Microbial Microbial NutritionNutrition

Bio3124Lecture # 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 RequirementsNutritional 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 LifeMystery 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 FactorsGrowth 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 factorsClasses of growth factors

• amino acids

• needed for protein synthesis

• purines and pyrimidines

• needed for nucleic acid synthesis

• vitamins

• function as coenzymes

Nutritional classification of MicroorganismsNutritional classification of Microorganisms

• Nutritional classes :Nutritional classes :

• Based on carbon source:• autotrophsautotrophs

• use carbon dioxide as their sole or principal carbon source

• heterotrophsheterotrophs

• use organic molecules as carbon sources which often also serve as energy source

• Based on energy source• phototrophsphototrophs use light

• chemotrophschemotrophs obtain energy from oxidation of chemical compounds

• Based on electron source• lithotrophslithotrophs use reduced inorganic substances

• organotrophsorganotrophs obtain electrons from organic compounds

Nutritional Resource ManagementNutritional 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 CellUptake 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 Passive Diffusion (simple 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 restrictedPassive diffusion is restricted

Substance Rate of intake

Water 100

Glycerol 0.1

Tryptophan 0.001

Glucose 0.001

Chloride ion 0.000001

Sodium ions 0.0000001

Facilitated DiffusionFacilitated 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

• 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

Facilitated Diffusion …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

Passive and Facilitated DiffusionPassive and Facilitated Diffusion

Note conformational change of carrier

Facilitated diffusion…Facilitated diffusion…

Examples

Members of major intrinsic

proteins (MIP) that form porin

Aquaporin: Aquaporin: channels to

transport water

glycerol transport channel

Animation: Facilitated diffusion

Active TransportActive 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 transportersABC 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 gradientActive 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 antiportCoupled transport: Symport and antiport

• Na-Sugar symporter• Na/Ca antiporter• Na increases in cytoplasm• How to balance?• Coupled to Na/K pump

Group TranslocationGroup Translocation

• chemically modifies molecules as it is brought into cells

• PEP sugar PEP sugar pphosphohosphottransferase ransferase ssystem (PTS): ystem (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 translocationActive 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

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 MediaTypes of Culture Media

Physical NaturePhysical Nature CompositionComposition ApplicationApplication

LiquidDefined

(synthetic) Supportive

Semi-solid Complex Enriched

Solid Differential

Selective

Defined or Synthetic MediaDefined or Synthetic Media

• all components and their concentrations are known

Complex MediaComplex 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…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. coliS. enterica

Techniques: Isolation of Pure CulturesTechniques: Isolation of Pure Cultures

Pure culturePure culture

Isogenic population of cells

arising from a single cell

Isolation techniquesIsolation 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

dispense cells onto

medium in a Petri dish

sterilize spreader by

dipping into 70% alcohol

followed by flaming

spread cells across surface

incubate plate

Spread plate technique

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