Mks chemotaxonomy

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Taxonomy of the bacteria by using chemicals present in the cell.

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Mks chemotaxonomy

  1. 1. Molecular Diagnostics Laboratory Department of Microbiology and Biotechnology Dr.M.K. SATEESH
  2. 2. Definition <ul><li>The classification of bacteria on the basis of their chemical composition. </li></ul><ul><li>Whole cell analysis </li></ul><ul><li>Cell parts </li></ul><ul><li>Physical & techniques development </li></ul><ul><li>Gas chromatography </li></ul>Whole cell analysis Cell parts Techniques development Physical development Gas chrom- atography Dr.M.K. SATEESH
  3. 3. Key Terms <ul><li>Isolation (culture) </li></ul><ul><ul><li>Agar plate/colonies </li></ul></ul><ul><ul><li>Liquid media test tube - bulk </li></ul></ul><ul><li>Identification & taxonomy </li></ul><ul><ul><li>Family </li></ul></ul><ul><ul><li>Genus </li></ul></ul><ul><ul><li>Species </li></ul></ul><ul><ul><li>Type </li></ul></ul><ul><ul><li>Strain </li></ul></ul><ul><li>Biochemical (physiological) tests </li></ul><ul><li>Molecular tests </li></ul><ul><ul><li>DNA-DNA homology </li></ul></ul><ul><ul><li>16S rRNA sequencing </li></ul></ul><ul><li>Chemical profiling </li></ul><ul><li>Non culture based detection </li></ul><ul><ul><li>Polymerase chain reaction- (PCR) </li></ul></ul><ul><ul><li>Agglutination (antigen detection) </li></ul></ul><ul><ul><li>Stain </li></ul></ul><ul><ul><li>Serology (antibody detection) </li></ul></ul>Dr.M.K. SATEESH
  4. 4. Classification <ul><li>Family: a group of related genera. </li></ul><ul><li>Genus: a group of related species. </li></ul><ul><li>Species: a group of related strains. </li></ul><ul><li>Type: sets of strain within a species (e.g. biotypes, serotypes). </li></ul><ul><li>Strain: one line or a single isolate of a particular species. </li></ul>Dr.M.K. SATEESH
  5. 5. Dr.M.K. SATEESH
  6. 6. Dr.M.K. SATEESH
  7. 7. Chemical analysis Sophisticated tools are available for studying the structural composition of bacteria (most commonly fatty acid , carbohydrate or ubiquinone profiling) of bacteria. Characterization of secreted metabolic products (e.g. volatile alcohols and short chain fatty acids ) is also helpful. Dr.M.K. SATEESH
  8. 8. Major Chemical compounds <ul><li>Cell wall composition </li></ul><ul><li>Lipid composition </li></ul><ul><li>Isoprenoid Quiniones </li></ul><ul><li>Cytochrome composition </li></ul><ul><li>Amino acids of proteins </li></ul><ul><li>Protein profiles </li></ul><ul><li>Enzyme characterization </li></ul><ul><li>Fermentation products profiles </li></ul>Dr.M.K. SATEESH
  9. 9. A model for bacterial speciation Dr.M.K. SATEESH
  10. 10. <ul><li>A single colony is placed on a slide and dried. </li></ul><ul><li>Stage1. Staining with crystal violet. </li></ul><ul><li>Stage 2. Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue. </li></ul><ul><li>Stage 3. Extraction with alcohol. Decolorizes some bacteria </li></ul><ul><li> ( Gram negative ) and not others ( Gram positive ). </li></ul><ul><li>Stage 4. Counterstaining with safranin. Gram positive bacteria are stained with crystal violet and remain purple. Gram negative bacteria are stained pink. </li></ul>Dr.M.K. SATEESH
  11. 11. Dr.M.K. SATEESH
  12. 12. Lipid composition <ul><li>Cellular FA are converted to methyl esters by treating with anhydrous methanolic Hcl, FAMEs are analyzed by using gas chromatography. </li></ul>R− CO − O−Glycerol R−CO−O−CH 3 Methanolic Hcl Mycolic acids, polar lipids, phospholipids, glycolipids, hopanoids, hydrocarbons, carotenoids Dr.M.K. SATEESH
  13. 13. Examples <ul><li>Mycobacterium </li></ul><ul><li>Corynebacterium </li></ul><ul><li>Nocardia </li></ul><ul><li>Rhodococcus </li></ul><ul><li>Micropolyspora </li></ul><ul><li>Bacterionema </li></ul><ul><li>Bacteroides </li></ul>phospholipids Steroids Dr.M.K. SATEESH
  14. 14. Dr.M.K. SATEESH
  15. 15. Fatty acid methyl ester (FAME) analysis Dr.M.K. SATEESH
  16. 16. Isoprenoid Quiniones <ul><li>Are a class of terpenoid lipids located in cytoplamsic membranes of many bacteria. They play important roles in ETC, oxidative phosphorylation and active transport. </li></ul><ul><li>Types 3 main; Ubiquinones, menaquinones, demethylmenaquinones. </li></ul><ul><li>Mycoplasma - menaquinones </li></ul><ul><li>Cyanobacteria- none ( phylloquinones, plastoquinones ) </li></ul><ul><li>Archea – none (Caldariellaquinones) </li></ul><ul><li>Streptococci- menaquinones </li></ul><ul><li>Strict aerobic G-ve- Ubiquinones; anaerobic G-ve- menaquinones </li></ul>Dr.M.K. SATEESH
  17. 17. Cytochrome composition <ul><li>G+ve; Cytochromes bcaa3o </li></ul><ul><li>Fac. Anaerobic G+ve; c is absent </li></ul><ul><li>Propionibacteria ; Cytochromes bda1 </li></ul><ul><li>Clostridium No Cytochromes </li></ul><ul><li>G-ve; Cytochromes bdoa1 </li></ul><ul><li>Phototroph; Cytochromes bc </li></ul>Cytochromes are membrane-bound hemoproteins that contain heme groups and carry out electron transport/redox processes-4 major types : a, b, c, d, o. Dr.M.K. SATEESH
  18. 18. Amino acids of proteins <ul><li>Comparison of specific kinds of proteins for their aa sequence: Cytochrome oxidase, superoxide dismutase, ferredoxin or other . </li></ul><ul><li>If sequence is similar between two they are similar E.g. Cytochrome c in non-S purple photosynthetic bacteria. </li></ul>Cytochrome oxidase Dr.M.K. SATEESH
  19. 19. Cytochrome oxidase Comparison of cytochrome oxidase from bacteria and bovine Dr.M.K. SATEESH
  20. 20. Sequence comparison of cytochrome oxidase from three species Dr.M.K. SATEESH
  21. 21. Protein profiles/fingerprints Total Cellular membrane proteins PAGE/ Isoelectric focusing ~ 10-30 proteins ( water soluble) Clostridium, Rhizobium, Haemophilus Dr.M.K. SATEESH
  22. 22. Enzyme characterization Citrate synthase is found in all living cells, so it has been a useful enzyme for comparing differences from organism to organism. Citrate synthase Dr.M.K. SATEESH
  23. 23. Citrate synthase.(monomer shown ) citrate is shown in green, and CoA is pink. Dr.M.K. SATEESH
  24. 24. Succinyl-CoA synthetase, ( succinate thiokinase) succinyl-CoA synthetase, sometimes called succinate thiokinase Dr.M.K. SATEESH
  25. 25. Fermentation products profile <ul><li>End products of carbohydrate and protein metabolism is analysed by using gas liquid chromatographic methods especially for </li></ul><ul><li>Clostridium </li></ul><ul><li>Bacteroides </li></ul><ul><li>Eubacterium </li></ul>Dr.M.K. SATEESH
  26. 26. Oxidation - Reduction Reactions are Always Coupled Oxidation = A B H + H { { { Reduction = gain of e - & H + loss of e - & H + NAD + NADH + H = electron Dr.M.K. SATEESH B A
  27. 27. MKS Use of Intact Cell MALDI Fingerprints to Identify Bacillus and Pseudomonas Isolates
  28. 28. Introduction <ul><li>Identification of bacteria by traditional molecular and biochemical methods is relatively slow and time consuming (and, in the case of molecular techniques, expensive). </li></ul><ul><li>An alternative to these is chemotaxonomy – the identification of bacteria by protein markers. Mass spectrometry is a powerful tool for such identification. </li></ul><ul><li>Classic mass spectrometry has an upper limit of 1000 Daltons. Compounds larger than this fracture under the laser power used to ionise the sample. </li></ul><ul><li>Matrix Assisted Laser Desorption/Ionisation Time of Flight (MALDI ToF) mass spectromterry overcomes this problem by using a matrix to buffer the laser power, thus reducing unwanted fragmentation . </li></ul>Dr.M.K. SATEESH
  29. 29. Principals of MALDI ToF Mass Spectrometry <ul><li>M atrix A ssisted L aser D esorption/ I onisation T ime o f F light mass spectrometry ( MALDI ToF MS ) was developed in 1987 by Hillenkamp, Karas and Tanaka . </li></ul><ul><li>This technique uses a biomolecule embedded in a co-precipitate of an UV-light absorbing matrix which is irradiated by a nanosecond laser pulse. </li></ul>Dr.M.K. SATEESH
  30. 30. Principles of MALDI ToF Mass Spectrometry <ul><li>Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. </li></ul><ul><li>The ionized biomolecules are accelerated in an electric field and enter the flight tube. </li></ul><ul><li>During the flight in this tube different molecules are separated according to their mass to charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. </li></ul>Dr.M.K. SATEESH
  31. 31. Principles of MALDI ToF Mass Spectrometry: Bacterial Analysis <ul><li>MALDI analysis of bacterial isolates uses unique components, or biomarkers, to differentiate at the species level. </li></ul><ul><li>Early work focused on phospholipids and other small molecules as principal biomarkers. </li></ul><ul><li>Using proteins as biomarkers, MALDI analysis allows the use of crude cellular fractions or cellular suspensions and the resulting data from such complex mixtures provides evidence for identification. </li></ul>Dr.M.K. SATEESH
  32. 32. Principles of MALDI ToF Mass Spectrometry: Bacterial Analysis <ul><li>In terms of speed, using whole bacterial eliminates all but the simplest sample preparation. </li></ul><ul><li>The spectral patterns obtained from this whole cell analysis gives characteristic fingerprints, which are thought to be from the desorbed components of the cell envelope. </li></ul><ul><li>The resulting fingerprints are then entered into a data base which allows unknown isolates to be cross referenced. </li></ul>Dr.M.K. SATEESH

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