BIOSYNTHESIS AND MICROBIAL GROWTH: ANABOLISM

Chapter 6Fall 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– 6.13 assembly of cellular structure– 6.14 growth

NOT COVERING: 6.4 – 6.7, 6.10 – 6.12

ANABOLISM – 3 STEPS

1. Monomer biosynthesis fatty acids, nucleotides, amino acids, sugars

2. Polymerization of monomers lipids, polysaccharides, glycogen, peptidoglycan, protein, RNA, DNA

3. Polymer assembly into cellular structures inclusion bodies, envelope, flagella, pili, cytosol, polyribosomes, nucleoid

Monomer polymerization

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• Why: nitrogen found in cellular components, amino acids and nucleic

acids; various redox states -5 to +3WHAT 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

• Nitrogen-fixing bacteria assimilate N2 and transform it into NH3

– free-living soil bacteriaand

– in bacteria Rhizobium living symbiotically in the roots of legume plants

Nitrogen Fixation done by Nitrogenase Complex

• Complex enzyme– Subunit 1 = azoferredoxin– Subunit 2 = molybdoferredoxin

REQUIRED TO VIEW & LEARN FROM ANIMATION

• Nitrogen-fixing bacteria assimilate N2 and transform it into NH3

– free-living soil bacteriaand– in bacteria Rhizobium living 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 N2

Azoferrodoxin carries 1 e-

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

• Nitrification – oxidation of nitrogen by bacteriaNH3 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 occursNO3

- NO2- NO N2O N2

– nitrogen is lost from the systems

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

• Nitrification: fixed nitrogen goes to gaseous nitrogen (Kim and Gadd, 10.2)

• Denitrification:

Where does NH3 go?

• Glutamine and glutamate donate amino groups in various synthetic reactions catalyzed by transaminases

Sulfur

• Found in – methionine and cysteine– Coenzymes– ETC in iron-sulfur proteins

• Sulfate = major source of inorganic sulfur• Sulfate actively transported into cellSulfate + ATP adenine-5’phosphosulfate + PPi

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 Walls:– Gram+: murein, teichoic acid, lipoteichoic acid,

lipoglycan– Gram-: murein– Archaea: pseudomurein, sulfonated

polysaccharide, glycoprotein

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

• 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

UDP-N-acetylmuramylpentapeptide

• Teichoic Acid biosynthesis: very similar to murein biosynthesis

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 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

• 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 Membrane

• Lipopolysaccharide– Composed of lipid A, core polysaccharide and O-

antigen• Phospholipids moved from CM by flippase

CELL DIVISION • Asexual propagation – binary fission• BINARY FISSION: cell number doubles with each cell

division1+1 = 22+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)

Y = log of # bacteria

X = time (hr)Phases• Lag• Log• Stationary• Death

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 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 ÷

1. Remove 1 ml at arrow during decline phase

2. Place in new liquid culture with nutrients

WHAT HAPPENS?

BACTERIAL GROWTH CALCULATIONS