Microbiology 4000

The study of microscopic organisms, such as bacteria, viruses, archaea, fungi, and protozoa, is what?

Exam 1 Study Guide

Chapter 1: Humans and the Microbial World

1/10/2018

A. Microbiology

a. The study of microscopic organisms, such as bacteria, viruses, archaea, fungi, and protozoa

b. Microbes are everywhere and on almost everything

B. Earth’s History

a. Microbes are the foundation for all life on earth

b. Life still depends on them today

C. Discovery

a. Hooke

i. 1665

ii. First to observe a “microscopic mushroom” (mold) with a simple microscope

b. Leeuwenhoek

i. 1674 If you want to learn more check out the major trial courts in texas are

ii. Made better lenses, observed bacteria, protozoa, algae, etc

What recycle vital elements in the environment?

iii. Wrote the Royal Society of London

c. Spontaneous generation (Bogus)

i. Louis Pasteur

1. 1861

2. Refutes spontaneous generation and provides support for

biogenesis and the germ theory

3. Also studies yeast fermentation and develops pasteurization

methods

a. Found CO2 and alcohol was produced from yeast

fermentation

b. Pasteurization- heating up the liquid enough to kill off all

of the dangerous microorganisms

4. Flask experiment

a. Trapped microbes from the air in a flask

b. Tipped flask as a positive control to show that it was

Why endospores made a pasteur’s experiment hard to repeat??

possible for microbes to grow in the broth If you want to learn more check out alan peel umd

c. Conclusion: the broth and air do NOT contain some “vital

force” and the microbial growth only comes from living

microbes (biogenesis)

5. Endospores made Pasteur’s experiment hard to repeat

a. Endospores are tough/resistant and would probably

survive his initial boiling, contaminating the initial broth

d. Infectious disease are still a concern:

i. Especially in developing parts of the world

ii. New emerging infectious diseases

iii. Drug resistant strains of known pathogens

iv. New information about old epidemics

e. Normal microbiota

i. Characteristic communities of microbes colonizing a particular location, our body

ii. More microbial cells on you than your own cells

1. About 1.3 to 1 If you want to learn more check out so, what is this caused by precisely?

iii. Includes “good” microorganisms and some pathogens

iv. Bacteria, some archaea, and eukaryotes (often fungi) and viruses v. Protection, development of immunity, make compounds (vitamins) vi. Composition linked or determined by many factors

1. Genetics, diet, lifestyle, geography, birth (bacteria in mother’s

vaginal fluid; microbio differs between vaginally delivered and c

section babies)

2. Social behaviors are key in some animals

a. Termites use fecal transfer to give their young the

microbes that allow them to digest wood

vii. Correlated with many aspects of health and disease

1. Digestion, weight, cancer, allergies, cardiovascular, and mental Don't forget about the age old question of econ 3030

health

G. Microbial Ecology If you want to learn more check out utsa environmental engineering
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a. Microbes recycle vital elements in the environment

b. Nitrogen Fixation- only certain species of bacteria and archaea! i. Turn nitrogen into ammonia, etc to be utilized

c. Carbon Fixation- microbes can be primary producers

i. Fixing carbon dioxide from the atmosphere into usable carbon for other things

d. Photosynthetic microbes – some produce O2

e. Degradation- microbes have unique and diverse enzymes to break down macromolecules

i. Digestion, bioremediation, composting, sewage treatment

ii. Cows need microbes in their gut to allow them to break down cellulose 1. They would starve without them

H. Top Hat questions

a. If all prokaryotes were eliminated from the planet, all animals would die i. Animals rely on prokaryotes for:

1. Nitrogen fixation

2. Digestion in some

3. The efficiency of the immune system

4. Turning CO2 into oxygen

I. Model Organisms

a. Similar metabolic and genetic processes

b. Easy and inexpensive to grow

i. Replicate very quickly

ii. Less ethical concerns

c. Industry? How can we make money off of microbes?

i. Food

ii. Enzymes

iii. Water treatment

iv. Biofuels

v. Nutritional supplements

vi. Antibiotics

vii. Solvents

viii. Herbicides

J. Microbial Diversity

a. Include all 3 domains of life:

i. Bacteria, archaea, and eukarya

b. The key difference between eukaryotes and prokaryotes:

i. Prokaryotes do NOT have a nucleus

ii. Eukaryotes DO have a defined nucleus

c. Some parasites have microscopic life-stage

d. Also includes non-living biological agents

i. Viruses, viroids, prions

e. Only about <1% of microorganisms have been grown and studied in the lab f. Prokaryotic cell types: 

i. Single celled, binary fission, no nucleus

ii. Bacteria (Bacterium)

1. Many are motile (flagella)

2. Commonly round, rod-shaped, or spiral

3. Most have rigid cell walls(peptidoglycan)

4. Organic and Inorganic food sources; some are photosynthetic 5. Found in soil, water, and in our microbiota

6. Most are beneficial to life, some cause infectious diseases

iii. The DNA polymerase in bacteria looks a lot different from archaea 1. And the DNA polymerase in archaea much more closely

resembles that of eukarya

2. Archaea and eukarya are more closely related than archaea and bacteria

iv. Archaea (archeon)

1. Similar to bacteria in size, shape, and motility (flagella)

2. Rigid cell walls (chemically different from bacteria)

3. Organic and Inorganic food sources; some are photosynthetic 4. Found in soil, water, and a few in our microbiota

5. Several are able to survive in extreme environments

a. Volcanoes, salt lake

6. Only associated with some diseases (no known pathogens) a. Are not known to actually cause any disease

b. Are not considered pathogens

g. Eukaryotic cell types 

i. Nucleus, organelles

ii. Algae

1. Diverse group of photosynthetic eukaryotes

2. Single-celled (microscopic) and multicellular (macroscopic) 3. Many different shapes and sizes, some are motile (flagella) 4. Cell walls

5. Often near the surface of aquatic or moist terrestrial

environments

iii. Protozoa

1. Single-celled; mostare motile (flagella, cilia, pseudopodia) 2. No cell wall

3. Ingest organic food sources

4. Both aquatic and terrestrial

5. Some cause disease (e.g. Plasmodium-malaria)

a. Malaria, african sleeping sickness

6. Are considered to be more animal-like

iv. Fungi

1. Single (yeast) and multicellular (molds)

2. Gain their energy from degrading organic materials

3. Cell walls

4. Primarily found on land

5. Some can cause infection (e.g. Candida yeasts-Candidiasis) v. Helminths

1. Multicellular parasitic worms

2. Eggs and larvae are microscopic

a. But become macroscopic when full grown

3. EX: tapeworms, Ascariasis

h. Non-living “Acellular” microbes

i. Viruses

1. Outside the host they are metabolically inactive

2. Simply nucleic acid packaged in a protein coat

3. No ribosomes (no gene expression on their own)

ii. Viroids

1. Simpler than viruses, only a short piece of RNA

2. Replicate only inside the host

3. Known examples are in plants

a. Only known to cause disease in plants

iii. Prions

1. Infectious proteins that cause neurodegenerative diseases

2. Misfolded versions of normal proteins

K. Nomenclature

a. Escherichia coli O157:H7 or Escherichia coli K12

i. Escherichia = genus

ii. Coli = species

iii. H7/K12 = strain

b. Different strains can be the difference between being infectious or not i. Or being resistant to an antibiotic or not

ii. All flavors of ice cream are still ice cream, but mint chocolate chip is not the same as vanilla

L. Sizing

a. Atoms → small molecules → lipids → proteins → ribosomes → viruses → smallest bacteria → prion → mitochondria → most bacteria → nucleus → most eukaryotic cells → adult roundworm (much larger) → human height (MUCH larger)

Chapter 3: Microscopy and Cell Structure

1/12/2018

A. Microscope techniques

a. Leeuwenhoek’s microscope in 1673 had 300x magnification

b. Today’s transmission electron microscopes have 100,000x magnification c. 3 parameters:

i. Magnification

ii. Resolution

1. Clarity or amount of detail that can be observed

iii. Contrast

1. Difference in color intensity

d. Light microscopy

i. Visible light passes through a series of lenses to produce a magnified image (About 1000x)

ii. Bright-field 

1. Most common

2. Illuminates field evenly

iii. Dark-field 

1. Uses dark background to increase contrast of that and specimen iv. Phase contrast 

1. -Amplifies slight variations in refractive index

2. -Can be live samples

v. Fluorescence

iii. 5 layers (surface layers)

1. Sheets of protein or glycoprotein

b. Eukaryotes

i. Diverse cell walls

ii. Protozoa do NOT have cell walls

iii. Algae

1. Polysaccharides (often cellulose or pectins) or even silica or

calcium

2. Diatoms

iv. Fungi

1. Polysaccharides (like chitin) and glycoproteins

v. What common types of macromolecules are in all cell walls?

1. Polysaccharides, peptides

H. Morphology of Prokaryotic Cells

a. Cell envelope- capsule (if present), cell wall, and cytoplasmic membrane b. Also contain:

i. Ribosomes

ii. Pilus

iii. Cytoplasm

iv. Chromosomes (DNA)

v. Nucleoid

vi. Flagellum

c. Capsules and slime layers

i. Gel-like layer outside cell wall

1. Bacteria

ii. Attachment and protection 

1. Attachment allows the formation of biofilm

iii. Avoid host immune cells (capsule) 

1. Capsule prevents phagocytes from engulfing them

iv. Vary in chemical composition, but often polysaccharides

v. Glycocalyx - “sugar shell”

vi. Capsule = distinct and gelatinous 

vii. Slime layer = diffuse and irregular 

viii. One strain could be able to make a capsule while another strain can not I. Filamentous protein appendages

a. Bacterial flagella

i. Long protein structures (flagellin)

ii. Used in most prokaryotic motility

iii. In bacteria, they spin like propellers 

iv. Powered by proton motive force 

v. Mobility helps with chemotaxis (moving toward/away from a chemical) vi. Flagellin subunits

vii. Polar

a. Electrochemical gradient of H+ (protons)

b. Source of potential energy 

i. Just a different source of energy

c. How is it generated:

i. Proton pumps

ii. Electron transport chain (redox)

1. Cellular respiration

d. What is it used for:

i. Cotransport

ii. motility

iii. ATP synthesis

L. Top Hat

a. Which of the following describe gram negative bacteria?

i. Have an outer membrane

ii. Generally less sensitive to various classes of antibiotics

iii. Thin peptidoglycan layer

iv. Contain lipopolysaccharides (LPS) in outer membrane

b. Why do ONLY gram positive cells appear purple during gram staining? i. Gram positive cells have a thick cell wall that becomes dehydrated during the alcohol (decolorization) step preventing the release of the stain

M. Internal Structures- Prokaryotes

a. Chromosome

i. Single, circular, double stranded (ds) DNA

ii. Tightly packed and supercoiled 

iii. Found in the nucleoid 

1. Not a membrane bound nucleus, but still contains the DNA

iv. Usually only 1 in bacteria

b. Plasmids

i. Smaller, singular, circular, dsDNA molecules

ii. Non-essential, but can contain beneficial genes

iii. Horizontal gene transfer (HGT)

c. Ribosomes

i. Site of protein synthesis (translation)

ii. Similar:

1. Made of proteins and RNA

2. 2 subunits

iii. Different in eukaryotes:

1. Size

2. Sequence

iv. Large subunit = 50S

v. Small subunit = 30S

vi. Prokaryote = 70S

vii. Eukaryote = 80S 

d. Cytoskeleton

i. Less extensive than eukaryotes but homologous

ii. Less is known

iii. Cell division and cell shape 

e. Storage granules

i. Is NOT membrane bound

1. Usually just has a protein barrier

ii. Accumulate and store large carbon polymers or other nutrients 1. EX: Glycogen or phosphate

f. Gas vesicles

i. Aquatic bacteria

ii. Protein bound compartment than provides buoyancy

iii. Controlled density 

iv. Allows the bacteria to float/sink in water to come closer to sunlight for an energy source

N. Endospores and Sporulation

a. Differentiated cells produced by some bacterium

b. Very resistant to heat, chemicals, radiation, etc

c. Formed during a distinct phase of growth (sporulation) and cued by environment (starvation, stress, etc)

d. Germination- back to vegetative growth

i. When environmental conditions are good again, the spore germinates and can then rapidly divide again

e. Sporulation is a lot slower than germination

f. Before endospores were discovered, some scientists had trouble replicating Pasteur’s flask experiment

O. Staining techniques

a. Special stains for cell structure

b. Capsule stain

c. Endospore stain

d. Flagella stain

P. Microbial Diversity (Table 3.7) 

a. Prokaryotic- Bacteria and Archaea

i. Polypeptide channels for secretion

ii. Smaller

iii. Different flagella

iv. Archaea have cell walls, but they are NOT made of peptidoglycan v. Bacteria cell walls are made of peptidoglycan

vi. Etc…

b. Eukaryotic- Eukarya

i. Membrane bound organelles

ii. Endomembrane system for secretion

iii. Larger

iv. Different flagella

v. Etc…

Chapter 4: Dynamics of Prokaryotic Growth

1/22/2018

A. Introduction

a. Robert Koch

i. Studied disease causing bacteria

ii. Germ Theory, Nobel prize

iii. Methods of cultivating bacteria (solid media, pure culture)

b. Prokaryotic can grow almost anywhere, even in the extreme

i. Each species requires appropriate nutrients and conditions

ii. Understanding growth is important because it allows you to understand how a pathogen grows and then you can figure out how to stop it

1. Disease prevention 

c. Microbial growth- increase in the number of cells in a population d. Mitosis = eukaryotes 

e. Binary fission = prokaryotes 

i. Bacteria and archaea 

B. Binary Fission

a. Cell division in bacteria

i. Replication of chromosome and it moves to ends; cell elongates ii. Plasma membrane pinches inward with the help of a protein ring iii. Cytoplasm is split, new cell membrane and cell wall are made

iv. Two Daughter Cells result 

b. Like all cells… timing is regulated!

c. Exponential growth

i. Generation (doubling) time- the time it takes for a population to double in number

ii. 1 → 2 → 4 → 8 → 16 = 4 doublings

iii. Doubling time is determined by both the species/strain and the growth media/environment 

iv. Number of cells originally present x 2n = number of cells at a given time (minutes) 

1. n = number of generations

v. If a strain has a generation time of 20 minutes, how many more cells will there be after 2 hours?

1. 120 minutes/20 minutes = 6

2. 26 = 64 cells

a. 2 because it is doubling

vi. If the doubling time is 60 minutes, how long would it take for the population to increase by 8x?

1. 1 → 2 → 4 → 8 (3 doublings)

2. 3 x 1 hour = 3 hours 

C. Growth Curve (closed system- no nutrients coming in and no nutrients going out) a. Lag– begin making enzymes for growth

i. What happens immediately after inoculation

b. Log/exponential- growing at a constant rate

c. Stationary– nutrients run out; growth plateaus

d. Death- cells are dying at a constant rate

e. Prolonged decline- few fit or tolerant cells survive

f. Metabolite = captured and made during metabolism

i. Used in industry

ii. Primary Metabolites – made during active growth and metabolism 1. e.g. amino acids, ethanol, citric acid, etc.

iii. Secondary Metabolites – not essential for growth; often made for defense or survival

1. e.g. antibiotics

2. Often made/used for competitive interactions

3. In stationary phase

D. Pure Culture

a. A population descended from a single cell therefore separated from other species or even strains

i. Petri dish- allows air, excludes contaminants

ii. Agar- solid, can be sterilized, few microbes can degrade in agar b. Streak plate method

c. Colony

i. Visible population (about 1 million cells) of pure culture of solid media 1. Cells are clones on each other

2. But they are physically doing different things

ii. Growth ranges from exponential at edges to death phase in center E. Growth in Nature

a. Mixed microbial communities 

i. In nature, microbes are almost always found in mixed, dynamic communities

b. Cooperative interactions

i. Metabolic waste of one organism is a nutrient for another

ii. Growth of species who are otherwise unable to survive

c. Competitive interactions

i. Compete for nutrients

ii. Synthesize toxic compounds to inhibit competitors

1. Toxins, antibiotics, ethanol, etc…)

2. Secretion

3. Fermentation (a lot of microbes don’t grow well in the presence

of ethanol)

iii. Can be direct/indirect competition

d. Biofilms

i. Polymer-encased communities

1. Secretion of polymers is tightly regulated through Quorum

Sensing

a. NOT random, have to have the right chemical signals to

initiate

2. EPS- extracellular polymeric substances

a. EX: polysaccharides and DNA

b. Helps molecules stick together so microbes can transport

nutrients more efficiently

i. Allows the coherence of cells 

ii. Can be more than one species, or domain

iii. Enable cells to resist disinfectants, antibiotics, host defenses, etc. 1. Antibiotics can’t reach the bottom of the biofilm

iv. Pass nutrients and waste through channels between cells

v. Content is different at the top vs bottom of the biofilm

1. Oxygen is more present at the surface

vi. Allow for adherence/attachment 

vii. EX: dental plaque, ear infections, UTIs; can develop on contact lenses, medical implants

F. Factors that Affect Growth

a. Preferred growth conditions and sources of energy and carbon are used to categorize different microbial species

b. Environmental factors

i. Temperature 

1. Psychrophile

a. 5°C to 15°C (very cold to frozen)

2. Psychrotroph

a. 15°C to 30°C (cool, room temp)

b. But can also grow in refrigeration) 

c. Listeria monocytogenes

i. Refrigerating food will not prevent this bacteria

from growing

3. Mesophile

a. 25°C to 45°C (body temp is 37°C)

4. Thermophile

a. 45°C to 70°C (very warm)

b. Thermus aquaticus→ Isolated from hot springs

5. Hyperthermophile

a. >70°C (very hot; even boiling)

6. (will not test on specific numbers, will test on cold/very cold, room temp/body temp, ect..)

7. We control temperature to control microbial growth

ii. Atmosphere (e.g. oxygen content)

1. Obligate Aerobe:

a. Must have O2 

b. In a test tube, there is only growth at the top

c. Produces superoxide dismutase and catalase

2. Facultative Anaerobe

a. Has the “option” to grow without O2 

b. Grows best with oxygen, but has other metabolic

pathways to still grow in its absence

c. In a test tube, bacteria grows throughout but there is

more growth towards the top

d. Produces superoxide dismutase and catalase

3. Obligate Anaerobe

a. Must have NO O2 

b. In a test tube, bacteria only grows at the bottom

c. Does NOT produce superoxide dismutase or catalase

4. Microaerophile

a. Likes a specific, low [O2] 

b. In a test tube, produces bacteria only at a specific point near the top

c. Produces some superoxide dismutase and catalase

5. Aerotolerant Anaerobe

a. Doesn’t care 

b. In a test tube, bacteria grows equally well throughout the whole tube

c. Metabolism doesn’t require oxygen and they are not

poisoned by it

d. Produces superoxide dismutase but NOT catalase

6. Reactive Oxygen Species (ROS) – produced as by products of some metabolic reactions

a. Chemically damage DNA, proteins, membrane, etc.

b. EX: ᐧOH, H2O2, O2-

7. Superoxide dismutase and Catalase– enzymes that break down ROS to O2 and water

8.

iii. pH 

1. Alkalophile

a. pH > 8.5

2. Neutrophile

5. Fastidious microbes are limited compounds that can use

6. eg. B. fastidiosus can only use urea

d. Table 4.2 in textbook

G. Top Hat

a. A sample of cells grows exponentially in nutrient rich media. How would the growth curve change if you were to put this sample in minimal media? i. The slope of the log phase would decrease

ii. Generation time increases

iii. The time in the lag phase would increase

1. This is the time period when the bacteria are collecting the

material/nutrients for them to grow, if there is less nutrients

available then this process is going to take longer

b. Match the organism with the best description

i. Bacteroides thetaiotaomicron (dominates the human gut- little to no oxygen) 1. Mesophilic obligate anaerobe 

2. Body temperature, no oxygen 

ii. Thermoanaerobacter kivui (found in deep sediments of an African lake- ~66 degrees C; no oxygen) 

1. Thermophilic obligate anaerobe 

2. Likes heat, no oxygen 

iii. Listeria Monocytogenes (can grow without oxygen if needed; can grow in cured meat and cheese- high salt) 

1. Halotolerant facultative anaerobe 

2. Can grow in high solute conditions, prefer to grow with oxygen but 

can grow without it if needed 

iv. Blastochloris virdis (photosynthetic when oxygen is absent; prefers to obtain carbon from others when there is oxygen) 

1. Facultative anaerobe- photoautotrophic and heterotrophic 

2. Can switch metabolism between photo and hetero 

v. Aquifex aeolicus (needs low amounts of oxygen; grows near volcanoes (95-100 degrees C); uses H2 (inorganic) for energy) 

1. Microaerophilic hyperthermophilic chemolithoautotroph 

2. Likes extreme heat, get energy from chemical sources 

vi. Mycobacterium tuberculosis (pathogen that infects and thrives in our oxygen rich lungs) 

1. Obligate aerobic mesophilic heterotroph 

2. Needs oxygen, body temperature 

Chapter 6: Bacterial Metabolism 

Part 1: Enzymes and Energetics 

A. Metabolism: Fueling Cell Growth

a. Metabolism- total of ALL the chemical reactions occuring in a cell b. Catabolism- breaking things down

i. Energy released

ii. Source of electrons (reducing power)

iii. Generates precursors for biosynthesis

c. Anabolism- building things up 

i. Energy consumed

ii. Needs electrons (reducing power)

iii. Synthesis of complex organic molecules from simpler ones

iv. Uses energy to power

v. Biosynthesis

d. Metabolite- product or substrate of metabolism; general term

B. Energetics

a. Energy is the capacity to do work 

b. 2 types:

i. Potential: stored energy

1. chemical bonds, concentration gradient, proton motive force

ii. Kinetic: energy of movement

1. movement of molecules, light energy, or electrons

c. Energy in the universe cannot be created or destroyed, only converted or transferred 

i. Light energy (kinetic) → chemical energy (potential) by photoautotrophs d. All cells obtain energy from their environment and convert it into a useful form. e. Some microbes harvest energy from:

i. Organic compounds

1. chemoorganoautotrophic

ii. Inorganic compounds

1. Chemolithotrophic

f. Free energy- energy available to do work

i. The difference between the amount of free energy in the reactants vs the products is the change in free energy of a reaction

ii. Exergonic reaction- reactants have more free energy

1. Energy is released 

2. Energetically favorable

iii. Endergonic reaction- products have more free energy

1. Requires an input of energy 

2. Energetically unfavorable

iv. This is the same regardless of the number of steps involved or how fast it goes

C. ATP (Adenosine triphosphate)

a. ATP is a common energy currency of the cell

b. ADP (adenosine diphosphate) can accept free energy

c. ADP + P → ATP; ATP → ADP + P

d. ATP has MORE free energy than ADP because it has an additional phosphate e. How Cells Transfer Energy to ATP:

i. Substrate-level Phosphorylation: exergonic reactions provide energy directly

1. Happens during glycolysis and TCA cycle 

2. A → B is exergonic

3. The direct transfer of a phosphate group

4. Free energy is used to phosphorylate ADP to ATP

ii. Oxidative phosphorylation: Redox rxns create PMF; drives ATP synthase 

1. Respiration

2. ATP synthase is used to make ATP

iii. Photophosphorylation: Light energy used to create a PMF; drives ATP synthase 

1. Photosynthesis

D. Redox Reactions and Electron Carriers

a. Electron Energy can be moved through series of oxidation-reduction reactions or Redox reactions

b. Substance that loses electrons is oxidized (X)

c. Substance that gains electrons is reduced (Y)

d. Gain/loss of a proton (H+) occurs as well

i. This is why NAD+ becomes NADH

e. NAD+ + 2 e- + H2 → NADH + H+

i. NAD+ is reduced to NADH

f. Electron carriers can carry reducing power (electrons) and transfer this energy to molecules with higher affinities for the electrons

i. Help the cell harvest or transfer energy in a controlled and stepwise manner

ii. Electron transport chain (respiration)

g. Oxidized form accepts electrons and reduced form donates electrons E. Redox and Central Catabolism

a. ?????

F. Metabolic Pathways

a. Cells often use multiple steps when degrading or synthesizing compounds i. This makes it easier to regulate 

ii. Allows for a more efficient use of energy

1. Controlled release of energy 

b. Starting compound → intermediate 1 → intermediate 2 → end product i. Linear metabolic pathway

c. Starting compound → intermediate 1 → intermediate 1a → end product 1 and Starting compound → intermediate 1 → intermediate 1b → end product 2 i. Branched metabolic pathway

d. Starting compound → intermediate 1 → intermediate 2 → intermediate 3 → end product OR intermediate 4 +staring compound → intermediate 1 → etc… i. Cyclic metabolic pathway

G. Enzymes

a. Biological catalysts that speed up reactions

i. These reactions would still occur without enzymes, just very slowly b. Do NOT change the difference in free energy

i. Will not change an exergonic to an endergonic reaction

c. Proper folding and shape are critical 

i. Peptide chains fold into proteins

d. How do Enzymes Work?

i. Highly specific for their substrate(s) → Active Site

ii. Lower the activation energy of a reaction

1. orient substrates, destabilize existing bonds, microenvironment, or help the chemistry of the reaction

iii. Not used up in a reaction

e. Name often reflects function; ends in –ase 

i. “isomerase” or “DNA polymerase”

f. Substrate attaches to active site of enzyme → enzyme-substrate complex formed → products get released and enzyme is unchanged

i. Enzymes are specific to the substrates they work with because they have specifically shaped active sites

g. Can be present in low concentrations; can be recycled and used again in different reactions

h. Factors that affect activity:

i. Environmental factors

1. Temperature

2. pH

3. self concentration (salt)

4. (affects the folding) (denature = unfold)

ii. Cofactors

1. Assist some enzymes’ activity (non-protein)

2. EX: Mg, Zn, electron carriers like FAD and NAD+)

iii. Allosteric regulation

1. Regulatory molecule binds the enzyme (not the active site)

2. Distorts shape; prevents OR enhances substrate binding

3. Often the end product of a pathway (Feedback regulation)

iv. Inhibition

1. Non-competitive - inhibitor binds somewhere other than the 

active site 

a. EX: Allosteric regulation → Inhibition is reversible

b. Some are also NOT reversible → mercury

i. Causes permanent damage

2. Competitive - inhibitor binds directly to the active site 

a. Competes with the substrate

b. EX: sulfa drugs (antibiotics) competitively inhibit an

enzyme in folate biosynthesis

i. Drug binds to active site INSTEAD OF substrate

H. Top Hat

a. Which reaction has more free energy is its products than its reactants? i. Endergonic

b. NADH + H+ → NAD+ and Pyruvate → lactic acid

i. Pyruvate is reduced

1. It gains an electron

ii. Lactic acid is the reduced product

1. Pyruvate is reduced to lactic acid

iii. NADH is oxidized

1. It loses an electron

iv. NAD+ is the oxidized product

1. NADH is oxidized to NAD+

c. Penicillin irreversibly binds the active site of the transpeptidase enzyme in bacteria, blocking access to its substrates. Penicillin is a _____

i. Competitive inhibitor

Chapter 6: Bacterial Metabolism 

Part 2: Sections 6.3-6.9 

A. Central catabolism - the breakdown of glucose

a. Includes:

i. Glycolysis

ii. Transition step

iii. TCA cycle

b. Glucose → glycolysis → pyruvate x2 → transition to CO2 or acetyl CoA x2 to the TCA cycle x2

i. As chain proceeds, glucose is oxidized (broken down) to CO2 ii. Each step has reducing power

c. Glycolysis

i. Glucose goes in; pyruvate comes out 

1. Glucose is broken down to pyruvate

ii. Many steps, 2 halves

1. Investment 

a. Start with glucose

b. ATP input

c. In bacteria, this step can occur via group translocation

2. Payoff 

a. ATP and NADH are produced

b. Get 2 pyruvates

3. In between these halves, G3P is produced as an intermediate

i. Series of electron carriers (enzyme complexes) that pass electrons to each other (REDOX) and use this energy to move protons (H+) across a membrane, generating Proton Motive Force (PMF) 

ii. Reducing power of NADH/FADH2 → electrons from the energy source move down the ETC from high energy to low energy → terminal electron acceptor at the end

1. Energy is released and used to generate a PMF along the way 2. Electron energy is gradually released as it is passed along the chain and is used to pump protons (H+)

3. Transfers energy to electrochemical gradient → PMF

iii. Prokaryotes → cytoplasmic membrane

1. Prokaryotes also use PMF to power membrane transport and flagella 

iv. Eukaryotes → inner mitochondrial membrane

v. In E. coli:

1. NADH dehydrogenase accepts e’s from NADH 

a. pumps H+

2. Succinate dehydrogenase accepts e’s from FADH2 

a. doesn’t pump H+

3. Ubiquinone moves freely within membrane to transfer e’s

4. Ubiquinol oxidase transfers the e’s to the Terminal O2 (aerobic respiration)

c. NADH generates more ATP per electron than FADH2

d. Aerobic - O2 is the final electron acceptor

i. Very efficient 

ii. Make the most ATP per molecule of glucose

e. Anaerobic - something other than O2 is used as the final acceptor i. Less efficient 

ii. Produce less ATP

f. Energy yield

i. In bacteria, theoretical maximum amount of ATP generated is 38 ATP/glucose 

1. 4 ATP from substrate-level phosphorylation

a. 2 ATP from glycolysis

b. 2 ATP from TCA cycle

2. 34 ATP from aerobic respiration/oxidative phosphorylation

a. 18 +4 ATP from NADH/FADH2 from TCA

b. 6 ATP from transition state NADH

c. 6 ATP from glycolysis NADH

ii. What is the maximum number of ATP molecules that can be made per glucose if:

1. You are an aerobic bacteria with a mutation in citrate

synthetase, an enzyme in the TCA cycle, that now renders it

non-functional

a. 14

b. You wont get 2 directly from TCA cycle or the 18 +4 from

the NADH/FADH2 produced from the TCA cycle

c. 38-2-18-4 = 14

2. You are an aerobic bacteria that encounters a chemical toxin.

This toxin alters your inner membrane so it is “leaky” to

protons

a. 4

b. 2 from glycolysis and 2 directly from TCA cycle

3. You are a bacteria grown with a competitive inhibitor of

glyceraldhyde3-phosephate (G3P) dehydrogenase, the enzyme

that catalyzes the first step of the pay-off phase of glycolysis

a. 0 (or technically -2)

b. You inhibit everything after you initially put in ATP

iii. In terms of maximum ATP produced, why does ‘1 glucose = 38 ATP’ not hold true for all microbes?

1. Anaerobic respiration is less efficient than aerobic respiration

(using oxygen as the final electron acceptor) so anaerobic

produces less ATP

C. Respiration Diversity

a. Prokaryotes are very diverse

i. Can have several different ETC electron carriers in 1 species

b. Anaerobic respiration

i. Terminal e-acceptor other than O2 

1. EX: nitrate (NO3-) or sulfate (SO42–)

2. these have lower affinities for electrons

ii. Harvests less energy than aerobic respiration, but enables growth without O2 

iii. Need a different enzyme to transfer the final e

c. ETC Diversity

i. Alternative ETC enzymes exist in some microbes to harvest electron energy directly from other carbon sources

1. formate, lactate, malate, glutamate

2. inorganic sources, like Fe, H2 or H2S

a. Would be used by chemolithotrophs (use inorganic

compounds)

ii. Light reactions of photosynthesis also use ETCs

1. Would be used by phototrophs 

iii. Electron Transport Chain (ETC)

iv. Different Ubiquinol Oxidases

1. to utilize lower O2 concentrations

a. Would be used by microaerophiles 

v. Similar goal: harvest energy from the environment and transfer it to ATP D. Overview

a. Carbon flow

i. Glucose → 2 pyruvate → 2 Acetyl CoA → 4 CO2

ii. Glucose → 2 pyruvate → CO2

b. Electron flow

i. Glucose → NADH/FADH2 → ETC → O2 (final E acceptor)

c. Energy flow

i. Glucose → ATP (SLP)

ii. Glucose → NADH/FADH2 → ETC → O2

iii. Glucose → NADH/FADH2 → ETC → PMF → ATP (oxidative

phosphorylation)

iv.

E. Fermentation → transfer of electrons

a. Is NOT anaerobic respiration, but is another alternative to aerobic respiration b. Partial catabolism of glucose (or other carbon source) where electron energy is transferred to pyruvate(final e-acceptor) 

i. The partial oxidation of sugars that can occur without O2 (inefficient) c. NADH reduces pyruvate (or derivative), NOT the ETC 

d. Who uses it?

i. Organisms that can’t respire; the final electron acceptor in respiration isn’t available or they lack an ETC altogether

1. anaerobic conditions

ii. E. coli can use aerobic and anaerobic respiration and fermentation iii. Streptococcus pneumonia can only use fermentation (no ETC) e. Regenerates NAD+

i. Coupled with glycolysis, cells are able to produce ATP without O2(or other ETC final electron acceptor)

f. Net result: 2 ATP/glucose 

g. 2 pyruvate → (2 NADH → 2 NAD+) → reduced product

i. Pyruvate gets reduced

h. Lactic acid fermentation

i. Lactate is the reduced end product, with no CO2

ii. Gram-positives in cheese, yogurt, pickles

1. Lactobacillus, Streptococcus

iii. Our microbiota

1. oral and vaginal bacteria

2. Yeast infections

iv. Human muscle cells

v. 2 pyruvate → (2 NADH → 2 NAD+) → 2 lactate

i. Alcohol fermentation

i. Two steps: CO2 and ethanol are final products

ii. 2 pyruvate → (2 CO2) and 2 acetaldehyde → (2 NADH → 2 NAD+) → 2 ethanol

iii. Brewing, winemaking, and baking (Saccharomyces)

iv. Yeast are very good at alcohol fermentation

j. Often also leads to food spoilage

k. How does fermentation benefit microbes?

i. Useful way to make ATP in the absence of oxygen

ii. Gives you a competitive advantage (allows you to produce ethanol, lactic acid, etc)

l. How does fermentation benefit humans?

i. Useful for food

ii. Useful in identifying microbes by identifying their byproducts

F. Carbon Catabolism- Variations

a. Alternative pathways (different enzymes) to breakdown glucose to pyruvate b. Microorganisms might have one or several of these 3 pathways

c. Pentose Phosphate Cycle

i. INPUT:

1. Glucose

ii. OUTPUT:

1. NADPH-reducing power used in biosynthesis

2. Precursor Metabolites(ex. nucleotide biosynthesis)

3. Glyceraldehyde-3-phosphate (G3P) –intermediate of

glycolysis; broken down to pyruvate

iii. Makes reducing power and components needed for some biosynthesis pathways