L25&26 fundamental concept (biochemistry)

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L25&26 fundamental concept (biochemistry)

  1. 1. FUNDAMENTAL CONCEPTS IN MICROBIOLOGY AEROBIC METABOLISM Metabolism : All the biochemical reactions that take place in cell Metabolic task Function Bringing nutrient into the cell To transport nutrient across the cytoplasmic membrane and concentrate them in the cytoplasm Catabolism To process the major nutrient and produce the 12 precursor metabolites, ATP and reducing power Biosynthesis To synthesis all necessary small molecules, including building blocks for macromolecules from precursor metabolites Polymerisation To link together building block, forming macromolecules, Eg. RNA, DNA, protein, polysaccharide and peptidoglycan Assembly To assemble macromolecu;les into organelles Catabolism Assembly Bringing in nutrients Cell membrane Biosynthesis Polymerisation New cell
  2. 2. Bringing nutrients into the cells • All nutrients pass through tiny water filled pores in the outer membrane formed by proteins called porin • Nutrient of concentration higher than inside the cells will be passed through (taking across the cell envelope) • Transporter protein (permease, facilitator or carrier)- bind to the nutrient in the periplasm • Mechanism of transportation i. Transporter mediated facilitated diffusion II. Active transport – action of transporter via pump requiring ATP (proton gradient). III. Energy requiring process that concentrates nutrient in the cell - group translocation
  3. 3. Porin Cytoplasm Transporter Low concentration of nutrient High concentration of nutrient
  4. 4. Catabolism Chemical changes/set of reaction that carbon or energy source undergo • Catabolite reactions produce 12 precursor metabolites for synthesis Precursor metabolites Glucose-6-phosphate Fructose-6-phosphate Triose phosphate 3-phosphoglycerate Phosphoenolpyruvate Pyruvate Acetyl Co-A Α-ketoglutarate Succinyl Co A Oxaloacetate Ribose 5-phosphate Erythrose 4-phosphate Catabolic pathway that leads to its synthesis Glycolysis Glycolysis Glycolysis Glycolysis Glycolysis Glycolysis TCA cycle TCA cycle TCA cycle TCA cycle Pentose phosphate Pentose phosphate Reducing power : redox reaction ATP : stored energy; compound that stores chemical energy (Adenosine triphosphate) Energy used during other steps of metabolism ATP ADP Energy conserved during catabolism
  5. 5. Figure 1: Glycolysis. Glycolysis is a pathway of central metabolism that converts a molecule of glucose into two molecules of pyruvate with a net yield of 2 molecules of ATP and 2 molecules of NADH, along with 6 precursor metabolites (shown in colored boxes)
  6. 6. Figure 2: The TCA cycle. The tricarboxylic acid TCA cycle converts pyruvate into CO2, reducing power, ATP (by substrate-level phosphorylation), and 4 precursor metabolites, shown in colored boxes. (FADH2 is a carrier of reducing power capable of converting NAD+ into NADH.
  7. 7. Pentose phosphate Figure 3: The pentose phosphate pathway. The pentose phosphate pathway is a part of central metabolism that forms 2 precursor metabolites, shown in boxes. The pathway begins with 1 intermediate of glycolysis and ends with another.
  8. 8. Biosynthesis – metabolic factory uses 3 products of catabolism • Precursor metabolites • ATP • Reducing power Building blocks for macromolecules Eg. Biosinthesis pathway : asparagine TCA glycolysis pentose-phosphate pathway a oxaloacetate pyruvate lysine Ribose-5 phosphate b methionine aspartate threonine pyruvate isoleucine histidine Driving force that fuels biosynthesis – reducing power stored mostly in the form of NADPH
  9. 9. a) A branched biosynthesis pathway converts 2 precursor metabolites (pyruvate and oxaloacetate from glycolysis and the TCA cycle, respectively) into 6 amino acids (red) by 22 enzyme-catalyzed reactions (arrows). The pathway uses 3 molecules of ATP (yellow arrows) and 4 molecules of NADPH (green arrows). b) An unbranched biosynthesis pathway converts a single precursor metabolite (ribose-5-phosphate from the pentose phosphate pathway) to a single amino acid (histidine) by 11 reactions. This pathway uses 1 ATP and 1 NADPH.
  10. 10. Polymerisation Molecular building blocks made by biosynthesis are joined together to form macromolecules Eg. Synthesis of DNA, RNA, proteins, polysaccharide and peptidoglycan Macromolecules Building blocks Protein 20 amino acids Nucleic acid Nucleotides RNA Adenine, guanine, cytosine, uracil, phosphate, ribose DNA Adenine, guanine, cytosine, thymine, phosphate, deoxyribose Polysaccharide Sugars Peptidoglycan N-acetyl muramic acid, N-acetyl glucosamine, 5 amino acids Lipid Fatty acid and other building blocks,
  11. 11. Polymerisation – catalysed by enzymes (protein) and protein structure is determined directly by DNA Therefore, Polymerisation reaction indirectly determined by DNA. • Polymerisation occurs by expanding chemical energy in the form of ATP
  12. 12. Assembly  Macromolecules assembled into cellular structures  Assembly may occur spontaneously (self-assembled), or may be the result of reactions catalysed by enzymes Eg.: Self – assembled : Formation of flagella (from flagellin) Reaction catalysed by enzymes : Formation of bacterial cell wall. short unit of peptidoglycans are released in periplasm assembled into intact cell wall
  13. 13. ANAEROBIC METABOLISM  The critical difference between aerobic and anaerobic metabolism lies in how ATP is generated.  In aerobic metabolism, E.coli makes most of its ATP by aerobic respiration, producing a proton gradient by an electron transport chain with oxygen as its terminal electron acceptor.  In the absence of oxygen the electron transport chain cannot function in this way.  Thus, aerobic respiration is impossible.  There are 2 ways that cells can make ATP from organic nutrients in the absence of oxygen.  One, called anaerobic respiration – uses an electron transport chain with a compound other than oxygen as the terminal electron acceptor.  The second, called fermentation, depends entirely on substratelevel phosphorylation.
  14. 14. Anaerobic Respiration  In aerobic respiration, oxygen accepts electrons and is reduced to water.  In anaerobic respiration, another compound is reduced by accepting these electrons.  Compound that can act as a terminal electron acceptor in anaerobic respiration include sulfate, nitrate, fumarate and trimethylamine oxide.  E. Coli, for example ,can use nitrate, fumarate or trimethylamine oxide as an electron acceptor if oxygen is not available.
  15. 15. Table 1: Some Terminal Electron Acceptor of Bacterial Electron Transport Chains Type of Respiration Terminal Electron Acceptor Reduced Product Aerobic Respiration Oxygen (O2) Water (H2O) - Sulfate reduction Sulfate (SO42-) Hydrogen sulfide (H2S) - Nitrate reduction Nitrate (NO3-) Nitrite (NO2-) - Fumarate reduction Fumarate (HOOC-CH=CH- Succinate (HOOO-CH2COOH) CH2-COOH) -Denitrification Nitrate (NO3-) Nitrogen gas (N2) Trimethylamine oxide reduction Trimethylamine oxide Trimethylamine Anaerobic Respiration
  16. 16. Fermentation  Fermentation is a form of anaerobic metabolism in which all ATP is generated by substrate-level phosphorylation.  Fermentation generates fewer molecules of ATP per molecule of substrate than do aerobic and anaerobic respiration.  For example, E.coli derives about 28 ATP molecules from glucose by aerobic respiration but only about 3 by fermentation.
  17. 17. - 1 molecule of glucose is metabolized to produce 2 molecules of pyruvate, 2 molecules of ATP & 2 of NADH. - In order to reoxidize the 2 molecules of NADH (and thus allow fermentation to continue), pyruvate is reduced to lactic acid.
  18. 18. Nutritional classes of microorganism Source of energy (ATP) Source of C atoms Chemical Rxn Light Energy Organic compounds Chemoheterotrophs Photoheterotrophs CO2 Chemoautotrophs Photoautotrophs Heterotroph organic compounds as a source of carbon Autotroph uses CO2 as a source of carbon
  19. 19. Genetic of microorganism • DNA structure • Replication of DNA • Regulation of gene expression Genotype / Phenotype • Genotype • Phenotype cell genetic plan cell appearance and function Mutation – Any chemical change in cell’s DNA • Base substitution mutation – changes a single pair of bases to different pair • Deletion mutation – removes a segment of DNA • Inversion mutation – reverses the order of a segment of DNA • Transposition mutation – moves a segment of DNA to a different position on the genome • Duplication mutation – adds an identical new segment of DNA next to the original one
  20. 20. Incidence of Mutation • Spontaneous mutation • Induced mutation Natural course of microbial growth resulted mutation Intentional chemical, physical or biological treatments Induced mutation - treated with mutagens Chemical mutagens : Eg. Nitrosoguanidine Physical mutagen UV light, X rays, gamma radiation UV stimulates adjacent pyrimidine bases, usually T, to react with one another thymine dimer Biological mutagen many carry fragments of DNA within their genome that are mutagenic, which moves from one part of the genome to another (transposable elements)
  21. 21. Selecting Mutants Direct selection – create conditions that favour growth of the desired mutant strain Indirect selection – counter selection, create conditions to prevent the growth of desired mutant. The growing cells are killed. The mutant will survive the lethal treatment which are isolated. Site–directed mutagenesis – product of recombinant DNA technology (mutate one particular gene)
  22. 22. Genetic Exchange Among Bacteria Genetic exchange – transfer of genes from one cell to another In bacteria – a portion of the DNA of one cell (the donor cell) is transferred to the other (recipient cell) merozygote 3 forms of genetic exchange in bacteria: • Transformation : During transformation, DNA leaves one cell and exists for a time in the extracellular environment. Then it is taken into another cell, become incorporated into the genome. DNA fragment can become part of the resident chromosome. • Conjugation : Carried out conjugative plasmids (plasmids able to transfer themselves to another cell) Eg.: F-plasmid (13 genes). One of the genes encodes a special pilus called sex pilus or the F-pilus F pilus allows F+ cells attach to F- cells
  23. 23. • Transduction : Transfer of chromosomal genes via virus that infect bacteria called bacteriophages, reproduce themselves. 2 kinds of transduction : Virulent phages (kills the host) Temperate phages (carried passively in the host without harming) Virulent – infect bacteria by attaching themselves to the surface of victim cells and rejecting their DNA Temperate – lysogenic cycle, phage DNA (prophage) exists as plasmid, incorporated in the host cell chromosome • Transduction mediated by virulent phage – generalised transduction because it transfers any portion of the bacteria chromosome from one cell to another • Prophage mediated specialised transduction – prophages are inserted only at a specific site on the bacterial chromosome
  24. 24. Conjugation Transduction
  25. 25. Recombinant DNA Technology • Techniques involve taking DNA from a cell, manipulate in vitro and putting it into another cell. • Recombination : processes of forming a new combination of genes by any means Gene cloning – fundamental tool of recombinant DNA technology • A process of obtaining a large number of copies of a gene from a single copy of the gene. Gene cloning involves 5 steps : 1. Obtaining a piece of DNA that carries the gene to be cloned 2. Splicing that DNA into a cloning vector (a DNA molecule that a host cell will replicate) 3. Putting the recombinant DNA (in this case the cloning vector with the desired gene spliced into it in an appropriate host cell) 4. Testing to ensure that the gene has actually been put into the host cell 5. Propagating the host cell to produce a clone of cells that carries the clone of genes
  26. 26. 1. Obtaining a piece of DNA that carries the gene to be cloned cell DNA DNA containing the gene to be cloned Is purified from intact cells
  27. 27. 2. Splicing that DNA into a cloning vector (a DNA molecule that a host will replicate) DNA molecules DNA fragments • Purified DNA is cut into pieces and spliced into cuts made in cloning vector DNA molecules • Cutting DNA using restriction endonuclease cloning vector molecules Eg. plasmid Recombinant DNA
  28. 28. 3. Putting the recombinant DNA (in this case the cloning vector with the desired gene spliced into it in an appropriate host cell) Recombinant DNA Recombinant DNA molecules are put into host cells by transformation
  29. 29. 4. Testing to ensure that the gene has actually been put into the host cell - host cells containing the gene to be cloned are identified by testing them for the presence of the gene product 5. Propagating the host cell to produce a clone of cells that carries the clone of genes - the colonies carrying the desired gene is propagated producing a clone of cells with the clone genes
  30. 30. Some application of recombinant DNA Field Application Importance Basic Biology DNA sequencing Directed mutagenesis Gene structure, function and relatedness between gene and microorganism Medicine Therapeutic proteins Gene therapy Improved vaccines Diagnosis Veterinary medicine Protein for treatment of diseases, genetic disorder Effective vaccines Rapid and accurate diagnosis Industry Altering microorganisms Improve production Agriculture Altering plants/farm animals Rapid breeding and disease resistance criminal DNA fingerprinting Identify individual DNA
  31. 31. Genomics – study of an organism as revealed by the sequence of bases in its DNA DNA sequencing- sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA. DNA Assembling- to aligning and merging fragments of a much longer DNA sequence in order to reconstruct the original sequence Annotation- The process of assigning function to DNA sequences Microarray Technology- a means of determining which of an organism’s genes are expressed under various conditions
  32. 32. THE END

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