1. Chapter 5
Microbial Nutrition

2. The Common Nutrient Requirements
Macroelements (macronutrients)
C, O, H, N, S, P, K, Ca, Mg, and Fe
required in relatively large amounts
Micronutrients (trace elements)
Mn, Zn, Co, Mo, Ni, and Cu
required in trace amounts
often supplied in water or in media components

3. Autotroph and Heterotroph
All organisms require Carbon, Hydrogen, and Oxygen. Carbon is needed for the backbone of all organic molecules.
In addition all organisms require a source of electrons. Electrons are involved in oxidation-reduction reactions in the cell, electron transport chains, and pumps that drive molecules against a concentration gradient on cell membranes.
Organic molecules that supply, carbon, hydrogen, and oxygen are reduced and donate electrons for biosynthesis.

4. Requirements for Carbon, Hydrogen, and Oxygen
often satisfied together
carbon source often provides H, O and electrons
autotrophs
use carbon dioxide as their sole or principal carbon source
heterotrophs
use organic molecules as carbon sources

5. Autotrophs CO2 is used by many microorganisms as the source of Carbon.
Autotrophs have the capacity to reduce it , to form organic molecules.
Photosynthetic bacteria are Photoautotrophs that are able to fix CO2 and use light as their energy source.

6. Heterotrophs
Organisms that use organic molecules as their source of carbon are Heterotrophs. The most common heterotrophs use organic compounds for both energy and their source of carbon.
Microorganisms are versatile in their ability to use diverse sources of carbon. Burkholderia cepacia can use over 100 different carbon compounds.
Methylotrophic bacteria utilize methanol, methane, and formic acid.

7. Comparison of Nutritional Modes
Major Nutritional Types of Microorganisms
Major Nutritional Type
Source of Energy, Hydrogen, electrons, and Carbon
Representative microorganisms
Photolithotrophic autotrophy
Light energy
Inorganic hydrogen/electron( H+/e-) donor
CO2 carbon source
Algae
Purple and green sulfur bacteria
Cyanobacteria
Photoorganotrophic heterotrophy
Organic H+/e- donor
Organic carbon source or CO2
Purple and Green non-sulfur bacteria
Chemolithotrophic autotrophy
Chemical energy source – inorganic
Sulfur oxidizing bacteria
Nitrifying bacteria
Iron oxidizing bacteria
Chemoorganotrophic heterotrophy
Chemical energy source( organic)
Organic( H+/e-) donor
Organic carbon source
Most non photosynthetic bacteria including pathogens.
Protozoans
Fungi
Study table adapted from Microbiology, Prescott, Chapter Five.

8. Photolithotrophic autotrophs
Use light energy and have CO2 as their carbon source.
Cyanobacteria uses water as the electron donor and release oxygen
Purple and green sulfur bacteria use inorganic donors like hydrogen and hydrogen sulfide for electrons

9. Chemoorganotrophic heterotrophs
Use organic compounds as sources of energy,hydrogen, electrons and carbon
Pathogenic organisms fall under this category of nutrition

10. Photoorganoheterotrophs
Common inhabitants of polluted streams. These bacteria use organic matter as their electron donor and carbon source.
They use light as their source of energy
Important ecological organisms

11. Chemolithotrophic autotrophs
Oxidize reduce inorganic compounds such as iron, nitrogen, or sulfur molecules
Derive energy and electrons for biosynthesis
Carbon dioxide is the carbon source

12. Requirements for Nitrogen
Nitrogen is required for the synthesis of amino acids that compose the structure of proteins, purines and pyrimidines the bases of both DNA and RNA, and for other derivative molecules such as glucosamine.
Many microorganisms can use the nitrogen directly from amino acids. The amino group ( NH2) is derived from ammonia through the action of enzymes such as glutamate dehydrogenase.
Most photoautotrophs and many nonphotosynthetic microorganisms reduce nitrate to ammonia and assimilate nitrogen through nitrate reduction. A variety of bacteria are involved in the nitrogen cycle such as Rhizobium which is able to use atmospheric nitrogen and convert it to ammonia. ( Found on the roots of legumes like soy beans and clover) These compounds are vital for the Nitrogen cycle and the incorporation of nitrogen into plants to make nitrogen comounds.

13. Phosphorous
Phosphorous is present in phospholipids( membranes), Nucleic acids( DNA and RNA), coenzymes, ATP, some proteins, and other key cellular components.
Inorganic phosphorous is derived from the environment in the form of phosphates. Some microbes such as E. coli can use organophosphates such as hexose – 6-phosphates .

14. Mixotrophy Chemical energy – source organic Inorganic H/e- donor
Organic carbon source

15. Requirements for Nitrogen, Phosphorus, and Sulfur
Needed for synthesis of important molecules (e.g., amino acids, nucleic acids)
Nitrogen supplied in numerous ways
Phosphorus usually supplied as inorganic phosphate
Sulfur usually supplied as sulfate via assimilatory sulfate reduction

16. Sources of nitrogen organic molecules ammonia
nitrate via assimilatory nitrate reduction
nitrogen gas via nitrogen fixation

17. Growth Factors 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

18. Classes of growth factors
amino acids
needed for protein synthesis
purines and pyrimidines
needed for nucleic acid synthesis
vitamins
function as enzyme cofactors

19. Amino acids
Proteins

20. Bases of nucleic acids
Adenine and guanine are purines
Cytosine, thymine, and uracil are pyrimidines
Also found in energy triphosphates( ATP and GTP)

23. Practical importance of growth factors
development of quantitative growth-response assays for measuring concentrations of growth factors in a preparation
industrial production of growth factors by microorganisms

24. Uptake of Nutrients by the Cell
Some nutrients enter by passive diffusion
Most nutrients enter by:
facilitated diffusion
active transport
group translocation

25. Passive Diffusion
molecules move from region of higher concentration to one of lower concentration because of random thermal agitation
H2O, O2 and CO2 often move across membranes this way

26. Facilitated Diffusion
Similar to passive diffusion
movement of molecules is not energy dependent
direction of movement is from high concentration to low concentration. With the concentration gradient
size of concentration gradient impacts rate of uptake

27. Facilitated diffusion
These proteins exist in plants, animals, fungi, bacteria, and protists.

28. Facilitated diffusion…
Differs from passive diffusion
uses carrier molecules (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

29. rate of facilitated diffusion increases more rapidly and at a lower
concentration
diffusion rate
reaches a plateau
when carrier
becomes
saturated
carrier saturation
effect
Figure 5.1

30. note conformational change of carrier
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
Figure 5.2

31. Active Transport energy-dependent process
ATP or proton motive force used
moves molecules against the gradient
concentrates molecules inside cell
involves carrier proteins (permeases)
carrier saturation effect is observed

32. Transporters
“Molecular Properties of Bacterial Multidrug Transporters” – Monique Putnam, Hendrik van Veen, and Wil Konings – PubMed Central. Full Text available .
Microbiol Mol Biol Review. 2000 December; 64 (4): 672–693

33. ABC transporters ATP-binding cassette transporters
observed in bacteria, archaea, and eucaryotes
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
Figure 5.3

34. antiport symport Figure 5.4
Figure 5.4: active transport using proton and sodium gradients
symport
Figure 5.4

35. Group Translocation
molecules are modified as they are transported across the membrane
energy-dependent process
Figure 5.5

36. Fe uptake in pathogens
The ability of pathogens to obtain iron from transferrins, ferritin, hemoglobin, and other iron-containing proteins of their host is central to whether they live or die
Some invading bacteria respond by producing specific iron chelators - siderophores that remove the iron from the host sources. Other bacteria rely on direct contact with host iron proteins, either abstracting the iron at their surface or, as with heme, taking it up into the cytoplasm

37. Iron and signalling
Iron is also used by pathogenic bacteria as a signal molecule for the regulation of virulence gene expression. This sensory system is based on the marked differences in free iron concentrations between the environment and intestinal lumen (high) and host tissues (low)
Listeria Pathogenesis and Molecular Virulence Determinants
José A. Vázquez-Boland,1,2* Michael Kuhn,3 Patrick Berche,4 Trinad Chakraborty,5 Gustavo Domínguez-Bernal,1 Werner Goebel,3 Bruno González-Zorn,1 Jürgen Wehland,6 and Jürgen Kreft3

38. Pathogens and Iron uptake
Burkholderia cepacia
Campylobacter jejuni
Pseudomonas aeruginosa
E. coli
Listeria monocytogenes

39. Iron Uptake ferric iron is very insoluble so uptake is difficult
microorganisms use siderophores to aid uptake
siderophore 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)
Figure 5.6

40. Listeriosis
One involves the direct transport of ferric citrate to the bacterial cell
Another system involves an extracellular ferric iron reductase, which uses siderophores
The third system may involve a bacterial cell surface-located transferrin-binding protein

41. Iron bacteria in the environment
There are several non-disease producing bacteria which grow and multiply in water and use dissolved iron as part of their metabolism. They oxidize iron into its insoluble ferric state and deposit it in the slimy gelatinous material which surrounds their cells.
These filamentous bacteria grow in stringy clumps and are found in most iron-bearing surface waters. They have been known to proliferate in waters containing iron as low as 0.1 mg/l.
In order to function, these aerobic bacteria need at least 0.3 ppm of dissolved oxygen in the water.

42. Culture Media
preparations devised to support the growth (reproduction) of microorganisms
can be liquid or solid
solid media are usually solidified with agar
important to study of microorganisms

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

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

45. 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

46. Types of Media general purpose media enriched media
support the growth of many microorganisms
e.g., tryptic soy agar
enriched media
general purpose media supplemented by blood or other special nutrients
e.g., blood agar

48. Types of media… Selective media
Favor the growth of some microorganisms and inhibit growth of others
MacConkey agar
selects for gram-negative bacteria
Inhibits the growth of gram-positive bacteria

49. Beta Hemolysis

50. Types of media… Differential media
Distinguish between different groups of microorganisms based on their biological characteristics
Blood agar
hemolytic versus nonhemolytic bacteria
MacConkey agar
lactose fermenters versus nonfermenters

51. Selective and differential media
Selects for Gram –
Differentiates between bacteria based upon fermentation of lactose( color change)

52. Mannitol Fermentation
Organism
Salt Tolerance
Mannitol Fermentation
 1. S. aureus
Positive - growth
Positive (yellow)
 2. S. epidermidis
Positive*- growth
Negative( color does not change) – no fermentation of mannitol with production of acid
 3. M. luteus
Negative
N/A**

53. Web References on Media
- General Reference
- MacConkey Agar
- Blood Agar

54. 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)

55. Spread-plate technique
1. dispense cells onto
medium in petri dish
4. spread cells
across surface
sterilize spreader
Figure 5.7

56. Streak plate technique
inoculating
loop
Figure 5.8

58. Isolation of Pure Cultures
population of cells arising from a single cell
Spread plate, streak plate, and pour plate are techniques used to isolate pure cultures

59. The Pour Plate Sample is diluted several times
Diluted samples are mixed with liquid agar
Mixture of cells and agar are poured into sterile culture dishes

60. Figure 5.9: pour-plate technique

61. Colony Morphology and Growth
individual species form characteristic colonies
Figure 5.10b

62. Terms 1. Colony shape and size: round, irregular, punctiform (tiny) 2
Terms 1. Colony shape and size: round, irregular, punctiform (tiny) 2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed) 3. Elevation: convex, umbonate, flat, raised 4. Color: color or pigment, plus opaque, translucent, shiny or dull 5. Texture: moist, mucoid, dry (or rough).

63. Figure 5.10a

64. Colony growth Most rapid at edge of colony Slowest at center of colony
oxygen and nutrients are more available at edge
Slowest at center of colony
In nature, many microorganisms form biofilms on surfaces