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Microbial Physiology_Lec2(Parts and Functions).pptx
- 1. Microbial Physiology BIO 63 jay-ard. malana, Rmicro
- 2. Syllabus • Introduction • Definitionand Scope of Microbial Physiology • Phylogenetic Tree of Life • Bacterial Cell • Major Cellular Structures • Chemistry and Synthesis of Cellular Structures • Microbial Growth • Definition of Growth • Measurement of Growth • Growth Physiology • Factors Affecting Growth and Stress Responses • Microbial Nutrition and Solute Transport • Nutritional Reqs of Mcgs • Mechanisms of Transport • Bioenergetics in the Cytosol • High Energy Molecules • Mechanisms of ATP Synthesis • Prokaryotic Metabolism • Central Metabolic Pathways • Fermentation • Photosynthesis • Metabolism of Lipids, Nucleotides, AA, and Hydrocarbons • Inorganic Metabolism • C1 Metabolism
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- 4. The E.coli Paradigm •modelorganism •Gr – •Fastidious •Easy to manipulate, easy to culture •low pathogeneticity
- 5. Cell Structure •three basicshapes: coccus, bacillus, spiral •Some pleiomorphic •No nucleus= •Specialized forms: budding sheathed and mycelial
- 6. Major Cell Structuresof a Bacteria
- 7. The Cell Surface •Interface •Protectscell interior •Maintains integrity •Transport molecules •Cell wall, cell membrane, glycocalyx, flagella, pili, fimbriae
- 8. Cell Wall • Maintainscell shape • Prevents lysis • Anchors appendages • Acts as a selective barrier • Pathogenicity and immune response
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- 10. Gram + CellWall • Thick peptidoglycan layer • NAG and NAM • Peptide bridges • Prevents osmotic lysis • Teichoic acids • Polymers of ribitol or glycerol phosphate • Lipoteichoic acids
- 11. Gram + TeichoicAcids • Polymers of repeating units glycerol or ribitol joined by phosphates • Highly antigenic • Provides high density of regularly oriented charges • Storage of phosphorous • Facilitates attachment of bacteriophages • Inhibits activity of autolytic enzymes that can hydrolyze the murein
- 12. Gram + TeichoicAcids Function WTA LTA Anchoring Structural Support Cell wall ____________ Cell wall ______________ Ion Regulation Maintains metal ion homeostasis Immune Recognition Highly antigenic, triggers HIR Also antigenic/ immunogenic Bacteriophage Attachment Recognized by bacteriophages Less involved Autolysin Regulation Prevents ____________________ Controls _____________________ Virulence Factor Important for pathogenic adhesion and immune evasion contributory
- 13. Gram- Cell Wall •Thin PG (2-7 nm) • Few layers • NAM and NAG • Less cross-linking • Outer membrane • Bilayer • Inner leaflet • Phospholipids • Outer leaflet • LPS • permeability barrier against harmful substances
- 14. Gram- Cell Wall •LPS • Lipid A • Core polysaccharide • O-antigen • Periplasmic space • Contains exoenzymes (e.g. beta- lactamases) • Porins • Channels; regulate nutrient uptake • Braun’s Lipoprotein • Anchors outermembrane to pg
- 15. Gram- Lipopolysaccharide • LIPIDA (toxic moiety) • Hydrophobic • Membrane-anchoring • Phosphorylated glucosamine disaccharides linked to FA • Triggers TLR4 (Toll-like receptor 4) signaling, leading to inflammatory cytokine production • Core Polysaccharide • short sugar chain • Outer core: high to moderate variability (hexoses) • Inner core: low structural variability • Composed of KDO, heptose, ethanolamine, and phosphate
- 16. Gram- Lipopolysaccharide • O-Antigen •Variable, antigenic sugar chain • Highly diverse • Helps bacteria evade immune system • Determines serotype classification • Not essential for viability
- 17. Lipopolysaccharide Synthesis Part Synthesis LipidA Synthesized from UDP-NAG Made in the CM by peripheral membrane proteins Core Synthesized on Lipid A Outer Core: each sugar is added one at a time to Lipid A O-antigen Synthesized on a separate lipid carrier (undecaprenyl-P) completed oligosaccharide chain is transferred to lipid A core
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- 19. Peptidoglycan in CellWalls Feature Gram-Positive Gram-Negative Thickness Thick (~20–80 nm) Thin (~2–7 nm) Layering Multilayered, heavily cross-linked Single-layered, less cross-linked Cross-Linking Peptide interbridge (pentaglycine bridge in S. aureus) Direct cross-links between tetrapeptides Teichoic Acids Yes – Provides negative charge and binding sites for ions Absent Outer Membrane Absent Present, contains LPS Periplasmic Space Minimal or absent Large periplasmic space Lysozyme Sensitivity Highly sensitive (lysozyme hydrolyzes β-1,4 bonds in NAG-NAM) Less sensitive (outer membrane prevents lysozyme access) Antibiotic Sensitivity Sensitive to penicillin, vancomycin (targets transpeptidases) Less sensitive due to outer membrane barrier Examples Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa
- 20. Peptidoglycan Synthesis • Cytoplasmic Stage •MurA and MurB • MurC-F • MraY • MurG
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- 22. Peptidoglycan Synthesis • Extracellular Transport •PBP (TGase and TPase) • Transglycosylase • Transpeptidase • Amidase, DD- Carboxypeptidas e and Lytic Transglycosylase • LdcA and NagZ
- 23. Peptidoglycan Synthesis • Recyclingand Regulation • AmpG • AmpD
- 24. Substances vs PeptidoglycanSynthesis Substance Effect on PG Synthesis Phosphonomycin Prevents synthesis of UDP-NAM from UDP-NAG Cycloserine Inhibition of pentapeptide formation Bacitracin Inhibits lysine incorporation into the PG; prevents dephosphorylation of the carrier lipid Vancomycin, Tunicamycin, Ristocetin Inhibits translocation step of the PG synthesis Penicillin Prevents cross-linking
- 25. Mechanism of Action(MOA) of β-Lactam Antibiotics • Penicillin: weak penetration in the periplasmic space of Gram neg • Inhibition of PBPs (Transpeptidases) • Penicillin mimics D-Ala-D-Ala, binds irreversibly to PBPs, and blocks cross- linking of peptidoglycan. • Weakened Peptidoglycan Structure • PG layer becomes unstable, making Gram-positive bacteria especially vulnerable. • Autolysin Activation → Cell Lysis
- 26. Penicillin-Binding Proteins PBP Molecules/CellActivity Function 1A or 1B 100 Transglycosylase, transpeptidase Murein synthesis (cell elongation) 2 20 Transpeptidase Growth in rod shape; cell elongation 3 50 Transglycosylase, transpeptidase Murein synthesis (septation) 4 110 DD-endopeptidase, DD- carboxypeptidase Cross-link hydrolysis 5 1800 DD-carboxypeptidase Destruction of unutilized pentapeptide 6 600 DD-carboxypeptidase Destruction of unutilized pentapeptide 1C, 7, 8 Not known Not known Not known
- 27. Periplasm • Separate compartmentbet CM and OM in Gram neg • A space in electron micrographs • ACTIVITIES: • Redox reactions • Osmotic regulation • Solute transport • Protein secretion • hydrolysis
- 28. Periplasm Component Function Oligosaccharides Involvedin osmotic regulation Solute-Binding Proteins Bind to solutes and deliver them to specific transporters in the CM Cytochrome c Involved in ETC and redox rxn Hydrolytic enzymes Degrade nutrients to smaller molecules that can be transported across the CM Detoxifying agents e.g. B-lactamase TonB proteins Required for the uptake of solutes (iron siderophores, vit B12) that do not diffuse through the porin
- 29. Archaeal Cell Wall •Lacks peptidoglycan • May contain polysaccharides, protein (S-layer) or ____________________ • Exhibit different cell wall profiles • Lacks outermembrane • Polysaccharides: glucose, glucuronic acid, acetate, galactosamine • e..g Methanosarcina spp
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- 31. Archaeal Cell Wall-S-Layer • S-Layer (Surface Layer) • Made of protein or glycoprotein subunits arranged in a paracrystalline pattern. • Forms a rigid, protective outer shell attached directly to the plasma membrane. • Example: Sulfolobus (thermoacidophile). • Structure: • Single-layer lattice of hexagonal, tetragonal, or linear symmetry. • Pores allow nutrient exchange while blocking large molecules. • Provides structural integrity and environmental resistance.
- 32. Archaeal Cell Wall-Pseudomurein • Pseudomurein (Pseudopeptidoglycan) • Found in some methanogenic archaea (Methanobacterium, Methanothermus). Similar to bacterial peptidoglycan, but with key differences: • N-acetyltalosaminuronic acid (NAT) instead of N-acetylmuramic acid (NAM). • β-1,3 glycosidic bonds instead of β-1,4 (resistant to lysozyme). • Peptide cross-links use L-amino acids, not D-amino acids like bacteria. • Structure: • Repeating units of NAT and NAG (N-acetylglucosamine) linked via β-1,3 bonds. • Cross-linked peptide bridges provide strength.
- 33. Mollicutes • Mycoplasma andSpiroplasma • Mostly parasitic and pathogenic • Cell wall-less • Gram-neg appearance • pleiomorphic • Have internal protein cytoskeleton • Some contain unusually tough cytoplasmic membrane due to the presence of sterols
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Editor's Notes
- #11 Explanation of Teichoic Acids ✅ Polymers of Repeating Units (Glycerol or Ribitol) Joined by Phosphates Teichoic acids are long-chain polymers composed of either glycerol phosphate or ribitol phosphate. These phosphate groups provide a negative charge, making the bacterial cell surface highly anionic. ✅ Highly Antigenic Teichoic acids are highly immunogenic, meaning they can elicit strong immune responses in the host. This antigenicity plays a role in host-pathogen interactions and bacterial virulence. ✅ Anchors __ to __ Wall teichoic acids (WTAs) are anchored to the peptidoglycan layer. Lipoteichoic acids (LTAs) are anchored to the cytoplasmic membrane. ✅ Provides High Density of Regularly Oriented Charges Due to their phosphate-rich structure, teichoic acids contribute to the electrostatic properties of the bacterial surface. This influences interactions with metal ions, host cells, and antimicrobial peptides. ✅ Storage of Phosphorus Since teichoic acids are phosphate-containing polymers, they can act as a reservoir of phosphorus when needed. This is useful for bacterial survival under conditions of phosphorus limitation. ✅ Facilitates Attachment of Bacteriophages Some bacteriophages (viruses that infect bacteria) recognize and bind to teichoic acids during infection. This helps bacteriophages attach to and infect Gram-positive bacteria. ✅ Inhibits Activity of Autolytic Enzymes That Can Hydrolyze the Murein Autolysins are enzymes that degrade peptidoglycan and regulate bacterial cell wall turnover. Teichoic acids inhibit autolysins, preventing premature cell wall breakdown.
- #18 Direction Type of Bond Function X-Direction (Horizontal) β-1,4 Glycosidic Bond (NAG-NAM) Forms backbone, attacked by lysozyme Y-Direction (Vertical) Peptide Bonds (NAM-NAM cross-links) Provides strength, targeted by β-lactam antibiotics Basic Structure of the Pentapeptide The pentapeptide attached to NAM typically follows this pattern: 🔹 L-Ala – D-Glu – meso-DAP (or L-Lys) – D-Ala – D-Ala This sequence varies between Gram-positive and Gram-negative bacteria but usually includes: L-Alanine (L-Ala) D-Glutamic acid (D-Glu) A diamino acid (meso-diaminopimelic acid (DAP) in Gram-negative, L-Lysine (L-Lys) in Gram-positive) Two terminal D-Alanine (D-Ala – D-Ala) The last D-Ala is lost during cross-linking, reducing it to a tetrapeptide in mature peptidoglycan.
- #20 Cytoplasmic Stage: MurA & MurB: Convert UDP-NAG into UDP-NAM. MurC-F: Sequentially add amino acids to form the pentapeptide. MraY: Links UDP-NAM-pentapeptide to undecaprenol (C55-P), forming Lipid I. MurG: Adds NAG, forming Lipid II. Membrane Transport: MurJ (Flippase): Transports Lipid II across the membrane. Extracellular Stage (Periplasmic Remodeling & Cross-Linking): PBP (TGase & TPase): Transglycosylase (TGase) polymerizes the NAG-NAM backbone. Transpeptidase (TPase) catalyzes cross-linking between pentapeptides. Amidase, DD-Carboxypeptidase (DD-CPase), and Lytic Transglycosylase (LT): Modify the peptidoglycan mesh for controlled degradation and remodeling. LdcA & NagZ: Recycle old peptidoglycan fragments. Recycling & Regulation: AmpG & AmpD: Regulate peptidoglycan turnover and response to β-lactam antibiotics.
- #21 Cytoplasmic Stage: MurA & MurB: Convert UDP-NAG into UDP-NAM. MurC-F: Sequentially add amino acids to form the pentapeptide. MraY: Links UDP-NAM-pentapeptide to undecaprenol (C55-P), forming Lipid I. MurG: Adds NAG, forming Lipid II. Membrane Transport: MurJ (Flippase): Transports Lipid II across the membrane. Extracellular Stage (Periplasmic Remodeling & Cross-Linking): PBP (TGase & TPase): Transglycosylase (TGase) polymerizes the NAG-NAM backbone. Transpeptidase (TPase) catalyzes cross-linking between pentapeptides. Amidase, DD-Carboxypeptidase (DD-CPase), and Lytic Transglycosylase (LT): Modify the peptidoglycan mesh for controlled degradation and remodeling. LdcA & NagZ: Recycle old peptidoglycan fragments. Recycling & Regulation: AmpG & AmpD: Regulate peptidoglycan turnover and response to β-lactam antibiotics.
- #22 Cytoplasmic Stage: MurA & MurB: Convert UDP-NAG into UDP-NAM. MurC-F: Sequentially add amino acids to form the pentapeptide. MraY: Links UDP-NAM-pentapeptide to undecaprenol (C55-P), forming Lipid I. MurG: Adds NAG, forming Lipid II. Membrane Transport: MurJ (Flippase): Transports Lipid II across the membrane. Extracellular Stage (Periplasmic Remodeling & Cross-Linking): PBP (TGase & TPase): Transglycosylase (TGase) polymerizes the NAG-NAM backbone. Transpeptidase (TPase) catalyzes cross-linking between pentapeptides. Amidase, DD-Carboxypeptidase (DD-CPase), and Lytic Transglycosylase (LT): Modify the peptidoglycan mesh for controlled degradation and remodeling. LdcA & NagZ: Recycle old peptidoglycan fragments. Recycling & Regulation: AmpG & AmpD: Regulate peptidoglycan turnover and response to β-lactam antibiotics.
- #23 Cytoplasmic Stage: MurA & MurB: Convert UDP-NAG into UDP-NAM. MurC-F: Sequentially add amino acids to form the pentapeptide. MraY: Links UDP-NAM-pentapeptide to undecaprenol (C55-P), forming Lipid I. MurG: Adds NAG, forming Lipid II. Membrane Transport: MurJ (Flippase): Transports Lipid II across the membrane. Extracellular Stage (Periplasmic Remodeling & Cross-Linking): PBP (TGase & TPase): Transglycosylase (TGase) polymerizes the NAG-NAM backbone. Transpeptidase (TPase) catalyzes cross-linking between pentapeptides. Amidase, DD-Carboxypeptidase (DD-CPase), and Lytic Transglycosylase (LT): Modify the peptidoglycan mesh for controlled degradation and remodeling. LdcA & NagZ: Recycle old peptidoglycan fragments. Recycling & Regulation: AmpG & AmpD: Regulate peptidoglycan turnover and response to β-lactam antibiotics.
- #25 1. Inhibition of Transpeptidase Activity (PBPs) Penicillin binds to penicillin-binding proteins (PBPs), which function as transpeptidases. PBPs normally cross-link the peptidoglycan peptide chains by forming covalent bonds between D-Ala-D-Ala of one PG unit and meso-DAP (or Lys) of another. Penicillin mimics the D-Ala-D-Ala structure, irreversibly binding to PBPs and blocking cross-link formation. 2. Weakening of Peptidoglycan Structure Without proper cross-linking, the PG layer becomes unstable and fragile. This is especially fatal for Gram-positive bacteria, which rely on thick peptidoglycan for cell integrity. In Gram-negative bacteria, β-lactams need to penetrate the outer membrane via porins to reach PBPs. 3. Activation of Autolysins → Cell Lysis Many bacteria naturally produce autolysins that remodel peptidoglycan during cell growth. Normally, PBPs counterbalance autolysins by continuously repairing the PG network. When β-lactams block PBPs, autolysins continue to degrade PG, leading to osmotic lysis.
- #27 Key Activities of the Periplasm Redox Reactions Facilitates oxidation and reduction processes. Contains cytochromes and enzymes like Dsb proteins, which promote disulfide bond formation. Osmotic Regulation Maintains cell stability under changing osmotic conditions. Contains osmoregulatory proteins (e.g., OsmY, MDOs) that adjust periplasmic composition based on external environment. Solute Transport Houses periplasmic binding proteins (PBPs) that aid in active transport via ABC transporters. Acts as a buffer zone where molecules transition between the environment and cytoplasm. Protein Secretion Participates in Type II secretion system (T2SS) and Twin-arginine translocation (Tat) pathway to transport folded proteins. Chaperones like SurA assist in outer membrane protein (OMP) folding. Hydrolysis & Enzymatic Breakdown Enzymes like β-lactamases degrade antibiotics, contributing to resistance. Proteases (e.g., DegP) eliminate misfolded proteins. Hydrolases break down complex molecules before transport into the cytoplasm.
- #34 1. Carbon Linkage of Lipids Bacteria & Eukarya: Lipids in their cell membranes have ester linkages. Archaea: Use ether linkages instead. Significance: Ether bonds are more chemically stable than ester bonds, allowing archaea to thrive in extreme environments (e.g., high temperatures, acidity, or salinity). 2. Phosphate Backbone of Lipids Bacteria & Eukarya: Use glycerol-3-phosphate as the backbone for their membrane lipids. Archaea: Use glycerol-1-phosphate instead. Significance: The difference in lipid backbones reflects a fundamental divergence in membrane chemistry, supporting the hypothesis that archaea and bacteria evolved separately from a common ancestor. 3. Metabolism Bacteria & Archaea: Have a bacterial-like metabolism, meaning they can perform diverse metabolic processes, including anaerobic respiration, fermentation, and nitrogen fixation. Eukarya: Metabolism is distinct and includes specialized organelles (mitochondria and chloroplasts) for energy production. 4. Presence of a Nucleus Bacteria & Archaea: No nucleus; their DNA is free-floating in the cytoplasm. Eukarya: Has a nucleus, where DNA is enclosed by a nuclear membrane. Significance: The presence of a nucleus is a major feature distinguishing eukaryotic cells from prokaryotic cells (Bacteria and Archaea). 5. Presence of Organelles Bacteria & Archaea: No organelles. Eukarya: Contains organelles such as mitochondria, endoplasmic reticulum, and Golgi apparatus. Significance: Organelles allow compartmentalization of cellular functions, increasing efficiency. 6. Presence of Spliceosomal Introns (Non-coding DNA Sequences) Bacteria & Archaea: No introns in their genes. Eukarya: Has introns, which are removed during RNA processing. Significance: Introns allow for gene regulation and alternative splicing, increasing genetic complexity in eukaryotes. 7. Presence of Telomeres Bacteria & Archaea: No telomeres, as they have circular chromosomes. Eukarya: Has telomeres, which protect the ends of linear chromosomes from degradation. Significance: Telomeres prevent DNA loss during replication, a key feature in eukaryotic cell division and aging. 8. Chromosome Shape Bacteria: Mostly circular chromosomes. Archaea: Circular chromosomes. Eukarya: Linear chromosomes. Significance: Circular chromosomes are more stable and commonly found in prokaryotes, whereas linear chromosomes require telomeres for maintenance in eukaryotes. 9. DNA Replication Bacteria: Uses bacterial-type replication enzymes. Archaea: Uses eukaryotic-like replication enzymes. Eukarya: Uses eukaryotic replication mechanisms. Significance: This similarity between Archaea and Eukarya supports the hypothesis that Eukarya evolved from an archaeal ancestor. 10. Transcription (RNA Synthesis) Bacteria: Uses bacterial transcription machinery. Archaea: Uses eukaryotic-like transcription machinery. Eukarya: Uses eukaryotic transcription. Significance: Archaea and Eukarya share similarities in transcription enzymes, further indicating a close evolutionary relationship. 11. Translation (Protein Synthesis) Bacteria: Uses bacterial ribosomes for translation. Archaea: Uses eukaryotic-like ribosomes for translation. Eukarya: Uses eukaryotic ribosomes. Significance: Archaea's translation system resembles Eukarya’s more than Bacteria’s, reinforcing their evolutionary link.