Microbial Nutrition Bio3124 Lecture # 4

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

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 Rate of intake Water 100 Glycerol 0.1 Tryptophan 0.001 Glucose 0.001 Chloride ion 0.000001 Sodium ions 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 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

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 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

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

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 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

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

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 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)

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)

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 Composition Application Liquid Defined (synthetic) Supportive Semi-solid Complex Enriched Solid 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 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

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 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