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Microbial Metabolism. Unit 2: 7 days. February 3 rd and 4 th : Microbial Metabolism . The sum of all chemical reactions in a living organism is called metabolism. Microbial Metabolism.

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microbial metabolism

Microbial Metabolism

Unit 2: 7 days

february 3 rd and 4 th microbial metabolism
February 3rd and 4th: Microbial Metabolism
  • The sum of all chemical reactions in a living organism is called metabolism
microbial metabolism1
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 metabolism2
Microbial Metabolism
  • Anabolism refers to chemical reactions in which simpler substances are combined to form more complex molecules
  • These reactions usually require energy
microbial metabolism3
Microbial Metabolism
  • The energy of catabolic reactions is used to drive anabolic reactions
  • The energy for chemical reactions is stored in ATP
enzymes
Enzymes
  • Proteins produced by living cells, that catalyze chemical reactions by lowering the activation energy
  • Generally globular proteins with characteristic shapes
naming enzymes
Naming Enzymes
  • Usually end in – ase
  • Six different classes, defined based on the type of reactions they catalyze
energy production
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 production1
Energy Production
  • Glucose is a reduced molecule
  • Energy is released during a cell’s oxidation of glucose
energy production2
Energy Production
  • Energy release can be trapped to form ATP from ADP and phosphate
  • Addition of a phosphate is called phosphorylation
energy production3
Energy Production
  • A series of enzymatically catalyzed chemical reactions called metabolic pathways store energy in and release energy from organic molecules
carbohydrate catabolism
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
alternatives to glycolysis
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
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 review1
Aerobic Respiration Review
  • The Electron Transport Chain:
aerobic respiration review2
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
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
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
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 review1
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
Photosynthesis Review
  • Conversion of light energy from the Sun into chemical energy
  • This chemical energy is then used for carbon fixation
metabolic diversity
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 anoxygenicphototrophs
metabolic diversity1
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 diversity2
Metabolic Diversity
  • Chemoheterotrophs use complex organic molecules as their carbon and energy sources
february 6 th microbial growth
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
Physical Requirements
  • Temperature:
    • Psychrophiles (cold-loving)
    • Mesophiles (moderate-loving)
    • Thermophiles (heat-loving)
physical requirements1
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 requirements2
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
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 requirements1
Chemical Requirements
  • Oxygen:
    • Obligate aerobes
    • Facultative anaerobes
    • Obligate anaerobes
    • Aerotolerant anaerobes
    • Microaerophiles
  • Other chemicals:
    • S, P, trace elements
culture media
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 media1
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
culture media2
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 media3
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 media4
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
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 growth1
Phases of Growth
  • During the stationary phase equilibrium between cell division and death exists
  • During the death phase cell death outpaces cell replication
measuring 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 growth1
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
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 10 th control of growth
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. botulinumwith heat
control of growth
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 growth1
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
control of growth2
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
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
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 control1
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 control2
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 control3
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 control4
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 control5
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 control6
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 control7
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
Conditions Influencing Control
  • The effectiveness of chemical disinfectants depends on the microorganism and the physical environment
conditions influencing control1
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 control2
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
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 methods1
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 11 th microbial genetics
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
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
dna and chromosomes
DNA and Chromosomes

Eukaryotes Prokaryotes

in prokaryotes
In Prokaryotes…
  • Translation can begin before transcription is complete
  • The two processes occur in the same location
regulation of bacterial gene expression
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 expression1
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 expression2
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
regulation of bacterial gene expression3
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
mutations1
Mutations
  • Mutagens
    • Chemicals
    • Radiation
  • Frequency of mutation
  • Identifying mutants
  • Identifying carcinogens
genetic transfer and recombination
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
Conjugation
  • Donor and recipient cells
transformation
Transformation
  • The process of transferring genes as ‘naked’ DNA in solution
transduction
Transduction
  • DNA is transferred with the help of a bacteriophage
plasmids and transposons
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 transposons1
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
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 12 th and 13 th recombinant dna and biotechnology
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
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 recombination1
Overview of Recombination
  • Large quantities of the gene or the gene product can then be harvested from the clone
biotechnology
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
biotechnology1
Biotechnology
  • Prepackaged kits are available for many genetic engineering techniques
restriction enzymes
Restriction Enzymes
  • Recognizes and cuts only one specific sequence of DNA
  • May produce sticky ends
  • Fragments can then spontaneously rejoin
vectors
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
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
sources of dna
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
selecting a clone
Selecting a Clone
  • Many genes are given markers so that they can be easily identified later
making a gene product
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 product1
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
Applications
  • Cloned DNA is used to:
    • Produce products
    • Study the cloned DNA
    • Alter the phenotype of an organism
applications1
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
applications2
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
applications3
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
applications4
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
Ethical Issues
  • Avoidance of release
  • Some are modified and cannot survive outside of a laboratory
  • Organisms used in the environment may contain ‘suicide genes’