Chapter 5Chapter 5

Microbial NutritionMicrobial Nutrition

The Common Nutrient The Common Nutrient RequirementsRequirements

MacroelementsMacroelements (macronutrients) (macronutrients)– C, O, H, N, S, P, K, Ca, Mg, and FeC, O, H, N, S, P, K, Ca, Mg, and Fe– required in relatively large amountsrequired in relatively large amounts

MicronutrientsMicronutrients ( (trace elementstrace elements))– Mn, Zn, Co, Mo, Ni, and CuMn, Zn, Co, Mo, Ni, and Cu– required in trace amountsrequired in trace amounts– often supplied in water or in media often supplied in water or in media

componentscomponents

Autotroph and HeterotrophAutotroph and Heterotroph

All organisms require Carbon, Hydrogen, and All organisms require Carbon, Hydrogen, and Oxygen. Carbon is needed for the backbone of all Oxygen. Carbon is needed for the backbone of all organic molecules.organic molecules.

In addition all organisms require a source of In addition all organisms require a source of electrons. Electrons are involved in oxidation-electrons. Electrons are involved in oxidation-reduction reactions in the cell, electron transport reduction reactions in the cell, electron transport chains, and pumps that drive molecules against a chains, and pumps that drive molecules against a concentration gradient on cell membranes.concentration gradient on cell membranes.

Organic molecules that supply, carbon, hydrogen, Organic molecules that supply, carbon, hydrogen, and oxygen are reduced and donate electrons for and oxygen are reduced and donate electrons for biosynthesis.biosynthesis.

Requirements for Carbon, Requirements for Carbon, Hydrogen, and OxygenHydrogen, and Oxygen

often satisfied togetheroften satisfied together– carbon source often provides H, O and carbon source often provides H, O and

electronselectrons

autotrophsautotrophs– use carbon dioxide as their sole or principal use carbon dioxide as their sole or principal

carbon sourcecarbon source

heterotrophsheterotrophs– use organic molecules as carbon sourcesuse organic molecules as carbon sources

AutotrophsAutotrophs

CO2 is used by many microorganisms CO2 is used by many microorganisms as the source of Carbon. as the source of Carbon.

AutotrophsAutotrophs have the capacity to reduce have the capacity to reduce it , to form organic molecules. it , to form organic molecules.

Photosynthetic bacteria are Photosynthetic bacteria are PhotoautotrophsPhotoautotrophs that are able to fix that are able to fix CO2 and use light as their energy CO2 and use light as their energy source.source.

HeterotrophsHeterotrophs

Organisms that use organic molecules as their Organisms that use organic molecules as their source of carbon are source of carbon are Heterotrophs. Heterotrophs. The most The most common heterotrophs use organic common heterotrophs use organic compounds for both energy and their source compounds for both energy and their source of carbon.of carbon.

Microorganisms are versatile in their ability to Microorganisms are versatile in their ability to use diverse sources of carbon. use diverse sources of carbon. Burkholderia Burkholderia cepaciacepacia can use over 100 different carbon can use over 100 different carbon compounds. compounds. Methylotrophic bacteria utilize methanol, Methylotrophic bacteria utilize methanol, methane, and formic acid.methane, and formic acid.

Comparison of Nutritional Comparison of Nutritional ModesModes

Major Nutritional Types of Microorganisms

Major Nutritional Type Source of Energy, Hydrogen, electrons, and Carbon

Representative microorganisms

Photolithotrophic autotrophy

Light energyInorganic hydrogen/electron( H+/e-) donorCO2 carbon source

AlgaePurple and green sulfur bacteriaCyanobacteria

Photoorganotrophic heterotrophy

Light energyOrganic H+/e- donorOrganic carbon source or CO2

Purple and Green non-sulfur bacteria

Chemolithotrophic autotrophy

Chemical energy source – inorganicOrganic H+/e- donorCO2 carbon source

Sulfur oxidizing bacteriaNitrifying bacteriaIron oxidizing bacteria

Chemoorganotrophic heterotrophy

Chemical energy source( organic)Organic( H+/e-) donorOrganic carbon source

Most non photosynthetic bacteria including pathogens. ProtozoansFungi

Study table adapted from Microbiology, Prescott, Chapter Five.

Photolithotrophic autotrophsPhotolithotrophic autotrophs

Use light energy and have CO2 as their carbon Use light energy and have CO2 as their carbon source.source.

Cyanobacteria uses water as the electron donor Cyanobacteria uses water as the electron donor and release oxygenand release oxygen

Purple and green sulfur bacteria use inorganic Purple and green sulfur bacteria use inorganic donors like hydrogen and hydrogen sulfide for donors like hydrogen and hydrogen sulfide for electronselectrons

Chemoorganotrophic heterotrophsChemoorganotrophic heterotrophs

Use organic compounds as sources of Use organic compounds as sources of energy,hydrogen, electrons and carbonenergy,hydrogen, electrons and carbon

Pathogenic organisms fall under this category of Pathogenic organisms fall under this category of nutritionnutrition

PhotoorganoheterotrophsPhotoorganoheterotrophs

Common inhabitants of polluted streams. These Common inhabitants of polluted streams. These bacteria use organic matter as their electron bacteria use organic matter as their electron donor and carbon source.donor and carbon source.

They use light as their source of energyThey use light as their source of energy

Important ecological organismsImportant ecological organisms

Chemolithotrophic autotrophsChemolithotrophic autotrophs

AutotrophsAutotrophs

Oxidize reduce inorganic compounds such as iron, Oxidize reduce inorganic compounds such as iron, nitrogen, or sulfur molecules nitrogen, or sulfur molecules

Derive energy and electrons for biosynthesisDerive energy and electrons for biosynthesis

Carbon dioxide is the carbon sourceCarbon dioxide is the carbon source

Requirements for NitrogenRequirements for Nitrogen

Nitrogen is required for the synthesis of amino acids that Nitrogen is required for the synthesis of amino acids that compose the structure of proteins, purines and pyrimidines the compose the structure of proteins, purines and pyrimidines the bases of both DNA and RNA, and for other derivative molecules bases of both DNA and RNA, and for other derivative molecules such as glucosamine.such as glucosamine.

Many microorganisms can use the nitrogen directly from amino Many microorganisms can use the nitrogen directly from amino acids. The amino group ( NH2) is derived from ammonia acids. The amino group ( NH2) is derived from ammonia through the action of enzymes such as glutamate through the action of enzymes such as glutamate dehydrogenase.dehydrogenase.

Most photoautotrophs and many nonphotosynthetic Most photoautotrophs and many nonphotosynthetic microorganisms reduce nitrate to ammonia and assimilate microorganisms reduce nitrate to ammonia and assimilate nitrogen through nitrate reduction. A variety of bacteria are nitrogen through nitrate reduction. A variety of bacteria are involved in the nitrogen cycle such as involved in the nitrogen cycle such as RhizobiumRhizobium which is able which is able to use atmospheric nitrogen and convert it to ammonia. to use atmospheric nitrogen and convert it to ammonia. ( Found on the roots of legumes like soy beans and clover) ( Found on the roots of legumes like soy beans and clover) These compounds are vital for the Nitrogen cycle and the These compounds are vital for the Nitrogen cycle and the incorporation of nitrogen into plants to make nitrogen incorporation of nitrogen into plants to make nitrogen comounds.comounds.

PhosphorousPhosphorous

Phosphorous is present in Phosphorous is present in phospholipids( membranes), Nucleic acids( phospholipids( membranes), Nucleic acids( DNA and RNA), coenzymes, ATP, some DNA and RNA), coenzymes, ATP, some proteins, and other key cellular proteins, and other key cellular components. components.

Inorganic phosphorous is derived from the Inorganic phosphorous is derived from the environment in the form of phosphates. environment in the form of phosphates. Some microbes such as Some microbes such as E. coliE. coli can use can use organophosphates such as hexose – 6-organophosphates such as hexose – 6-phosphates . phosphates .

MixotrophyMixotrophy

Chemical energy – source organicChemical energy – source organic

Inorganic H/e- donorInorganic H/e- donor

Organic carbon sourceOrganic carbon source

Requirements for Requirements for Nitrogen, Phosphorus, and Nitrogen, Phosphorus, and

SulfurSulfurNeeded for synthesis of important Needed for synthesis of important molecules (e.g., amino acids, nucleic molecules (e.g., amino acids, nucleic acids)acids)Nitrogen supplied in numerous waysNitrogen supplied in numerous waysPhosphorus usually supplied as inorganic Phosphorus usually supplied as inorganic phosphatephosphateSulfur usually supplied as sulfate via Sulfur usually supplied as sulfate via assimilatory sulfate reductionassimilatory sulfate reduction

Sources of nitrogenSources of nitrogen

organic moleculesorganic molecules

ammoniaammonia

nitrate via assimilatory nitrate nitrate via assimilatory nitrate reductionreduction

nitrogen gas via nitrogen fixationnitrogen gas via nitrogen fixation

Growth FactorsGrowth Factors

organic compoundsorganic compounds

essential cell components (or their essential cell components (or their precursors) that the cell cannot precursors) that the cell cannot synthesizesynthesize

must be supplied by environment if must be supplied by environment if cell is to survive and reproducecell is to survive and reproduce

Classes of growth factorsClasses of growth factors

amino acidsamino acids– needed for protein synthesisneeded for protein synthesis

purines and pyrimidinespurines and pyrimidines– needed for nucleic acid synthesisneeded for nucleic acid synthesis

vitaminsvitamins– function as enzyme cofactorsfunction as enzyme cofactors

Amino acids

Proteins

Bases of nucleic acidsBases of nucleic acids

Adenine and guanine Adenine and guanine are purinesare purines

Cytosine, thymine, Cytosine, thymine, and uracil are and uracil are pyrimidinespyrimidines

Also found in energy Also found in energy triphosphates( ATP triphosphates( ATP and GTPand GTP))

Practical importance of Practical importance of growth factorsgrowth factorsdevelopment of quantitative growth-development of quantitative growth-response assays for measuring response assays for measuring concentrations of growth factors in a concentrations of growth factors in a preparationpreparation

industrial production of growth industrial production of growth factors by microorganisms factors by microorganisms

Uptake of Nutrients Uptake of Nutrients by the Cellby the Cell

Some nutrients enter by Some nutrients enter by passive passive diffusiondiffusion

Most nutrients enter by:Most nutrients enter by:– facilitated diffusionfacilitated diffusion– active transportactive transport– group translocationgroup translocation

Passive DiffusionPassive Diffusion

molecules move from region of molecules move from region of higher concentration to one of lower higher concentration to one of lower concentration because of random concentration because of random thermal agitationthermal agitation

HH22O, OO, O22 and CO and CO22 often move across often move across membranes this waymembranes this way

Facilitated DiffusionFacilitated Diffusion

Similar to Similar to passive diffusionpassive diffusion– movement of molecules movement of molecules is notis not energy energy

dependentdependent– direction of movement is from high direction of movement is from high

concentration to low concentration. concentration to low concentration. With the concentration gradientWith the concentration gradient

– size of concentration gradient impacts size of concentration gradient impacts rate of uptakerate of uptake

Facilitated diffusionFacilitated diffusion

http://biocyc.org/ECOLI/new-image?type

=ENZYME&object=GLPF-MONOMER. These proteins exist in plants, animals, fungi, bacteria, and protists.

Facilitated diffusion…Facilitated diffusion…

Differs from passive diffusion Differs from passive diffusion – uses carrier molecules (uses carrier molecules (permeasespermeases))– smaller concentration gradient is required for smaller concentration gradient is required for

significant uptake of moleculessignificant uptake of molecules– effectively transports glycerol, sugars, and effectively transports glycerol, sugars, and

amino acidsamino acids

more prominent in eucaryotic cells than in more prominent in eucaryotic cells than in procaryotic cellsprocaryotic cells

Figure 5.1

•rate of facilitateddiffusion increasesmore rapidly andat a lowerconcentration

•diffusion ratereaches a plateau when carrier becomessaturated

carrier saturationeffect

Figure 5.2

note conformational changeof carrier

Active TransportActive Transport

energy-dependent processenergy-dependent process– ATP or proton motive force usedATP or proton motive force used

moves molecules against the moves molecules against the gradientgradient

concentrates molecules inside cellconcentrates molecules inside cell

involves carrier proteins (permeases)involves carrier proteins (permeases)– carrier saturation effect is observedcarrier saturation effect is observed

TransportersTransporters

“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 transportersABC transporters

ATP-binding ATP-binding cassette cassette transporterstransporters

observed in observed in bacteria, bacteria, archaea, and archaea, and eucaryoteseucaryotes

Figure 5.3

Figure 5.4

antiport

symport

Group TranslocationGroup Translocationmolecules are molecules are modified as modified as they are they are transported transported across the across the membranemembrane

energy-energy-dependent dependent processprocess

Figure 5.5

Fe uptake in pathogensFe uptake in pathogens

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

Iron and signallingIron and signalling

Iron is also used by pathogenic bacteria as Iron is also used by pathogenic bacteria as a signal molecule for the regulation of a signal molecule for the regulation of virulence gene expression. This sensory virulence gene expression. This sensory system is based on the marked differences system is based on the marked differences in free iron concentrations between the in free iron concentrations between the environment and intestinal lumen (high) environment and intestinal lumen (high) and host tissues (low)and host tissues (low)

ListeriaListeria Pathogenesis and Molecular Virulence Determinants Pathogenesis and Molecular Virulence Determinants

José A. Vázquez-Boland,1,2* Michael Kuhn,3 Patrick Berche,4 Trinad Chakraborty,5 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 Gustavo Domínguez-Bernal,1 Werner Goebel,3 Bruno González-Zorn,1 Jürgen Wehland,6 and Jürgen Kreft3Wehland,6 and Jürgen Kreft3

Pathogens and Iron uptakePathogens and Iron uptake

Burkholderia cepaciaBurkholderia cepacia

Campylobacter jejuniCampylobacter jejuni

Pseudomonas aeruginosaPseudomonas aeruginosa

E. coliE. coli

Listeria monocytogenesListeria monocytogenes

Iron UptakeIron Uptakeferric iron is very ferric iron is very insoluble so uptake insoluble so uptake is difficultis difficultmicroorganisms use microorganisms use siderophores to aid siderophores to aid uptakeuptakesiderophore siderophore complexes with ferric complexes with ferric ionioncomplex is then complex is then transported into celltransported into cell

Figure 5.6

ListeriosisListeriosis

One involves the direct transport of One involves the direct transport of ferric citrate to the bacterial cell ferric citrate to the bacterial cell

Another system involves an Another system involves an extracellular ferric iron reductase, extracellular ferric iron reductase, which uses siderophores which uses siderophores

The third system may involve a The third system may involve a bacterial cell surface-located bacterial cell surface-located transferrin-binding proteintransferrin-binding protein

Iron bacteria in the Iron bacteria in the environmentenvironment

There are several non-disease producing There are several non-disease producing bacteria which grow and multiply in water bacteria which grow and multiply in water and use dissolved iron as part of their and use dissolved iron as part of their metabolism. They oxidize iron into its metabolism. They oxidize iron into its insoluble ferric state and deposit it in the insoluble ferric state and deposit it in the slimy gelatinous material which surrounds slimy gelatinous material which surrounds their cells. their cells. These filamentous bacteria grow in stringy These filamentous bacteria grow in stringy clumps and are found in most iron-bearing clumps and are found in most iron-bearing surface waters. They have been known to surface waters. They have been known to proliferate in waters containing iron as low proliferate in waters containing iron as low as 0.1 mg/l. as 0.1 mg/l.

Culture MediaCulture Media

preparations devised to support preparations devised to support the growth (reproduction) of the growth (reproduction) of microorganismsmicroorganisms

can be liquid or solidcan be liquid or solid– solid media are usually solidified with solid media are usually solidified with

agaragar

important to study of important to study of microorganismsmicroorganisms

Synthetic or Defined MediaSynthetic or Defined Media

all all components components and their and their concentrations concentrations are knownare known

Complex MediaComplex Media

contain some contain some ingredients of ingredients of unknown unknown composition composition and/or and/or concentrationconcentration

Some media componentsSome media components

peptonespeptones– protein hydrolysates prepared by partial protein hydrolysates prepared by partial

digestion of various protein sourcesdigestion of various protein sources

extractsextracts– aqueous extracts, usually of beef or yeastaqueous extracts, usually of beef or yeast

agaragar– sulfated polysaccharide used to solidify liquid sulfated polysaccharide used to solidify liquid

mediamedia

Types of MediaTypes of Media

general purpose mediageneral purpose media– support the growth of many microorganismssupport the growth of many microorganisms– e.g., tryptic soy agare.g., tryptic soy agar

enriched mediaenriched media– general purpose media supplemented by blood general purpose media supplemented by blood

or other special nutrientsor other special nutrients– e.g., blood agare.g., blood agar

Types of media…Types of media…

Selective mediaSelective media– Favor the growth of some Favor the growth of some

microorganisms and inhibit growth of microorganisms and inhibit growth of othersothers

– MacConkey agarMacConkey agarselects for gram-negative bacteriaselects for gram-negative bacteria

Inhibits the growth of gram-positive bacteriaInhibits the growth of gram-positive bacteria

Beta Hemolysis

Types of media…Types of media…

Differential mediaDifferential media– Distinguish between different groups Distinguish between different groups

of microorganisms based on their of microorganisms based on their biological characteristicsbiological characteristics

– Blood agarBlood agarhemolytic versus nonhemolytic bacteriahemolytic versus nonhemolytic bacteria

– MacConkey agarMacConkey agarlactose fermenters versus nonfermenterslactose fermenters versus nonfermenters

Selective and differential media

Selects for Gram –

Differentiates between bacteria based upon fermentation of lactose( color change)

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 The Spread Plate and Streak PlateStreak Plate

Involve spreading a mixture of cells Involve spreading a mixture of cells on an agar surface so that individual on an agar surface so that individual cells are well separated from each cells are well separated from each otherother

Each cell can reproduce to form a Each cell can reproduce to form a separate separate colonycolony (visible growth or (visible growth or cluster of microorganisms)cluster of microorganisms)

Figure 5.7

1. dispense cells ontomedium in petri dish

2. - 3. sterilize spreader

4. spread cellsacross surface

Spread-plate techniqueSpread-plate technique

Figure 5.8

inoculatingloop

Streak plate techniqueStreak plate technique

Isolation of Pure CulturesIsolation of Pure Cultures

Pure culturePure culture– population of cells arising from a single population of cells arising from a single

cellcell

Spread plateSpread plate, , streak platestreak plate, and , and pour pour plateplate are techniques used to isolate are techniques used to isolate pure culturespure cultures

The Pour PlateThe Pour Plate

Sample is diluted several timesSample is diluted several times

Diluted samples are mixed with liquid Diluted samples are mixed with liquid agaragar

Mixture of cells and agar are poured Mixture of cells and agar are poured into sterile culture dishesinto sterile culture dishes

Figure 5.9

Colony Morphology and GrowthColony Morphology and Growthindividual individual species form species form characteristic characteristic coloniescolonies

Figure 5.10b

Terms1. Colony shape and size: round, irregular, punctiform (tiny)2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed)3. Elevation: convex, umbonate, flat, raised4. Color: color or pigment, plus opaque, translucent, shiny or dull5. Texture: moist, mucoid, dry (or rough).

Figure 5.10a

Colony growthColony growth

Most rapid at edge of colonyMost rapid at edge of colony– oxygen and nutrients are more available oxygen and nutrients are more available

at edgeat edge

Slowest at center of colonySlowest at center of colony

In nature, many microorganisms In nature, many microorganisms form biofilms on surfacesform biofilms on surfaces