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UNIVERSITY OF THE PHILIPPINES MANILA
COLLEGE OF ARTS AND SCIENCES
DEPARTMENT OF BIOLOGY
BIOLOGY 120 (General Microbiology)
Lecture 2.1 – Microbial Nutrition and Metabolism
By: Lani Manahan-Suyom, MSc
SS AY 2013-2014
Outline
1.0 Microbial Nutrition
1.1
Nutrition and Cell Chemistry
1.2
Nutrient Uptake1.3
Culture Media
2.0 Metabolism
Microorganisms require about ten elements in large quantities, because
they are used to construct carbohydrates, lipids, proteins, and nucleic acids.
Several other elements are needed in very small amounts and are parts of
enzymes and cofactors
Biochemical components of cells
Water: 80% of wet weight
Dry weight
o Protein 40-70%
o Nucleic Acid 13-34%
o
Lipid 9-15%o Also monomers, intermediates and inorganic ions
Macronutrients
Cells make proteins, nucleic acids and lipids
Macronutrients
o Macromolecules, metabolism
o CHON SP K Mg Fe
o Sources
Organic compounds
Inorganic salts
Micronutrients
Elements needed in trace quantities
o Co, Cu, Mn, Zn, V
o Enzymes
o Tap water
Most of them are part of enzymes
o Iron – cytochrome, carboxylases
Micronurients (trace elements) needed by microorganisms
Boron (B) Autoinducer for quorum sensing in bacteria also
found in some polketide antibiotics (creates biofilm) Chromium
(Cr)
Possible but not proven component for glucose
metabolism (necessary in mammals)
Cobalt (Co) Vitamin B12; transcarboxulase (only in propionic
acid bacteria)
Copper (Cu) In respiration, cytochrome oxidase; in
photosynthesis, plastocyanin, some superoxide
dismutases
Iron (Fe) Cytochromes; catalases; peroxidises; iron-sulfur
proteins; oxygenases; all nitrogenises
Manganese
(Mn)
Activator of many enzymes; component of certain
superoxide dismutases and of the water splitting
enzyme in oxygenis phototrophs (photosystem II)
Molybdenum
(Mo)
Certain flavin-containing enzymes; some
nitrogenises, nitrate reductases, sulphite oxidases,
DMSO-TMAO reductases; some formate
dehydrogenases
Nickel (Ni) Most hydrogenases; coenzymes F430 of
methanogens; carbon monoxide dehydrogenases;
urease
Selenium
(Se)
Dormate dehydrogenase; some hydrogenases; the
amino acid selenocysteine
Tungsten
(W)
Some formate dehydrogenases; oxotransferases of
hyperthermophiles
Vanadium(V) Vanadium nitrogenises; bromoperoxidase
Zinc (Zn) Carbonic anhydrase; alcohol dehydrogenase; RNA
and DNA polymerases and many DNA binding
proteins
A not every micronutrient listed is required by all cells some metals
listed are found in enzymes or cofactors present in only specific
microorganisms
B needed in greater amounts than other trace metals
Growth Factors
Biotin – Carboxylation (Leuconostoc)
Cyanocobalamin or Vit B12 – Molecular rearrangements (Euglena)
Folic Acid – one carbon metabolism (Enterococcus) Pantothenic Acid – fatty acid metabolism (Proteus)
Pyridoxine or Vit B6 – transamination (Lactobacillus)
Niacin – precursor of NAD and NADP (Brucella)
Riboflavin or Vit B2 – precursor of FAD and FMN (Caulobacter)
Thiamine or Vit B1 – aldehyde group transfer (Bacillus anthracis)
Groups of microorganisms: nutritional requirement
Energy Sources
Phototrophs Light
Chemotrophs Oxidation of organic/inorganic
compounds
Hydrogen and Electron Sources
Lithotrophs Reduced inorganic molecules
Organotrophs Organic molecules
Carbon Sources
Autotrophs CO2 sole or principal
biosynthetic carbon source
Heterotrophs Reduced, preformed, organic
molecules from other organisms
Major Nutritional
Types
Sources of Energy,
Hydrogen/Electrons
and Carbon
Representative
Microorganisms
Photolithotrophic
Autotrophy
Light energy
Inorganic
hydrogen/elect
ron donor CO2 carbon
source
Algae
Purple and
green sulphur
bacteria Blue-green
algae
(cyanobacteria)
Photoorganotrophic
Heterotrophy
Light energy
Organic
hydrogen/elect
ron donor
Organic carbon
source (CO2
may also be
used)
Purple non-
sulfur bacteria
Green non-
sulfur bacteria
Chemolithotrophic
Autotrophy
Chemical
energy source
(inorganic)
Inorganic
hydrogen/elect
ron donor
CO2 carbon
source
Sulfur oxidizing
bacteria
Hydrogenbacteria
Nitrifying
bacteria
Iron bacteria
Chemoorganotrophic
Heterotrophy
Chemical
energy source
(organic)
Organic
hydrogen/elect
ron donor
Organic carbon
source
Protozoa
Fungi
Most non-
photosynthetic
bacteria
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Chemoorganotrophs are by definition heterotrophs.
Most chemolithotrophs and phototrophs are autotrophs.
Autotrophs are sometimes called primary producers
o they synthesize new organic matter from CO2
Virtually all organic matter on Earth has been synthesized by primary
producers, in particular, the phototrophs
Properties of Microbial Photosynthetic Systems
Property Cyanobacteria Green and Purple
Bacteria
Purple Nonsulfur
Bacteria
Photopigment Chlorophyll Bacteriochlorophyll Bacteriochlorophyll
O2
Production
Yes No No
Electron
Donors
H2O H2, H2S, S H2, H2S, S
Carbon
Source
CO2 CO2 Organic/CO2
PrimaryProducts of
Energy
Conversion
ATP + NADPH ATP ATP
Chemoautotroph
Bacteria Electron donor Electron
acceptor
Production
Alcaligens and
Pseudomonas
sp.
H2 O2 H2O
Nitrobacter NO2 O2 NO3, H2O
Nitrosomonas NH4 O2 NO2, H2O
Desulfovibrio H2 SO4 H2O, H2S
Thiobacillusdenitrificans
S, H2S NO3 SO4, N2
Thiobacillus
ferooxidans
Fe2+
O2 Fe3+
, H2O
Nitrifying Bacteria
Uptake of Nutrients
Nutrient molecules frequently cannot cross selectively permeable
plasma membranes through passive diffusion and must be transported
by one of three major mechanisms involving the use of membrane
carrier proteins
1.
Phagocytosis – Protozoa
2. Permeability absorption – most microorganisms
Simple transport
o Passive transport or simple diffusion
o Facilitated diffusion*
Group translocation**
ABC transporter**
*could transport agains or with the concentration gradient
**active transports against concentration gradient
Passive Diffusion
Passive diffusion is the process in which molecules move from a region
of higher concentration to one of lower concentration as a result of
random thermal agitation. A few substances, such as glycerol, can cross
the plasma membrane by passive diffusion
Facilitated Diffusion
The rate of diffusion across selectively permeable membranes is greatly
increased by the use of carrier proteins, sometimes called permeases,
which are embedded in the plasma membrane. Since the diffusion
process is aided by a carrier, it is called facilitated diffusion. The rate of
facilitated diffusion increases with the concentration gradient much
more rapidly and at lower concentrations of the diffusing molecule
than that of passive diffusion
Can be against or with concentration gradient
A Model of Facilitated Diffusion
The membrane carrier can change conformation after binding an
external molecule and subsequently release the molecule on the cellinterior. It then returns to the outward oriented position and is ready
to bind another solute molecule
Because there is no energy input, molecules will continue to enter only
as long as their concentration is greater on the outside
Symport and Antiport
Active Transport
Active transport is the transport of solute molecules to higher
concentrations, or against a concentration gradient, with the use of
metabolic energy input
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Group Translocation
The best-known group translocation system is the
phosphoenolpyruvate: sugar phosphotransferase system (PTS), which
transports a variety of sugars into prokaryotic cells while
Simultaneously phosphorylating them using phosphoenolpyruvate
(PEP) as the phosphate donor
PEP + sugar (outside) pyruvate + sugar-P (inside)
Mechanism of the phosphotransferase system of E. Coli
o For glucose uptake, the system consists of five proteins:
Enzyme (Enz) I, Enzymes IIa, IIb, and IIc, and HPr. A phosphate
cascade occurs from phosphoenolpyruvate (PE-P) to Enzyme
IIc and the latter actually transports and phosphorylates the
sugar. Proteins HPr and Enz I are nonspecific and transportany sugar. The Enz II components are specific for each
particular sugar
ABC Transporter
ABC stands for ATP binding cassette
ABC transporters exist for the uptake of organic compounds (sugars
and amino acids, inorganic nutrients such as sulfate and phosphate,
and trace metals)
Proteins involved
o Periplasmic binding protein – high affinity to substrate even
at low concentration (less than 1 micromolar)
o Membrane spanning transporter – forms the transport
channel
o ATP hydrolyzing protein – supply energy
Has high affinity for to the molecule it needs to transport (even ifmolecule level is really low)
Simple Comparison of Transport Systems
Culture Media – nutrient solutions used to grow microorganisms
mostly for chemotroph organism
Despite the fact that microbiologists have been growing
microorganisms in laboratory cultures for over 125 years, most
microorganisms in nature have yet to be cultured, leaving many
challenges to the microbiologist today
Major Classes of Media
Defined
o Precise amounts of highly purified inorganic or organic
chemicals
o Exact composition (in both qualitative and quantitative sense)
is known
Complex
o Employ digests of microbial, animal or plant products, such as
casein (milk protein), beef (beef extract), soybeans (tryptic
soy broth), yeast cells (yeast extract), or any of a number of
other highly nutritious yet impure substances
Other Classification of Media
According to use
o Enriched
o Selective
o Differential
o General purpose
Fluidity
o Solid
o Semi solid
o Liquid
Development of Solid Medium
Before agar
o
Liquid medium
Potato slices
o Robert Koch (1881) used boiled potato, sliced
o Not all bacteria grew well
Gelatin
o Frederick Loeffler
o Meat extract medium + gelatine
o But gelatine liquid at RT
Agar
o Fannie Hesse (1882)
o Agar used for jams and jelly
o Generally not metabolized by microbes
o Liquefies at 100C, solidifies at 40C
Anaerobic Condition for Growth
Reducing media
o Contain chemicals (thioglycollate of oxyrase) that combine O2
o Heated to drive off or use up O2
Anaerobic Jar
Types of Anaerobic Bacteria
Facultative anaerobes
Obligate anaerobes
Microaerophyllic anaerobes – certain amount of oxygen tolerated
Anaerobic Chambers/Incubators
Anaerobic Jars
o
with anaerobic pack (releases CO2 and H and produceswater)
o gas generators
o candle jar – usually more effective in microaerophyllic (flame
uses up all the oxygen and flame dies)
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An Overview of Metabolism
Metabolism
total of all chemical reactions occurring in the cell
A simplified view of cell metabolism depicts how catabolic degradative
reactions supply energy needed for cell functions and how anabolic
reactions bring about the synthesis of cell components from nutrients
Note that in anabolism, nutrients from the environment or those
generated from catabolic reactions are converted to cell components,
whereas in catabolism, energy sources from the environment are
converted to waste products
Enzymes
Biological catalyst
o Lowers activation energy of a reaction
Enzymes are generally much larger than the substrates
Present in small amount because they are reusable
Active site – the portion of the enzyme to which the substrate binds
Catabolism
Fermentation
o Anaerobic catabolism
o Organic compound is both an electron donor and an electron
acceptor
o ATP is produced by substrate level phosphorylation
Respirationo Catabolism in which a compound is oxidized
o O2 (or an O2 substitute) as the terminal electron acceptor
o Usually accompanied by ATP production by oxidative
phosphorylation
more ATP is produced in respiration than in fermentation and thus
respiration is the preferred choice. But many microbial habitats lack O2
and in such habitats, fermentation is the only option for energy
conservation by chemoorganotrophs
Glycolysis (Embden-Meyerhof-Parnas pathway)
Stage 1 – preparatory reactions
o These are not redox reactions and do not release energy
o Lead to the production of a key intermediate of the pathway
Stage 2 – production of NADH, ATP and Pyruvate
o Redox reactions
o Energy is conserved in the form of ATP, and two molecules of
pyruvate are formed
o Redox balance has not yet been achieved
o Pyruvate formed here
Stage 3 – consumption of NADH and production of Fermentation
products
o
Redox reactions occur once againo Fermentation products are formed
Kreb Cycle (Citric Acid Cycle)
NADH – produces 2-3 ATPs
Respiration and Electron Transport
Respiration in which molecular oxygen or some other oxidant serves as
the terminal electron acceptor
The discussion of respiration deals with both the carbon and electron
transformations
o (1) The biochemical pathways involved in the transformation
of organic carbon to CO2
o (2) The way electrons are transferred from the organic
compound to the terminal electron acceptor, driving ATP
synthesis at the expense of the proton motive force
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Electron Transport
Electron transport systems are composed of membrane associated
electron carriers. These systems have two basic functions
o (1) to accept electrons from an electron donor and transfer
them to an electron acceptor
o (2) to conserve some of the energy released during electron
transfer for synthesis of ATP
In bacteria – ETC happens in plasma membrane not mitoch. membrane
Types of Oxidation-reduction enzymes involved in electron transport
1. NADH dehydrogenases
2.
Riboflavin containing electron carriers generally called flavoproteins3. Iron sulphur proteins
4. Cytochromes
*in addition, one class of nonprotein electron carriers is known, the lipid
soluble quinines
ATP and Cell Yield
The amount of ATP produced by an organism has a direct effect on cell
yield
Cell yield is directly proportional to the amount of ATP produced
Energy costs for assembly of macromolecules are much the same for all
microorganisms
Production of ATP is specific to a strain which is related to its cell yield
Energy Storage
Most microorganisms produce insoluble polymers that can later be
oxidized for the production of ATP
Importance of polymer formation
o Potential energy is stored in a stable form
o Insoluble polymers have little effect on the internal osmotic
pressure of cells
*Storage polymers make possible the storage of energy in a readily
accessible form that does not interfere with other cellular processes
The Proton Motive Force
Carriers in the Electron Transport Chain (ETC) are
o Arranged in increasing positive reduction potential
o
Oriented in such a way that as e- are transported, protons are
separated from e-
o The final donating the electrons plus protons to a terminal
electron acceptor such as O2
o H+ are extruded to the outer surface of the membrane
o H+ originate from two sources
NADH
Dissociation of water into H and OH2??
o The extrusion of H+ to the environment results in the
accumulation of OH2 on the inside of the membrane
o As a result the two sides of the membrane differ in both
charge and pH
o Formation of an electrochemical potential across the
membrane = PMF
ATP Synthase (ATPase)
The use of PMF to generate ATP
Catalyzed a reversible reaction between ATP and ADP + P
Two components
o F1 complex – carries out the chemical functions
o F0 complex – ion translocating functions
Composition
o F1 complex - α3β 3γδε
o F0 complex - a,b2, c12
F0 generates the torque that is transmitted to F1
Assignment: Read Similarities of the ATP Synthesis via ATPase and
flagellar locomotion
Anabolism: biosynthesis
Sugar
Amino Acids
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Fatty Acids
Regulation of the Activity of Enzymes: Feedback Inhibition
Reaction shut off because of an excess of the end product
Allosteric enzymes has 2 binding sites, the active site (where substrate
binds) and the allosteric site, where the end product of the pathwaybinds
Allosteric Site
With 2 protein binding sites
Without end product in the environment - active site can accept a
substrate, so chemical production will proceed
If end product is in excess – it will bind to the allosteric site and prevent
substrate from binding to active site
Regulation of the Activity of Enzymes: Covalent Modification
A) when cells are grown with excess ammonia, glutamine synthetase is
covalently modified by adenylylation as many as 12 AMP groups can be
added. When cells are ammonia-limited, the groups are removed,
forming ADP
B) Adenylylated GS subunits are catalytically inactive so the overall GS
activity decreases progressively as more subunits are adenylylated.
If end product is present, it will bind to the enzyme but when it binds,
the enzyme activity will just go down
The more product that binds, the less active the enzyme activity will be
Activity of enzyme not totally shut down