1. Microbial Nutrition
Bio3124
Lecture # 4

2. Reading: Ch. 4 (up to page 130) Lecture topics
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

3. 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

4. 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

5. 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

6. Classes of growth factors
amino acids
needed for protein synthesis
purines and pyrimidines
needed for nucleic acid synthesis
vitamins
function as coenzymes

7. 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

8. 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)

9. 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

10. 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

11. Passive diffusion is restricted
Substance Rate of intake Water 100 Glycerol 0.1 Tryptophan Glucose Chloride ion Sodium ions

12. 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

13. 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

14. 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

15. Facilitated diffusion…
Examples
Members of major intrinsic proteins (MIP) that form porin
Aquaporin: channels to transport water
glycerol transport channel
possible mechanism of action of permease used for facilitated diffusion; movement is down the gradient; note higher concentration of transported molecule on outside of cell
Note conformational change of carrier

16. Animation: Facilitated diffusion

17. 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

18. 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 system; solute-binding protein is located in periplasm of gram-negative bacteria but is attached to external surface of cytoplasmic membrane in gram-positive bacteria; solute-binding proteins may also participate in chemotaxis

19. 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
Figure 5.4: active transport using proton and sodium gradients

20. Coupled transport: Symport and antiport
Na-Sugar symporter
Na/Ca antiporter
Na increases in cytoplasm
How to balance?
Coupled to Na/K pump

21. Group Translocation PEP sugar phosphotransferase system (PTS):
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

22. Active transport by group translocation
energy-dependent 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

23. Active transport by group translocation
PEP-Phosphotransferase System (PTS)
Widely used for sugar uptake

24. 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
Figure 5.6: siderophore ferric iron complexes
Figure 5.6a: ferrichrome is a cyclic hydroxamat molecule formed by many fungi
Figure 5.6b: E. coli produces enterobactin, a cyclic catacholate derivative
Figure 5.6c: ferric iron probably complexes with three siderophore groups to form a six-coordinate, octahedral complex (enterobactin-iron complex is shown)

25. Iron Uptake Siderophores, eg. enterochelin
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+
Figure 5.6: siderophore ferric iron complexes
Figure 5.6a: ferrichrome is a cyclic hydroxamat molecule formed by many fungi
Figure 5.6b: E. coli produces enterobactin, a cyclic catacholate derivative
Figure 5.6c: ferric iron probably complexes with three siderophore groups to form a six-coordinate, octahedral complex (enterobactin-iron complex is shown)

26. Culture Media
most contain all the nutrients required by the organism for growth
classification
chemical constituents from which they are made
physical nature
function

27. Types of Culture Media Physical Nature Composition Application Liquid
Defined (synthetic)
Supportive
Semi-solid
Complex
Enriched
Solid
Differential
Selective

28. Defined or Synthetic Media
all components and their concentrations are known

29. Complex Media
contain some ingredients of unknown composition and/or concentration

30. Some media components peptones extracts agar
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

31. 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

32. 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

33. 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

34. Techniques: Isolation of Pure Cultures
Isogenic population of cells arising from a single cell
Isolation techniques
spread plate
streak plate
pour plate

35. 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)

36. 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

37. Animation: Streak plate technique

38. 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

39. 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?

40. 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