolism refers to chemical reactions in which simpler substances are combined to form more complex molecules

• These reactions usually require energy


• 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:


• The Electron Transport Chain:


• 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


• 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


• 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


• 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)


• 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


• 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


• 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


• Desiccation:– Absence of water– Microbe can not grow– May remain viable– Viruses and endospores resist desiccation


• Osmotic Pressure:– In high salt and sugar concentrations microbes

undergo plasmolysis– Molds and yeasts are more resistant


• 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


• 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


• 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


• 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


• 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


• 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’

Microbial Metabolism

Documents

cells energy

release energy

oxidized energy

atp molecules

activation energy

catalyzed chemical reactions

anabolic reactions

type of reactions