Transcript Freeman 1e: How we got there

CHAPTER 5
Nutrition, Laboratory Culture, and
Metabolism of Microorganisms
Nutrition and Culture of
Microorganisms
• The hundreds of chemical compounds
present inside a living cell are formed from
nutrients. Elements required in fairly large
amounts are called macronutrients, whereas
metals and organic compounds needed in very
small amounts are called micronutrients
(Table 5.2) and growth factors (Table 5.3),
respectively.
• Some prokaryotes are autotrophs, able to build all
of their cellular structures from carbon dioxide.
• Nitrogen is important in proteins, nucleic acids, and
several other cell constituents.
• Iron plays a major role in cellular respiration, being a
key component of cytochromes and iron-sulfur
proteins involved in electron transport.
•To obtain iron from various insoluble minerals, cells
produce agents called siderophores that bind iron and
transport it into the cell (Figure 5.1).
Siderophores - Iron-chelating
agents produced by
microorganisms
Anoxic conditions - iron is
Fe2+ (soluble)
Oxic conditions – iron is Fe3+
(insoluble minerals)
Ferric entrobactin produced
by E. coli and S.
typhimurium
Siderophore aquachelin produced by marine microorganisms (iron is low in
picogram conc. (10-12 g)
Tail assists in transferring iron through the membrane into the cell
How to grow cells in the Lab?
• Culture media supply the nutritional needs of
microorganisms and can be either chemically defined
(defined medium) or undefined (complex medium).
• Selective, differential, and enriched are terms that
describe media used for the isolation of particular
species or for comparative studies of microorganisms.
• Successful cultivation and maintenance of
pure cultures of microorganisms can be done
only if aseptic technique (Figure 5.3) is
practiced to prevent contamination by other
microorganisms.
Aseptic Technique
Medium containing growth factors
• Culture media (Table 5.4) are sometimes
prepared in a semisolid form by the addition
of a gelling agent (e.g. agar) to liquid media.
• Such solid culture media immobilize cells,
allowing them to grow and form visible,
isolated masses called colonies (Figure 5.2).
Confluent growth of
Serratia marcescens
on MacConkey agar
Isolated colonies
Pseudomonas aeruginosa on Trypticase-Soy agar
Shigella flexneri on MacConkey agar
Energetics and Enzymes
• Free energy (G) is the energy in a chemical
reaction that is available to do useful work.
The change in free energy during a reaction is
G0'. Table 5.5 shows the free energy of
formation for a few compounds of biological
interest.
Can not be formed spontaneously. It
can decompose to Nitrogen and Oxygen
• The chemical reactions of the cell are
accompanied by changes in energy, expressed in
kilojoules.
•A chemical reaction can occur with the release of
free energy (exergonic reactions, or catabolism) or
with the
consumption of free energy (endergonic reactions,
or anabolism).
Catalysis and Enzymes
• Activation energy is the energy required to
bring all molecules in a chemical reaction
into the reactive state (Figure 5.5).
• The reactants in a chemical reaction must first be activated
before the reaction can take place, and this requires a catalyst.
• Enzymes are catalytic proteins that speed up the rate of
biochemical reactions by raising the activation energy.
Enzymes are highly specific in the reactions they catalyze, and
this specificity is found in the three-dimensional structure of
the polypeptide(s) in the protein.
Catalytic cycle
of enzyme
Oxidation-Reduction and
Energy-Rich Compounds
Oxidation - removal of electron(s) from a substance
Reduction – addition of electron(s) to a substance
Energy (ATP) is released or consumed during oxidation or
reduction reactions
• Oxidation-reduction (redox) reactions (Figure 5.8)
involve the transfer of electrons from electron donor
to electron acceptor.
• The tendency of a compound to accept or
release electrons is expressed quantitatively
by its reduction potential, E0'.
• In a redox reaction
The substance oxidized is the electron donor.
The substance reduced is the electron acceptor.
• One way to view electron transfer reactions in
biological systems is to imagine a vertical tower. The
tower represents the range of reduction potentials
possible for redox couples in nature, from those with
the most negative E0's on the top to those with the
most positive at E0's on the bottom (Figure 5.9).
• Anaerobic (anoxic)
H2 + fumarate (e’-acceptor)
succinate
•Aerobic (oxic)
Succinate + ½ O2
fumarate + H2O
(e’- donor)
• Others
Succinate + NO3
fumarate + NO2 + H2O
NAD as a Redox Electron Carrier
• In a cell, the transfer of electrons from
donor to acceptor typically involves one or
more electron carriers. Some electron carriers
are membrane-bound, whereas others—such
as NAD+/NADH–are freely diffusible
(Figure 5.10), transferring electrons from one
place to another in the cell.
Nicotinamide adenine dinucleotide
• Coenzymes (NAD and NADP) increase the
diversity of redox reactions possible in a cell
by allowing chemically dissimilar molecules
to interact as primary electron donor and
terminal electron acceptor, with the coenzyme
acting as an intermediary (Figure 5.11).
Schematic of an oxidationreduction reaction
Glyceraldehyde 3-phosphate
dehydrogenase
NADH dehydrogenase
Quinone
Glyceraldehyde 3-phosphate (glycolysis)
or
Isocitrate or malate (citric acid cycle)
1,3-bisphosphoglycerate (glycolysis)
Alpha ketoglutarate or oxaloacetate
Quinol
Energy-Rich Compounds and Energy
Storage
• The energy released in redox reactions is conserved in the
formation of certain compounds that contain energy-rich
bonds.
1. phosphate bonds
2. sulfur bond
• The most common of these compounds is
adenosine triphosphate (ATP), the prime
energy carrier in the cell.
•Long-term storage of energy is linked to the
formation of polymers (e.g. glycogen, PHB),
which can be consumed to yield ATP.
Major Catabolic Pathways,
Electron Transport, and the
Proton Motive Force
Glycolysis as an Example of
Fermentation
• Glycolysis is a major pathway of
fermentation and is a widespread method of
anaerobic metabolism. The end result of
glycolysis is the release of a small amount of
energy that is conserved as ATP and the
production of fermentation products. For each
glucose consumed in glycolysis, two ATPs are
produced.
• Glycolysis is an anoxic process and can be
divided into three major stages, each
involving a series of individually catalyzed
enzymatic reactions (Figure 5.14).
Glycolysis (Embden-Meyerhof pathway)
Substrate level phosphorylation = ATP
is synthesized during catabolism of an organic compound
Oxidative phosphorylation = ATP is
produced at the expense of proton motive force
Photophosphorylation = ATP is produced during
photosynthesis using a mechanism similar to oxidative
phosphorylation
Respiration and MembraneAssociated Electron Carriers
• Electron transport systems consist of a series
of membrane-associated electron carriers that
function in an integrated way to carry
electrons from the primary electron donor to
oxygen as the terminal electron acceptor.
Energy Conservation from the
Proton Motive Force
• When electrons are transported through an
electron transport chain (Figure 5.19),
protons are extruded to the outside of the
membrane, forming the proton motive force
(Figure 5.20).
Electron Transport Chain
Generation of proton motive
force during aerobic respiration
Acidic environment
• Key electron carriers include flavins, quinones, the
cytochrome complex, and other cytochromes, depending on
the organism. The cell uses the proton motive force to make
ATP through the activity of ATP synthase (ATPase) (Figure
5.21), a process called chemiosmosis.
FMN
Quionone
ATP synthase (ATPase)
Complex V
Reversible reaction
Two part enzyme
F1 – multisubunit
F0 – proton conducting channel
Mechanism
Proton movement
Through F0 derives
Rotation of c
Proteins, resulting
torque is transmitted to εγ. This
causes a conformational change in
β to allow binding of ADP and Pi.
ATP is formed when β returns to
original conformation
Carbon Flow in Respiration
and Catabolic Alternatives
The Citric Acid Cycle
• Respiration involves the complete oxidation
of an organic compound with much greater
energy release than occurs during
fermentation. The citric acid cycle (Figure
5.22) plays a major role in the respiration of
organic compounds.
Citric acid cycle
Catabolic Alternatives
• In anaerobic respiration, electron acceptors
other than O2 can function as terminal
electron acceptors for energy generation.
• Chemolithotrophs use inorganic compounds
as electron donors, whereas phototrophs use
light to form a proton motive force. The
proton motive force is involved in all forms of
respiration and photosynthesis (Figure 5.23).
Energetics and carbon flow
Biosynthesis of Sugars and
Polysaccharides
• Polysaccharides are important structural
components of cells and are biosynthesized
from activated forms of their monomers.
• For organisms growing in culture media or
in nature that are not provided with these
building blocks, they must be biosynthesized
from simpler components, a process called
anabolism (Figure 5.24).
Summary
• Gluconeogenesis is the production of
glucose from nonsugar precursors (Figure
5.25).
Biosynthesis of polysaccharides
Glycogen biosynthesis
ADPG = adenosine diphosphoglucose
Gluconeogenesis
Glucose formation
Pentoses for nucleic acid synthesis
Biosynthesis of Amino Acids
and Nucleotides
• Amino acids are formed from carbon
skeletons generated during catabolism
(Figure 5.26).
Biosynthesis of amino acids
• Nucleotides (purines and pyrimidines) are
biosynthesized using carbon from several
sources (Figure 5.28).
Biosynthesis of
purines and
pyrimidines,
monomers of
nucleotides
(a) Precursors of purines
(b) Inosinic acid, precursor of purines
(c) Orotic acid and (d)
uridylate are precursors of
pyrimidines
Biosynthesis of Fatty Acids and
Lipids
• Fatty acids are synthesized two carbons at a
time and then attached to glycerol to form
lipids (Figure 5.29).
ACP = acyl carrier protein hold growing fatty acids
(b) Inosinic acid, precursor of purines
Biosynthesis of fatty acids
Fatty acids are synthesized 2 carbon at a time