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BIOSYNTHESIS AND MICROBIAL GROWTH: ANABOLISM

BIOSYNTHESIS AND MICROBIAL GROWTH: ANABOLISM. Chapter 6 Fall 2012. COVERING IN CLASS: Overview of pathways leading to cellular structures EMPHASIS on: 6.1-6.3: assimilation 6.8 Synthesis of saccharides and their derivatives 6.9 assembly of outer membrane

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BIOSYNTHESIS AND MICROBIAL GROWTH: ANABOLISM

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  1. BIOSYNTHESIS AND MICROBIAL GROWTH: ANABOLISM Chapter 6 Fall 2012

  2. COVERING IN CLASS: • Overview of pathways leading to cellular structures • EMPHASIS on: • 6.1-6.3: assimilation • 6.8 Synthesis of saccharides and their derivatives • 6.9 assembly of outer membrane • 6.13 assembly of cellular structure • 6.14 growth NOT COVERING: 6.4 – 6.7, 6.10 – 6.12

  3. ANABOLISM – 3 STEPS • Monomer biosynthesis  fatty acids, nucleotides, amino acids, sugars • Polymerization of monomers  lipids, polysaccharides, glycogen, peptidoglycan, protein, RNA, DNA • Polymer assembly into cellular structures  inclusion bodies, envelope, flagella, pili, cytosol, polyribosomes, nucleoid

  4. Monomer polymerization

  5. ANABOLISM • Needs more than carbon skeletons • Needs: nitrogen, sulfur, phosphorous • From where: assimilation - incorporation of inorganic chemicals into organic molecules • photosynthesis: CO2 -> sugar • Nitrogen fixation: conversion of N2 to NH3 (ammonia) by bacteria or lightning • Sulfate

  6. Nitrogen: Bacteria are Key • Why: nitrogen found in cellular components, amino acids and nucleic acids; various redox states -5 to +3 WHAT IS THE ADVANTAGE OF SO MANY REDOX STATES? • Bacteria prefer to obtain nitrogen from organic sources: organic nitrogen, ammonia, nitrate BUT THEY CAN’T ALWAYS, SO • How does nitrogen (N2) get into biological system • Lightning creates NO3- • Nitrogen fixation creates NH3 • Only some bacteria/archaea (prokaryotes) have the ability to “FIX” nitrogen see Table 6.2 • Eukaryotes do not fix nitrogen

  7. Nitrogen-fixing bacteria assimilate N2 and transform it into NH3 • free-living soil bacteria and • in bacteria Rhizobiumliving symbiotically in the roots of legume plants

  8. Nitrogen Fixation done by Nitrogenase Complex • Complex enzyme • Subunit 1 = azoferredoxin • Subunit 2 = molybdoferredoxin REQUIRED TO VIEW & LEARN FROM ANIMATION

  9. Nitrogen-fixing bacteria assimilate N2 and transform it into NH3 • free-living soil bacteria and • in bacteria Rhizobiumliving symbiotically in the roots of legume plants N2 + 6 H + large amount of ATP → 2 NH3 N2 + 8 H+ + 8 e− + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi

  10. Nitrogenase fixes N2 Azoferrodoxin carries 1 e-

  11. Electron movement: Fe-S cluster to P cluster to Mo-Fe cluster

  12. Nitrification – oxidation of nitrogen by bacteria NH3 NO2-  NO3- • energy-releasing reactions • nitrates can be used by plants, but they have to be reduced (requires energy) • In low-oxygen settings (oceans, soils, sediments), denitrification occurs NO3- NO2-  NO N2O  N2 • nitrogen is lost from the systems

  13. Where does NH3 go? • Made into glutamate 2-ketoglutarate + NH3 + NADPH + H+ glutamate + NADP+ + H2O Glutamate + NH3 + ATP  glutamine + ADP + Pi Glutamine + NADPH + H+ + 2-ketoglutarate  2 glutamate + NADP+

  14. OTHER TYPES OF NITROGEN METABOLISM • Nitrification: fixed nitrogen goes to gaseous nitrogen (Kim and Gadd, 10.2) • Denitrification:

  15. Where does NH3 go? • Glutamine and glutamate donate amino groups in various synthetic reactions catalyzed by transaminases

  16. Sulfur • Found in • methionine and cysteine • Coenzymes • ETC in iron-sulfur proteins • Sulfate = major source of inorganic sulfur • Sulfate actively transported into cell Sulfate + ATP  adenine-5’phosphosulfate + PPi

  17. Polysaccharides • Storage material – glycogen • Cell wall structural polymers – murein (peptidoglycan), teichoic acid • Outer membrane – lipopolysaccharide (LPS) • Precursors • made in cytoplasm • transported across cytoplasmic membrane

  18. CELL WALL REVIEW • Cell Walls: • Gram+: murein, teichoic acid, lipoteichoic acid, lipoglycan • Gram-: murein • Archaea: pseudomurein, sulfonated polysaccharide, glycoprotein

  19. Murein Monomers • Made from fructose-6-phosphate (EMP) Fig. 6-28 • Uses glutamine, acetyl-CoA, UTP, PEP, and NADPH + H+ • Products = UDP-N-acetylglucosamine and UDP-N-acetylmuramate

  20. Addition of Amino Acids • Non-ribosomal addition of peptides • ATP supplies energy to form peptide bond i.e., add amino acids • Uses L- and D- amino acids • L-amino acids  D-amino acids • 2nd and 3rd amino acids vary with species

  21. UDP-N-acetylmuramylpentapeptide

  22. Teichoic Acid biosynthesis: very similar to murein biosynthesis

  23. Gram+ Cell Wall Surface Proteins • Cell wall proteins include enzymes and virulence factors • Two processes for positioning in cell wall • Sorting: • Sortase • recognizes and cleaves a consensus sorting sequence, LPXTG • covalently attaches surface proteins to peptidoglycan at a penta-glycine crossbridge • Sequence found on > 100 proteins • M proteins of Streptococcus pyogenes • protein A of Staphylococcus aureus • internalins of Listeria monocytogenes

  24. Gram+ Cell Wall Surface Proteins • Targeting: noncovalent attachment of proteins to cell surface • via specialized binding domains • interact with secondary wall polymers • Teichoic acids • Polysaccharides • Proteins include: • muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes • surface S-layer proteins of bacilli and clostridia • virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections

  25. Gram- Outer Membrane • Proteins and phospholipids made in cytoplasm • Proteins - transported across CM and murein (PG) before assembling • Lipoproteins actually link to murein; transported via GSP (Sec) or ABC pathways • Integral proteins called OMP (outer membrane proteins) transported via GSP (Sec) and chaperone/usher pathways; OMPs have β- barrel structure; porins = example • Similar structure in mitochondria

  26. Gram- Outer Membrane • Lipopolysaccharide • Composed of lipid A, core polysaccharide and O-antigen • Phospholipids moved from CM by flippase

  27. CELL DIVISION • Asexual propagation – binary fission • BINARY FISSION: cell number doubles with each cell division 1+1 = 2 2+2 = 4 4+4 = 8, etc. • DNA synthesis – genome copied • Size of bacterial membrane, etc. increases; cells get longer • Cellular components increase • Cell divides in middle

  28. GROWTH PHASES (liquid culture) Y = log of # bacteria X = time (hr) Phases • Lag • Log • Stationary • Death

  29. GROWTH PHASES (liquid culture) • Lag: adaptation to new environment, synthesis of new genes, no cell ÷ • Log: cell division, exponential growth • Stationary: cell division = cell death; replacement; limiting environment (nutrients) • Death: decline;  living cells; no cell ÷

  30. GROWTH PHASES (liquid culture) • Lag: adaptation to new environment, synthesis of new genes, no cell ÷ • Log: cell division, exponential growth • Stationary: cell division = cell death; replacement; limiting environment (nutrients) • Death: decline;  living cells no cell ÷ Remove 1 ml at arrow during decline phase Place in new liquid culture with nutrients WHAT HAPPENS?

  31. BACTERIAL GROWTH CALCULATIONS

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