History of Microbiology

1. History of Microbiology

3. Domains of Life

4. Endosymbiotic Theory • Mitochondria and chloroplast have their own circular genome • Also have 70S ribosomes • This is evidence that organelles were originally bacteria taken up by a predatory eukaryote ancestor

5. Endosymbiotic Theory Eukaryotic predatory ancestor Begins to produced energy for host, evolving into current mitochondrion Digestion No Digestion Uptake into cell Bacteria

6. Plasma Membrane Polar Headgroup Hydrophobic Tail

7. Membrane O O C O C C O O

8. Membrane Proteins

9. Cell Wall

10. Bacterial Cell Wall Techoic Acid Peptidoglycan O- polysaccharide Core- polysaccharide LPS Endotoxin Porin Lipoprotein GRAM - GRAM +

11. Peptidoglycan and Lysozyme • Peptidoglycan is composed of β(14) linked N-acetylglycosamine (G) and N-Acetylmuramic acid (M), amino acids (lysine, diaminopimelic acid = DAP) • Crosslinking = Strength • Lysozyme breaks β(14) bonds-therefore selective for Cell Wall of Bacteria • Archaepseudomurein is composed of β(13) linked N-acetylglycosamine and N-Acetylalosaminuronic acid • Lysozyme insensitive

12. Cell Wall Synthesis • Divisome complex: Fts proteins for FtsZ ring defining division plane • FtsI synthesizes peptidoglycan (wall bands show border for new synthesis) • Glycanpentapeptide precursor must be transported across cell membrane first (Bactoprenol)

13. Cell Wall Synthesis M G M G M G M Transpeptidation crosslinks glycan layers – inhibited by Penicillin G M M G FtsI Bactoprenol M G

14. Endospores

15. Cell Mobility Whooo!

16. Flagellum Flagellin Hook LPS L P Peptidoglycan H+ MS C Cytoplasm -Powered by proton gradient -Flagellin added to terminus end

17. Chemotaxis Cells can either “run” to move or “tumble” to change orientation. Attractants increase frequency of “runs”, repellents increase frequency of “tumbles”

18. Regulation of Chemotaxis • 1)Response to signal • Need Membrane Protein to bind attractant/repellent (MCP = Methyl-accepting Chemotaxis Protein) • Need to relay the signal to flagellum: CheA which undergoes auto-phosphorylation to CheA-P • Also CheW involved

19. Chemotaxis (1) Attractant Repellent MCP MCP Slows auto-phosphorylation Speeds auto-phosphorylation CheA CheA-P CheA CheA-P

20. Regulation of Chemotaxis • 2)Control of Flagellar Rotation • - CheA-P transfers P to CheY CheY-P causes flagellar motor to Tumble = no movement (CW rotation) • CheZ removes P from CheY so that it cannot bind motor CCW rotation = Run

21. Chemotaxis (2) Attractant Repellent MCP MCP Slows auto-phosphorylation Speeds auto-phosphorylation CheA CheA-P CheA CheA-P CheZ CheY-P CheY Cannot Bind Motor Binds Motor CCW spin = Run CW spin = Tumble

22. Chemotaxis (3) Even in presence of attractant/repellent it is still desirable to have some random movement

23. Chemotaxis • 3) Adaptation (temporally controlled) • -CheR (think regulation) adds methyls to MCP continuosly • -CheB removes methyls from MCP  Becomes very active when Phosporylated • Methylated MCP are responsive to Repellents, insensitive to Attractants

24. Chemotaxis (3) CheR Adds Methyls to MCP Attractant MCP Does not slow auto-phosphorylation Slows auto-phosphorylation CheB (not very active) CheB-P (active) CheA CheA-P CheA CheA-P Demethylates MCP for a “reset” CheY-P CheY Cannot Bind Motor Binds Motor CW spin = Tumble CCW spin = Run

25. Taxis • A response to any number of stimuli • Phototaxis (light), aerotaxis (oxygen) • E.coli: Tar aspartate/malate attractants, Cobalt/Nickel repellents

26. Molecular Adaptations to Environment • Hyperthermophiles: Protein Stability • Amino acid substitutions for “heat tolerant folds”: Maximize ionic forces (between +/-ve charges) of acidic/basic amino acids -di-inositol phosphate, diglycerol phosphate, mannosylglycerate also produced to stabilize proteins - +

27. Molecular Adaptations to Environment • Hyperthermophiles: Membrane Stability • Bacteria have membranes enriched with SATURATED fatty Acids • Archae can have membrane monolayers (see before) All single bonds in tails allows for close stacking to Maximize attractive forces

28. Molecular Adaptations to Environment • Psychrophiles: Membrane Stability • have membranes enriched with UNSATURATED fatty Acids Cis Double Bond in tails causes bend which prevents close stacking increases fluidity

29. Salt Tolerance • Halophiles are adapted to high salt concentration • aw = vapour pressure of air in equilibrium with solution/vapour pressure of air above pure water • Many ions in solution attract polar water away from its gaseous state in air

30. Salt Tolerance • Halophiles are adapted to high salt concentration Organisms concentrate nutrients, therefore water diffuses into cell Osmotic Pressure Normal Conditions Osmotic Pressure High Salt concentrations draw water out of cell What is a cell to do? High Salt

31. Salt Tolerance • Halophiles are adapted to high salt concentration with COMPATIBLE SOLUTES Halophile Halophiles import/synthesize solutes to return osmotic pressure towards the cytoplasm “Compatible Solutes” since they do not interfere with cell metabolic reactions (not toxic at high concentrations) Osmotic Pressure High Salt

32. Oxygen Tolerance • Thioglycolate broth (reduces agent gets rid of molecular oxygen) • Resazurin indicates presence of O2 by turning Pink Obligate Anaerobe – Can only grow in absence of oxygen Obligate Aerobe – Can only grow in presence of oxygen Facultative Aerobe – Can grow in absence of oxygen, but grows better with oxygen Aerotolerant – Unaffected by oxygen content Microaerophilic – Can only grow in presence of low oxygen

33. Oxygen Tolerance Facultative Aerobe Obligate Aerobe O2 Present-Oxic Zone O2 Absent - Anoxic Zone Aerotolerant Microaerophilic Obligate Anaerobe

34. Genetic Regulation DNA Transcriptional Regulation-positive/negative regulation, riboswitches mRNA Translational Regulation-Attenuation, RNAi Enzyme Inhibition (allosterics)

35. Enzyme Inhibition Allosteric control

36. Transcriptional Regulation Positive Control-e.g. Maltose catabolism Negative Control Activator (protein) binds to Activator Binding Site(DNA sequence) Effector (protein) binds to Operator (DNA sequence) Induction-e.g. Lac operon Repression-e.g. Arginine synthesis Corepressors bind effector protein  Stop transcription (anabolic/biosynthetic operons) Inducers bind effector protein  Start transcription (catabolic/degradativeoperons)

37. Transcriptional Regulation Negative Control Positive Control RNA Polymerase RNA Polymerase Repressor protein prevents binding of RNA Polymerase  Transcription Blocked Activator protein promotes binding of RNA Polymerase  Transcription Proceeds

38. Transcriptional Regulation Negative Control - Induction Negative Control - Repression RNA Polymerase RNA Polymerase Aporepressor cannot bind DNA  Transcription Proceeds Repressor protein prevents binding of RNA Polymerase  Transcription Blocked RNA Polymerase RNA Polymerase Repressor protein binds inducer, binding of RNA Polymerase allowed Transcription Proceeds Co-repressor binds repressor protein, prevents binding of RNA Polymerase  Transcription Blocked

39. Quorum Sensing Example: AcylHomoserineLactone (AHL) Cells in population excrete AHL Quorum specific proteins expressed at critical AHL concentration AHL Synthase expressed produces more AHL AHL is taken up by neighbouring cells, binds AHL Activator protein AHL activator protein promotes transcription

40. Translational Control • mRNA is ssRNAcan base pair with itself or bind other molecules • antisense RNA can base pair with part of mRNA and can block ribosomes from binding • Riboswitches: RNA can bind small molecules to change mRNA structure free up Ribosome Binding Site for translation

41. Attenuation Tryptophan Operon encodes 5 genes needed to synthesize tryptophan DNA 5 genes mRNA 5 internal ribosome binding sites

42. Attenuation • Transcription and Translation can occur simultaneously in E.Coli. • Rho-independent termination of transcription can terminate the mRNA molecule before any of the biosynthetic genes are transcribed • Signalled by stem-loop in RNA followed by multiple U’s

43. Attenuation 1 2 3 4 uuuuu mRNA 4- can base pair with 3 Followed by multiple U’s 1- codes for trp and can base pair with 2 2- can base pair with 1 or 3 3- can base pair with 2 or 4

44. Attenuation Tryptophan not available in cell Trp genes needed to synthesize more RNA polymerase continues trp genes expressed DNA Ribosome Stalls since it can’t find the tryptophan “1” codes for 2 base pairs with 3 1 4 uuuuu RNA polymerase mRNA 2 3 1- codes for trp and can base pair with 2 3- can base pair with 2 or 4 4- can base pair with 3 Followed by multiple U’s 2- can base pair with 1 or 3

45. Attenuation Tryptophan available in cell Trp genes NOT needed to synthesize any more DNA 3 hybridizes with 4  signals attenuation Ribosome continues since it can find the tryptophan “1” codes for, stops at stop codon RNA polymerase drops off DNA uuuuu mRNA 1 2 3 4 1- codes for trp and can base pair with 2 3- can base pair with 2 or 4 4- can base pair with 3 Followed by multiple U’s 2- can base pair with 1 or 3

46. Practice Questions • Who was the first person to observe bacteria? Who founded the study of Bacteriology? a)van Leeuwenhoek/Hooke b)Hooke/Pasteur c)Koch/Cohn d) van Leeuwenhoek/Cohn e) Winogradsky/Koch

47. Practice Questions • A chemolithoautotroph is an organism which: a) Produces energy from light b)Oxidizes inorganic compounds for energy, and uses CO2 as a carbon source c) Produces energy from light and uses CO2 as a carbon source d) Oxidizes inorganic compounds for energy and has organic carbon sources

48. Practice Questions • A new organism is discovered and is found to have the DNA organized in a discrete region. It is classified as a: a) Bacteria b) Prokaryote c) Eukaryote d) Archae e) Could be any of the above

49. Practice Questions • The E. Coli genome is: a) Circular b) 2.7 million base pairs c) 4.68 million base pairs d) Has multiple Chromosomes e) A and C

50. Practice Questions • Phototrophs a) Are oxygenic b) Produce energy from Light c) Utilize Pigments d) Can be anoxygenic e) B, C, and D