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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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slide2

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

anabolism 3 steps
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

anabolism
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
nitrogen bacteria are key
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
slide7
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
nitrogen fixation done by nitrogenase complex
Nitrogen Fixation done by Nitrogenase Complex
  • Complex enzyme
    • Subunit 1 = azoferredoxin
    • Subunit 2 = molybdoferredoxin

REQUIRED TO VIEW & LEARN FROM ANIMATION

slide9
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

nitrogenase fixes n 2
Nitrogenase fixes N2

Azoferrodoxin carries 1 e-

slide13
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
where does nh 3 go
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+

other types of nitrogen metabolism
OTHER TYPES OF NITROGEN METABOLISM
  • Nitrification: fixed nitrogen goes to gaseous nitrogen (Kim and Gadd, 10.2)
  • Denitrification:
where does nh 3 go1
Where does NH3 go?
  • Glutamine and glutamate donate amino groups in various synthetic reactions catalyzed by transaminases
sulfur
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

polysaccharides
Polysaccharides
  • Storage material – glycogen
  • Cell wall structural polymers – murein (peptidoglycan), teichoic acid
  • Outer membrane – lipopolysaccharide (LPS)
  • Precursors
    • made in cytoplasm
    • transported across cytoplasmic membrane
cell wall review
CELL WALL REVIEW
  • Cell Walls:
    • Gram+: murein, teichoic acid, lipoteichoic acid, lipoglycan
    • Gram-: murein
    • Archaea: pseudomurein, sulfonated polysaccharide, glycoprotein
murein monomers
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
addition of amino acids
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
gram cell wall surface proteins
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
gram cell wall surface proteins1
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
gram outer membrane
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
gram outer membrane1
Gram- Outer Membrane
  • Lipopolysaccharide
    • Composed of lipid A, core polysaccharide and O-antigen
  • Phospholipids moved from CM by flippase
cell division
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
growth phases liquid culture
GROWTH PHASES (liquid culture)

Y = log of # bacteria

X = time (hr)

Phases

  • Lag
  • Log
  • Stationary
  • Death
growth phases liquid culture1
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 ÷
growth phases liquid culture2
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?