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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’