CSS 360Lecture 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. Discussion Questions from last week2. Review Reading assignment from week 2; Condron et al.3. Bacterial diversity4. Viruses and their role in natural systems5. Bacterial metabolism6. 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

• PhylumClassOrderFamilyGenusSpeciesStrain

8 Australian agricultural soils under different treatmentsClustered into groups within a 5% 16S rRNA gene sequence difference of one anotherDiversity was highest when stubble was incorporated into the soilDiversity 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

LEOthe Lion SaysGER

LEOLoss of Electrons is Oxidation

GERGain of Electrons is Reduction

Redox Reactions

• Chemicals that accept electrons are REDUCED• Chemicals that donate electrons are OXIDIZED

In aerobic respiration: C6H12O6 (aq) + 6 O2 (g) → 6 CO2 (g) + 6 H2O (l)

oxidation

reduction

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

O2 H2O

NO3- N2

Fe3+ Fe2+

SO42- HS-

CO2 CH4

Aerobic Respiration

Requires O2 for two purposes:1.A terminal acceptor for e- that are released during oxidation of organic substrates2.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

11

22

33

1. Nitrate is transported into the cell2. 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 cell4. Through a series of steps nitrite is converted to N2 (more in Nitrogen lecture)

2a2a

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

Anaerobic Metabolism

Organic MatterOrganic Matter

Simple Organic Compounds

Simple Organic Compounds

OxygenReductionOxygenReduction

NitrateReductionNitrateReduction

ManganeseReductionManganeseReduction

IronReductionIronReduction

SulfateReductionSulfateReduction

CO2

ReductionCO2

Reduction

Short-chainFatty AcidsHydrogen

Short-chainFatty AcidsHydrogen

CO2

BioavailableNutrients

CO2

BioavailableNutrients

CH4

BioavailableNutrients

CH4

BioavailableNutrients

FermentationFermentation

ExtracellularEnzymeActivity

ExtracellularEnzymeActivity

Decreasing Energy Yield

Fermentation

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 (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

Sulfate Respiration

• 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

4H2 + SO42- H2S + 2H2O + 2OH

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

Anaerobic Metabolism

Detrital Matter

Complex PolymersCelluloses; Hemicelluloses;Proteins: Lipids and Waxes;

Lignin

Complex PolymersCelluloses; Hemicelluloses;Proteins: Lipids and Waxes;

Lignin

ElectronAcceptorsElectron

Acceptors

End productsCO2 H2O, nutrients

H2S, CH4, and others

End productsCO2 H2O, nutrients

H2S, CH4, and others

MonomersSugars; Amino acids;

Fatty acids

MonomersSugars; Amino acids;

Fatty acids

End products Monomers+ energy [e- donor]

[Bacterial Cell]

ExtracellularEnzymeHydrolysis

ExtracellularEnzymeHydrolysis Leaching

Erosion

Catabolism

IV. Enzymes

Enzymes: Highly specialized proteins

S + E ES E+PS=Substrate E=Enzyme 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)

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

Properties of Enzymes

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

S

P

Transition state: peak of the energy barrier where decay to S or P is equally probable

Free

Ene

rgy,

G

Reactions Without Enzymes

ΔG: activation energy for the reaction, difference in energy between ground state and transition state – enzymes reduce the transition state!

S P

Substrate (S): acted upon by the enzymeProduct (P): product of the enzymeEnzyme (E): the catalyst

S

P

Transition state: peak of the energy barrier where decay to S or P is equally probable

Free

Ene

rgy,

G

Reactions With Enzymes

ΔGuncatalyzed

S P

ESEP

ΔGcatalyzed

Difference represents the lowering of the activation energy

E + S ES EP E + P

The active site is an enzyme’s catalytic centerIn 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 andare 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 ofoxidation/reduction

• small molecule can donate or accept electrons while the largerprotein remains unchanged

Factors Controlling Enzyme Activities

Enzyme concentration:

Rate

of r

eacti

on

Enzyme Concentration

Why: More enzymes available to encounter/react with substrate molecules

Rate

of r

eacti

on

Substrate Concentration

Factors Controlling Enzyme Activities

(enzyme concentration constant)

Vmax

½Vmax

Km

Vmax: Maximum reaction velocity

Km: Substrate concentration at which reaction rates are half maximal, also called the Michaelis-Menten constant

Substrate concentration

Temperature ºC

Perc

ent M

axim

um V

eloc

ity

0 20 40 600

20

40

60

80

100

Factors Controlling Enzyme Activities

Temperature

Rate

of r

eacti

on

0 2 4 6 8 10 10

pepsin trypsin Enzyme proteins are extremely sensitive to pH, although most enzymes are active near neutral pH there is variability

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

Factors Controlling Enzyme Activities

pH

Extracellular EnzymesEnzymes that have crossed the cytoplasmic membrane of the microbial cell

Can be:Exoenzymes: Released into the outside environment and not attached to its producerEctoenzymes: 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-1,4-glucosidase

Catalyzes the hydrolysis of terminal 1,4 linked β-D-glucose residues from β-D-glucosides, including short chain cellulose oligomers.

4-MUB-β-D-glucoside

3.2.1.21

α-1,4-glucosidase

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-D-glucoside

3.2.1.20

β-1,4-xylosidase Degrades xylooligomers (short xylan chains) into xylose. 4-MUB-β-D-xyloside

3.2.1.37

β-D-1,4-cellobiosidase

Catalyzes the hydrolysis of 1,4-β-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose.

4-MUB-β-D-cellobioside

3.2.1.91

β-1,4-N-acetyl-glucosaminidase

Catalyzes the hydrolysis of terminal 1,4 linked N-acetyl-beta-D-glucosaminide residues in chitooligosaccharides (chitin derived oligomers).

4-MUB-N-acetyl-β-D-glucosaminide

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-7-amino-4-methylcoumarin

3.4.11.1

Acid Phosphatase

Mineralizes organic P into phosphate by hydrolyzing phosphoric (mono) ester bonds under acidic conditions.

4-MUB-phosphate

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 AssignmentCondron et al., 87-94 (Website)Bardgett 57-62

Discussion Questions