Download Sept 19th Lecture 4

CSS 360
Lecture 4
Bacterial Diversity, Viruses, and
Introduction to Bacterial
Metabolism
Last Lecture
• We covered bacterial morphology, genetics, and
mechanisms of genetic exchange
• Any questions?
Outline
1.
2.
3.
4.
5.
6.
Discussion Questions from last week
Review Reading assignment from week 2; Condron et al.
Bacterial diversity
Viruses and their role in natural systems
Bacterial metabolism
Enzymes
I. Bacterial Diversity
Haeckel’s Tree of Life from
The Evolution of Man 1897
Anthropocentric
Bacteria
Bacterial Diversity
• Tree of Life circa 1997
• Tree of Life circa
2009
• Based on sequenced
genomes
• Based on the 16S rRNA gene sequence, there are now approximately 25 PHYLA of bacteria
• Aug 9, 2011 :: 1,921,179 16S rRNA sequences in the database
• 97% identity of 16S rRNA gene is considered to be the same species
• If this were applied to eukaryotes then all primates would be one species
• PhylumClassOrderFamilyGenusSpeciesStrain
8 Australian agricultural soils under different treatments
Clustered into groups within a 5% 16S rRNA gene sequence difference of one another
Diversity was highest when stubble was incorporated into the soil
Diversity was lowest when their was no stubble
Soil Bacteria
Same 8 ag samples
~100,000 sequences
Score on the left is an
indicator of % best match to
the bacterial group on the
top.
This compares between
what we have and have not
cultured
~9% have 100% match to a
cultured bacteria
What Controls Bacterial Diversity?
Lozupone and Knight, 2007
• Nothing has been proven
• Salinity and pH
• Moisture? Soil type? Temperature?
Fierer and Jackson, 2005
II. Viruses
• Cell-free element of genetic material
• Nucleic acid core (DNA or RNA)
• 20-300 nm
• Living cell?
• Can’t metabolize, grow or reproduce outside a host cell
• They are obligate intracellular parasites
• Can do all these within a host
• Contain the genetic capacity to enter and capture the
metabolic and reproductive machinery of the host
Viral Morphology
•Icosahedral (20 triangular faces)
• Helical-resemble long rods
• Enveloped
• External membrane composed of lipids, proteins, and
carbohydrates
• Complex
• Bacteriophage
• Think about:
• What are the similarities between a bacteriophage and a
plasmid?
Revisiting Bacteriophages
• Infect all common bacteria
• Up to 1011 g-1 soil
• Two life cycles
• lytic- takes over cell machinery, copies viral
DNA, constructs new viruses, lysis of cell
• lysogenic-”Hangs out”, does not enter lytic
cycle, function similar to a plasmid, enters
into bacterial chromosome
• Called a prophage
• When enters lytic cycle can take up
pieces of bacterial DNA flanking the viral
gene
• Bacterial DNA also packed into head
Revisiting Bacteriophages
• Host specificity is narrow
• Limited to 1 or a small group of species
• Widely distributed in soils, but can only be found when the
host bacterium is present
• Important in transfer of genetic material
• (lysogenic phase)
• Possible role in controlling bacterial population
• More important in aquatic systems than in soil
• Degraded by proteases, bound to OM or CEC
III. Bacterial Metabolism
Two main groups of bacteria based on where they get their C from
Where do Bacteria Get Their Energy?
(Fixes CO2)
Bacterial Metabolism
• Problem:
• Bacteria need C in order to build cell components
• Monomers  Polymers
• This is an endergonic reaction
• To get this energy bacteria need to somehow produce
ATP (energy) in order to grow, construct cell structures,
enzymes, etc.
• They need an exergonic reaction
• Carbon is in a reduced form in the environment
• Bacteria oxidize this carbon
• Every oxidation reaction is coupled to a reduction reaction
• Transference of electrons from a donor to an acceptor
• RED (reduction) OX (oxidation) reaction
Redox Reactions
LEO
LEO
the Lion Says
GER
Loss of Electrons
is Oxidation
GER
Gain of Electrons
is Reduction
• Chemicals that accept electrons are REDUCED
• Chemicals that donate electrons are OXIDIZED
reduction
In aerobic respiration: C6H12O6 (aq) + 6 O2 (g) → 6 CO2 (g) + 6 H2O (l)
oxidation
Redox “Power”
• Energy is released during these redox reactions
• Bacteria must “intercept” these electrons in order to
produce energy
• The goal of metabolism is to conserved the energy
released during these reactions and converting it to ATP
• There are different bacterial strategies for conserving
this energy
Redox “Power”
• In respiration most are
oxidizing organic matter
• They couple this with
reduction
• There are many different
electron acceptors and
thus different methods of
respiration
• A “hill” that electrons flow down. The higher the placement on the “hill”, the
higher the potential energy of that chemical when coupled with an oxidation
reaction
Oxidation of Organic Matter
• Electron acceptor used in soils is driven by the redox potential of a soil
• Respiration energy yield becomes lower as O2 is more limiting
• Anaerobic respiration
Oxidation of Organic Matter
• Microaggregate from Lecture 1
• Reducing conditions increase
towards the interior of the
aggregate
CO2  CH4
SO42-  HS-
O2  H2O
NO3-  N2
Fe3+  Fe2+
Aerobic Respiration
Requires O2 for two purposes:
1.A terminal acceptor for e- that are released during
oxidation of organic substrates
2.As a reactant during enzymatic attack of organic substrates
and inorganic compounds.
Greater energy yield during aerobic breakdown due to:
1.Complete oxidation of C atoms to CO2
2.A high positive reduction potential. A large difference
between e- donor and oxygen.
(the steepest electron “hill”)
Nitrate Respiration
Anaerobic Metabolism
3
1
2a
2
1. Nitrate is transported into the cell
2. Nitrate is reduced to nitrite (NO3-  NO2-)
2a. Electrons are transferred to NADH and protons move out of the cell (PMF),
generating ATP
3. Nitrite is toxic so it is transported out of the cell
4. Through a series of steps nitrite is converted to N2 (more in Nitrogen lecture)
Anaerobic Metabolism
Iron Respiration
Oxidation of acetate coupled with reduction of Fe3+ to Fe2+
• Prevalent in soils
• Oxidize organic compounds
• Forms oxidized root channels in soils that are reduced
• Siderophores for Fe chelation OR nanowires
Decreasing Energy Yield
Extracellular
Enzyme
Activity
Oxygen
Reduction
Nitrate
Reduction
Organic
Matter
Simple
Organic
Compounds
CO2
Bioavailable
Nutrients
Manganese
Reduction
Iron
Reduction
Fermentation
Short-chain
Fatty Acids
Hydrogen
Sulfate
Reduction
CO2
Reduction
CH4
Bioavailable
Nutrients
Fermentation
• Anaerobic metabolism (not respiration)
• Hydrogen gas is common by-product
• H2 consumed by sulfate reducers and methanogens
• Instead of pyruvate going into the Kreb’s cycle it is
reduced to lactate to regenerate NAD+
• 2 ATP produced vs 38 in aerobic respiration
C6H12O6 → 2 C2H5OH + 2 CO2 glucose to ethanol (beer, wine)
C6H12O6 → 2 CH3CHOHCOOH glucose to lactic acid (sour yogurt)
C12H22O11 + H2O → 4 CH3CHOHCOOH lactose to lactate (yogurt, cheese)
Anaerobic Metabolism
Sulfate Respiration
4H2 + SO42-  H2S + 2H2O + 2OH
• Among the oldest bacteria on Earth ~3 billion yrs ago
• Strict anaerobes
• Important in oceans and coastal wetlands
• May rely on fermentation produced H2 or organic acids
• H2S is very toxic to aerobic organisms
• Leads to death of roots in waterlogged soils
• Microbes responsible are strict anaerobes
• Produce a black layer under golf course greens by using
organic matter in root exudates to reduce soil sulfates to
ferrous sulfates
• H2S pollutes canals of Venice, so the water turns black
Anaerobic Metabolism
Methanogenesis
• Formation of methane (CH4)
• “Last” step in organic matter decay process
• O2 absent, other e- acceptors are used up
• H2 and CO2 concentrations increase
• Fermentation products are also available (short chain fatty acids)
•Without methanogenesis C would accrete in anaerobic environments
CO2 + 4 H2 → CH4 + 2H2O Example of CO2 as electron acceptor
CH3COOH → CH4 + CO2 Example of acetate as electron acceptor
IV. Enzymes
Detrital Matter
Extracellular
Enzyme
Hydrolysis
Leaching
Erosion
Complex Polymers
Monomers
Celluloses; Hemicelluloses;
Proteins: Lipids and Waxes;
Lignin
Sugars; Amino acids;
Fatty acids
Catabolism
Electron
Acceptors
End products
CO2 H2O, nutrients
H2S, CH4, and others
End products
+ energy
Monomers
[e- donor]
[Bacterial Cell]
Enzymes: Highly specialized proteins
S+E
S=Substrate
ES
E=Enzyme
E+P
P=Product
Catalysts for biological reactions
• Lower activation energy
• Increase the rate of reaction
• Activity lost if denatured
• May contain cofactors or coenzymes such as metal ions
or organic (vitamins)
Properties of Enzymes
1. High reaction rates
• rates of enzymatically catalyzed reactions are typically
106-1012 faster than uncatalyzed reactions
2. Mild reaction conditions
• temperatures below 100°C, atmospheric pressure,
nearly neutral pH
3. Specificity
• enzymes have a high degree of specificity for their
substrates
4. Regulation
• the catalytic activity of many enzymes is modulated by
concentrations of substances other than their products
---With some exceptions
Reactions Without Enzymes
Free Energy, G
Transition state: peak of the energy barrier where
decay to S or P is equally probable
ΔG: activation energy for the
reaction, difference in energy
between ground state and transition
state – enzymes reduce the
transition state!
S
P
S
P
Substrate (S): acted upon by the enzyme
Product (P): product of the enzyme
Active site: region of the enzyme where substrate binds
and is converted to product
Reactions With Enzymes
Transition state: peak of the energy barrier where decay to
S or P is equally probable
Free Energy, G
ΔGuncatalyzed
EP
ES
ΔGcatalyzed
S
P
S
E+S
P
ES
EP
E+P
Substrate (S): acted upon by the enzyme
Product (P): product of the enzyme
Enzyme (E): the catalyst
Difference represents the
lowering of the activation
energy
The active site is an enzyme’s catalytic
center
In most cases substrates are held in the active site by weak interactions
– interactions typically involve groups of a few amino acids on the active site
Catalyze the conversion of substrate to product.
– A single enzyme molecule can catalyze thousands or more reactions a
second.
– Enzymes are unaffected by the reaction and
are reusable.
Cofactors- increase chemical reactivity of enzymes
• Enzymes augment their chemical reactivity through the use of two types of
enzyme cofactors:
Small molecule coenzymes (loosely associated with enzyme structure) and
prosthetic groups, an integral part of enzyme structure
– adds chemical reactivity not present in 20 amino acids
• metal ions such as Fe(II)
– enables repeated cycles of
oxidation/reduction
• small molecule can donate or accept electrons while the larger
protein remains unchanged
Factors Controlling Enzyme Activities
Rate of reaction
Enzyme concentration:
Why: More enzymes available to
encounter/react with substrate
molecules
Enzyme Concentration
Factors Controlling Enzyme Activities
Substrate concentration
Rate of reaction
Vmax
Vmax: Maximum reaction velocity
½Vmax
Km: Substrate concentration at which
reaction rates are half maximal, also
called the Michaelis-Menten constant
Km
Substrate Concentration
(enzyme concentration constant)
Factors Controlling Enzyme Activities
Temperature
Percent Maximum Velocity
100
80
60
40
20
0
0
20
40
Temperature ºC
60
Factors Controlling Enzyme Activities
pH
Enzyme proteins are extremely
sensitive to pH, although most
enzymes are active near
neutral pH there is variability
trypsin
Rate of reaction
pepsin
0
2
4
6
8
10
10
What happens at low pH?
-enzymes are proteins and
their bonds (H-bonds) lose
stability when pH is not
optimum. This changes the
shape of the enzyme
molecule so substrate does
not fit
Extracellular Enzymes
Enzymes that have crossed the cytoplasmic membrane of
the microbial cell
Can be:
Exoenzymes: Released into the outside environment and
not attached to its producer
Ectoenzymes: Secreted from a cell but remain chemically
bound to the outer membrane of the producer
Most are:
Hydrolytic: Involve addition of water molecules across
the enzyme susceptible cleavage
Table 1. Soil enzyme functions, substrates and classification (Grandy et al., 2007)
Enzyme
Enzyme Function
Substrate
EC
β-1,4glucosidase
Catalyzes the hydrolysis of terminal 1,4 linked β-D-glucose
residues from β-D-glucosides, including short chain cellulose
oligomers.
4-MUB-β-Dglucoside
3.2.1.21
α-1,4glucosidase
Principally a starch degrading enzyme that catalyzes the
hydrolysis of terminal, non-reducing 1,4-linked α -D-glucose
residues, releasing α-D-glucose
4-MUB-α-Dglucoside
3.2.1.20
β-1,4-xylosidase
Degrades xylooligomers (short xylan chains) into xylose.
4-MUB-β-Dxyloside
3.2.1.37
β-D-1,4cellobiosidase
Catalyzes the hydrolysis of 1,4-β-D-glucosidic linkages in
cellulose and cellotetraose, releasing cellobiose.
4-MUB-β-Dcellobioside
3.2.1.91
β-1,4-N-acetylglucosaminidase
Catalyzes the hydrolysis of terminal 1,4 linked N-acetyl-betaD-glucosaminide residues in chitooligosaccharides (chitin
derived oligomers).
4-MUB-Nacetyl-β-Dglucosaminide
3.1.6.1
Leucine amino
peptidase
Catalyzes the hydrolysis of leucine and other amino acid
residues from the N-terminus of peptides. Amino acid
amides and methyl esters are also readily hydrolyzed by this
enzyme.
L-Leucine-7amino-4methylcoumarin
3.4.11.1
Acid
Phosphatase
Mineralizes organic P into phosphate by hydrolyzing
phosphoric (mono) ester bonds under acidic conditions.
4-MUBphosphate
3.1.3.2
Phenol oxidase
Also known as polyphenol oxidase or laccase. Oxidizes
benzenediols to semiquinones with O2.
L-DOPA
1.10.3.2
Peroxidase
Catalyzes oxidation reactions via the reduction of H2O2. It is
considered to be used by soil microorganisms as a lignolytic
enzyme because it can degrade molecules without a
precisely repeated structure
L-DOPA
1.11.1.7
Ratios between the activity of enzymes involved in C cycling and
those involved in N and P cycling can reflect the nutrient
conditions of the site
Reading Assignment
Condron et al., 87-94 (Website)
Bardgett 57-62
Discussion Questions