Chapter 5 Microbial Nutrition

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

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.

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

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.

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.

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.

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

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

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

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

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.

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 .

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

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

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

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

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

Amino acids Proteins

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

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

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

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

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

Facilitated diffusion http://biocyc.org/ECOLI/new-image?type=ENZYME&object=GLPF-MONOMER. These proteins exist in plants, animals, fungi, bacteria, and protists.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

Synthetic or Defined 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

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

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

Beta Hemolysis

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

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

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** http://www.austin.cc.tx.us/microbugz/20msa.html  

Web References on Media   http://www.jlindquist.net/generalmicro/102diff.html - General Reference http://medic.med.uth.tmc.edu/path/macconk.htm - MacConkey Agar http://www.indstate.edu/thcme/micro/hemolys.html - Blood Agar

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)

Spread-plate technique 1. dispense cells onto medium in petri dish 4. spread cells across surface 2. - 3. sterilize spreader Figure 5.7

Streak plate technique inoculating loop Figure 5.8

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

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

Figure 5.9: pour-plate technique

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

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).

Figure 5.10a

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