Transcript Chapter 5

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 oxidationreduction 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
Light energy
Organic H+/e- donor
Organic carbon source or CO2
Purple and Green non-sulfur
bacteria
Chemolithotrophic
autotrophy
Chemical energy source –
inorganic
Organic H+/e- donor
CO2 carbon source
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
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 – 6phosphates .
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 growthresponse 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/ECO
LI/newimage?type=ENZYM
E&object=GLPFMONOMER. 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
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
Figure 5.3
antiport
Figure 5.4
symport
Group Translocation
molecules are
modified as
they are
transported
across the
membrane
energydependent
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
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.
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
– 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
– 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)
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
Figure 5.7
4. spread cells
across surface
2. - 3. sterilize spreader
Streak plate technique
inoculating
loop
Figure 5.8
Isolation of Pure Cultures
Pure culture
– 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
Colony Morphology and Growth
individual
species form
characteristic
colonies
Figure 5.10b
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
– oxygen and nutrients are more available at
edge
Slowest at center of colony
In nature, many microorganisms form
biofilms on surfaces