Microbial
Nutrition
Bio3124
Lecture # 4
Lecture outline
• Reading: Ch. 4 (up to page 130)
• Lecture topics
• Nutritional requirements
• Nutritional classes of microorganisms
• Nutrient uptake mechanisms
• Culturing bacteria and culture media
• Culturing and pure colony isolation methods
Nutritional Requirements
• Nutrients are substances required for biosynthesis of
macromolecules, energy production and growth
• macroelements (macronutrients)
• required in relatively large amounts
• C, H, N, O, P, S and K, Mg, Fe and Ca
• micronutrients (trace elements)
• Mn, Zn, Co, Mo, Ni, and Cu
• required in trace amounts, used as cofactors by
enzymes
• often supplied in water or in media components
Mystery Behind Life
• Molecules of life are formed through reductive pathway
that requires:
• Source of electrons and protons
• Source of energy
• A carbon source at oxidized state
• Process:
• energy is used to release electrons from an
inroganic/organic source
• Transfer onto a carbon containing molecule
• The reduced form of carbon is used to build new
macrmolecular derivatives
Growth Factors
• Are organic compounds
• essential cell components (or their precursors) that
the cell cannot synthesize
• must be supplied by environment if cell is to survive
and reproduce
Classes of growth factors
• amino acids
• needed for protein synthesis
• purines and pyrimidines
• needed for nucleic acid synthesis
• vitamins
• function as coenzymes
Nutritional classification of Microorganisms
• Nutritional classes :
• Based on carbon source:
• autotrophs
• use carbon dioxide as their sole or principal carbon source
• heterotrophs
• use organic molecules as carbon sources which often also serve
as energy source
• Based on energy source
• phototrophs use light
• chemotrophs obtain energy from oxidation of chemical compounds
• Based on electron source
• lithotrophs use reduced inorganic substances
• organotrophs obtain electrons from organic compounds
Nutritional Resource Management
•
Depending on how carbon, energy and electron sources are used
microroganisms can be divided into five nutritional categories:
(auto/hetero) (photo/chemo) (litho/organo)
Uptake of Nutrients by the Cell
• Some nutrients enter by passive diffusion.
(Membranes are permeable for them)
• Most nutrients enter by:
• facilitated diffusion
• active transport
• group translocation
Passive Diffusion (simple diffusion)
• molecules move from region of higher concentration
to lower concentration because of random thermal
agitation
•
is not energy dependent
• H2O, O2 and CO2 often move across membranes this
way
Passive diffusion is restricted
Substance
Water
Glycerol
Tryptophan
Glucose
Chloride ion
Sodium ions
Rate of intake
100
0.1
0.001
0.001
0.000001
0.0000001
Facilitated Diffusion
• similar to passive diffusion e.g.,
• movement of molecules is not energy dependent
• direction of movement is from high concentration to
low concentration
• concentration gradient impacts rate of uptake
Facilitated Diffusion …
• differs from passive diffusion
• uses carrier molecules (transporters, eg. permeases)
• smaller concentration gradient is required for significant
uptake of molecules
• effectively transports glycerol, sugars, and amino acids
• more prominent in eucaryotic cells than in
procaryotic cells
Passive and Facilitated Diffusion
 rate of facilitated
diffusion increases
more rapidly
at a lower
concentration
 diffusion rate
reaches a plateau
when carrier becomes
saturated
 carrier saturation
effect not seen in PD
Facilitated diffusion…
Examples
 Members of major intrinsic
proteins (MIP) that form porin
 Aquaporin: channels to
transport water
 glycerol transport channel
Note conformational change of carrier
Animation: Facilitated diffusion
Active Transport
Bacteria use active transport to accumulate scarce
sources of nutrients from their natural habitat
• energy-dependent process
• ATP or proton motive force used
• moves molecules against the concentration gradient
• concentrates molecules inside cells
• involves carrier proteins
• carrier saturation effect is observed at high solute
concentrations
ABC transporters
•
•
ATP-Binding Cassette transporters
observed in bacteria, archaea, and
eucaryotes
•
Transports sugars like arabinose,
galactose, ribose etc
•
Cargo delivery by porins (OmpF) to
periplasmic space where:
•
Solute binds to a specific binding
protein (SBP) that delivers it to the
transporter
Transporter conformation changes
•
•
ATP binds to transporter subunits
in lumen side
•
Upon ATP hydrolysis the solute is
transferred into the cytoplasm
Active Transport using proton gradient
 PMF instead of ATP can be

indirectly utilized to
transport sugars into
bacterial cells
Sugars can be transported
by a symporter that is driven
by Na+ gradient outside the
cell
 Na+ gradient itself is
generated through H+
gradient coupled antiporter
that pumps the Na+ to the
periplasmic space
Coupled transport: Symport and antiport
•
•
•
•
•
Na-Sugar symporter
Na/Ca antiporter
Na increases in cytoplasm
How to balance?
Coupled to Na/K pump
Group Translocation
• chemically modifies molecules as it is brought into cells
• PEP sugar phosphotransferase system (PTS):
• best known system, transports a variety of sugars
• while phosphorylating them using phosphoenolpyruvate (PEP)
as the phosphate donor
• Found among the member of enterobacteriacae, clostridium,
staphylococcus, and lactic acid bacteria
Active transport by group translocation
• energydependent
process: PEP
• Phosphate is
carried via E1,
HPr to
cyotosolic
protein IIA
• IIB receives P
and passes to a
sugar molecule
that has been
transported into
the cell via IIC
protein
Active transport by group translocation
• PEP-Phosphotransferase System (PTS)
• Widely used for sugar uptake
Iron Uptake
• ferric iron is very
insoluble so uptake is
difficult
• Microorganisms chelate
Fe3+ using,
Hydroxamates
• Form complexes with
ferric ion
• complex is then
transported into cell
Iron Uptake
• ferric iron is very
insoluble so uptake is
difficult
• Microorganisms chelate
Fe3+ using,
Siderophores, eg.
enterochelin
• Pass through OM via FepA
• FebB (a SBP), delivers to
ABC (FepG,FepD, FepC)
delivery to cytoplasm
• Reductio to Fe2+
Culture Media
• most contain all the nutrients required by the
organism for growth
• classification
• chemical constituents from which they are made
• physical nature
• function
Types of Culture Media
Physical Nature
Application
Liquid
Composition
Defined
(synthetic)
Semi-solid
Complex
Enriched
Solid
Supportive
Differential
Selective
Defined or Synthetic Media
• all components
and their
concentrations
are known
Complex Media
• contain some
ingredients of
unknown
composition
and/or
concentration
Some media components
• peptones
• protein hydrolysates prepared by partial digestion
of various protein sources
• extracts
• aqueous extracts, usually of beef or yeast
• agar
• sulfated polysaccharide used to solidify liquid
media
Functional Type of Media
• supportive or general purpose media
• support the growth of many microorganisms
• e.g., tryptic soy agar, Nutrient broth, Luria Bertani
• enriched media
• general purpose media supplemented
by blood or other special nutrients
• e.g., blood agar
Types of media…
Selective media
• favor the growth of some microorganisms
and inhibit growth of others
• e.g., MacConkey agar
• selects for gram-negative bacteria
Types of media…
Differential media
• distinguish between different groups
of microorganisms based on their
biological characteristics
• e.g., blood agar
• hemolytic versus nonhemolytic
bacteria
• e.g., MacConkey agar
• lactose fermenters versus
nonfermenters
E. coli
S. enterica
Techniques: Isolation of Pure Cultures
Pure culture
Isogenic population of cells
arising from a single cell
Isolation techniques
 spread plate
 streak plate
 pour plate
The Spread Plate and Streak Plate
• involve spreading a mixture of cells on an
agar surface so that individual cells are well
separated from each other
• each cell can reproduce to form a separate
colony (visible growth or cluster of
microorganisms)
Streak plate technique

using a sterile loop transfer cells from solid or broth culture onto
an agar plate

streaking lines are made with an intermittent
flaming the loop

Cells are diluted on the streak lines and
separated as individual cells

Each cell grows and forms a colony after
proper incubation
Click for animation
Animation: Streak plate technique
Spread plate technique
 dispense cells onto
medium in a Petri dish
 sterilize spreader by
dipping into 70% alcohol
followed by flaming
 spread cells across surface
 incubate plate
Pour plate technique
 sample is diluted
several times, eg
10-fold dilution
series
 diluted samples
are mixed with
liquid agar
 mixture of cells
and agar are
poured into
sterile culture
dishes
 What is the cfu/ml
of culture?
Calculation of bacterial cell concentration
 Question: plating of triplicate 100 ul from 10-7 dilution of
an actively growing E.coli culture produced 37, 42 and
44 isolated colonies on nutrient agar plates following
ovenight incubation at 37⁰C. Calculate the number of
the colony forming units per milliliter of the original
culture.
Answer: 4.1x 109 cfu/ml