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Microbial Nutrition I. The Common Nutrient Requirements of Microbial Cells >95% of dry weight of bacterial cells is made up of 10 major components carbohydrates, nucleic acids proteins, lipids, – Carbon (C) – Oxygen (O) – Hydrogen (H) – Nitrogen (N) – Sulfur (S) – Phosphorous (P) mg/l – enzyme activity, heatresistance of spores, co-factors, cytochrome components – Potassim (K) – Calcium (Ca) – Magnesium (Mg) – Iron (fe) Minor components mcg/l (mg/l) – enzyme activity, co-factors, nitrogen fixation, vitamin components – – – – – – – Manganese (Mn) Zinc (Zn) Cobalt (Co) Molybdenum (Mo) Nickel (Ni) Copper (Cu) Others (B, Se, …) Usually enough in water sources to satisfy requirements Specialized Requirements Silica – Diatoms need silicic acid for silica walls (H4SiO4) High sodium concentrations – Halophiles II. Requirements for Carbon, Hydrogen, and Oxygen-often satisfied together Terms and Definitions: Categorization based on nutritional requirements Carbon Source Autotroph CO2 = principle carbon source Includes photosynthetic bacteria and those capable of oxidizing inorganic material for energy generation Heterotroph Utilize more reduced and complex carbon sources derived from other organisms (“nourished by others”) Organic compounds used to provide carbon Prototroph Utilizes same components as most members of the same species Auxotroph Mutated microbe that has lost the ability to synthesize critical precursors Must have nutritional precursors supplied Energy Source Phototroph Light energy harvested by photosynthetic processes Carbon from CO2 Chemotroph Organic or inorganic compounds provide energy by oxidative processes Hydrogen or Electron Source Lithotrophs Use reduced inorganic compounds as electron source “Rock eaters” Organotrophs Use organic compounds as H and electron donors III. Major Nutritional Microorganism Types Photolithhotrophic autotrophs (aka photautotrophs) Carbon and energy source: – CO2 – Light energy H/e- source = inorganic donor – e.g. H2O, hydrogen, H2S and elemental sulfur Examples – Algae (eukaryotic) – Cyanobacteria – Purple and green sulfur bacteria Photoorganotroic heterotrophs Carbon and energy source – CO2 and organic compounds – Light energy H/e- source – Organic donor Examples – Purple non-sulfur bacteria – Green non-sulfur bacteria Chemolithotrophic autotrophs (aka chemoautotrophs) Carbon and energy source – CO2 – Inorganic compounds – (a few chemolithotrophs get carbon from organic sources = chemolithotrophic heterotrophs = mixotrophic – inorganic energy, organic carbon) H/e- source – Oxidation of inorganic compounds H2S, S, NO2, H2, Fe2+ Examples – Sulfur oxidizers (Thiobacillus) – Hydrogen bacteria – Nitrifying bacteria (nitrites, ammonia) (Nitrobacter, Nitrosomonas) – Iron bacteria (Siderocapsa) Play major role in ecological transformation of compounds (ammonia to nitrate; sulfur to sulfate – NH3 NO3– S• SO42- Chemoorganotrophic heterotroph (aka chemoheterotrophs) Carbon and energy source – Organic H/e- source – Organic donor Examples – – – – Protozoa Fungi Most non-photosynthetic bacteria Most pathogens (medically important bacteria = chemoheterotrophs) IV. Nitrogen, Phosphorous and Sulfur are needed for the basic building blocks of cells Nitrogen Amino acids Nucleic acids (purines and pyrimidines) Some carbohydrates and lipids Enzyme co-factors Phosphorous ATP Co-factors Nucleic acids (phosphodiester bonds) Phospholipids (lipid bilayer) Some proteins Sulfur S-containing amino acids Some carbohydrates Thiamine Biotin Growth Factors Organic compounds required by microorganisms for growth and NOT synthesized by that mircoorganism Obtain compounds or their precursors from the external environment Three major classes – Amino acids, purines/pyrimidines, vitamins Minor classes – Heme (H. influenzae), cholesterol (some Mycoplasma) Nutrient Uptake – Specific Mechanimsms Utilizing Selective Permeability VI. Facilitated Diffusion Requires large concentration gradient for efficient transport Differs from passive diffusion which utilizes osmosis to achieve transfer of small substances (glycerol, H2O, O2, CO2) Facilitative diffusion employs carrier proteins called permeases to transfer components selectively across the PM No metabolic energy needed Works effectively even in low concentration gradients Requires concentration gradient to facilitate uptake – Equilibrium will be established – But substance is NOT accumulated against a gradient Probably involves a conformational change of carrier to deliver components across the lipid bilayer – Therefore effective for lipid-insoluble material Not utilized much by bacteria but it does occur (e.g glycerol uptake by E. coli) Active Transport – Transport of molecules AGAINST a concentration gradient Material is more concentrated on the inside of the cell than on the outside Ability to concentrate solutes in dilute environments – Metabolic energy required ATP hydrolysis or Proton motive forces (proton gradients generated by electron transport) – Carrier proteins utilized energy dependent in PM (ATP) Membrane-bouond Multi-subunit Form a pore AKA permeases May associate with substrate binding proteins in the periplasmic space of Gram-negative bacteria where substrate is handed over for entry across PM (e.g. arabinose, lactose, maltose, galactose, robose, glutamate, histidine, leucine) Types of active transport – Symport is the linked transport of two substances in the same direction – Antiport is the linked transport of two substances in opposite directions Group Translocation – Transfer of solutes coupled with chemical modification – Example: Phosphoenolpyruvate (PEP): Sugar phosphotransferase system (PTS) – Sugars are transported ad phosphorylated using PEP as the phosphate donor – Glucose, fructose, mannitol, sucrose, N-acetyl glucosamine, cellobiose, and other solutesn – PTS proteins cann also serve as chemoreceptors in chemotaxis Iron uptake – Ferric iron (Fe3+) is insoluble uner aerobic conditions – Bacteria must transport iron across PM to use in cytochromes and many enzymes – the organism secretes siderophores that complex with the very insoluble ferric ion, which is then transported into the cell – Siderophores = iron chelators Types of siderophores – Hydroxamates (e.g. ferrichrome used by fungi) – Catecholates (e.g. enterobactin used by E. coli) Iron handed off to the cell after siderophore binds to siderophore receptor protein on the microorganism VII. Types of Culture Media When media component are known = Defined Media (synthetic media) When exact composition of some components is not nown = Complex Media (enriched, artificial, crude) – Required by fastidious organisms Fastidious organisms are difficult to culture on ordinary media because of its need for secial nutritional factors (stringent physiological requirements for growth and survival) Complex media often contains blood or serum – Sometimes blood must be lysed (chocolate agar) to release hemin and NAD (e.g Haemohilus and Neisseria – which do not produce siderophores) Other undefined components: – Peptones (hydrolyzed protein) – Meat extracts or infusions (lean meat) – amino acids, peptides, nucleotdes, vitamins, mnerals and organic acids – Yeast extract (Brewer’s yeast – B vitamins, nitrogen and carbon compounds) Agar added if solid medium is required – Agar = complex polysaccharide from red algae General purpose media favors the growth of a variety of microbe types – Example: Tryptic soy broth – Can be enriched with blood components – Enriched media are supplemented by blood or other special nutrients to encourage the growth of fastidious heterotrophs Selective Media supports the growth of particular microorganisms while inhibiting the growth of others – Examples Bile salts and dyes – suppress Gram-positive bacteria while favoring the growht of Gramnegative bacteria Can select based on enzymes e.g. cellulose utilization requires cellulase Antibiotic resistance (plasmid-encoded, Rplasmid) Differential Media distinguished different bacterial groups – Examples: Blood agar – hemolysis (alpha, beta or gamma hemolysis) Eosin methylene blue agar (EMB) – used to identify lactose fermenters (colony turns dark purple) Some media can exhibit characteristics of more than one type – blood agar is enriched and differential, and distinguishes between hemolytic and nonhemolytic bacteria VIII. Culturing Techniques: Isolating Pure Cultures a population of cells arising from a single cell – can be accomplished from mixtures by a variety of procedures: – spread plates – streak plates – pour plates Spread plate – 100-200 bacterial cells are placed on the center of an agar surface and spread evenly with a glass rod – Every cell grows into a separate colony Each colony = pure culture – Useful for quantitative purposes Streak plate – Inoculating loop is used to streak cultures – Dilutions made with different streaks, flaming between streaks Pour plate – Diluted sample series is mixed with molten agar and poured immediately – Cells become embedded in the agar and on top forming discrete colonies Colonies are macroscopically visible growths or clusters of microorganisms on solid media Colony growth is most rapid at the colony's edge because oxygen and nutrients are more available; growth is slowest at the colony's center Colony morphology helps microbiologists identify bacteria because individual species often form colonies of characteristic size and appearance
