Microbiology Exam 3

Metabolism

Sum of all chemical reactions within a living organism

Catabolism

• breakdown of compounds

• release energy

• provide building blocks

Anabolism

• building compounds

• require energy

• use up building blocks

Enzymes

• Sum of all enzymes → metabolism of cell

• size = 300 amino acids

• 10 - 1,000 kilodaltons (110 Da = 1 a.a.)

• are proteins

• specific targets for antibiotics

• end is -ase

Catalyst

Lowers activation energy without being permanently altered (allows reaction to happen easier)

Enzyme composition (apoenzyme)

Protein (main part) may act on its own (depending on the enzyme)

Enzyme composition (coenzyme)

• Organic molecule (nonprotein) might be required

• ex: vitamins

Enzyme composition (cofactor)

• inorganic molecule (nonprotein) might be required

• ex: minerals

Enzyme composition (active site)

Where substrate binds and shape of sight is essential (interact with enzyme)

Enzyme structure (protein levels)

1. Primary

• Determined by DNA

• Held together by peptide bonds

2. Secondary

• Alpha (α) helix – coiled shape

• Beta (β) pleated sheet – folded, sheet-like shape

3. Tertiary

• Formed by interactions between R-groups (side chains)

• Includes hydrogen bonds, ionic bonds, disulfide bridges, and hydrophobic interactions

• Determines the protein’s specific function

4. Quaternary

• The structure formed when two or more polypeptide chains (subunits) combine

• Not all proteins have this level.

Enzyme denature

Loss in conformation or shape

• no longer active

• caused by high temperature or extreme pH

Enzyme production (constitutive)

Produced all the time and activity doesn’t change

Enzyme production (inducible)

Environment causes increased production or activity

Enzyme production (repressible)

Environment causes decreased production or activity

Exoenzymes

Virulence factors and bacteria secrete outside of cell

• ex: DNase, amylase

Enzyme activity

1. active site (can bind substrate)

2. forms enzyme (substrate complex)

3. substrate is transformed to products

4. products are released

5. enzyme is “recycled”

Factors influencing enzyme activity (temperature)

• Low temperature: molecules move more slowly; enzyme becomes rigid

• High temperature: protein denatures or unfolds because noncovalent (and hydrogen) binds are broken

Factors influencing enzyme activity (pH - high H+ or OH-)

Extreme pH can cause enzymes to denature

Factors influencing enzyme activity

1. temperature

2. pH

3. substrate

4. inhibitors

Factors influencing enzyme activity (substrate)

• substrate concentration

• saturation: all active sites are filled with substrate

Factors influencing enzyme activity (inhibitors)

• competitive

• noncompetitive

Competitive inhibitors

• binds the ACTIVE SITE

• does not undergo any reaction or change (the enzyme)

• Irreversible: an inhibitor that binds permanently to an enzyme

• Reversible: an inhibitor that binds temporarily to an enzyme

• antimicrobial agent sulfanilamide

Noncompetitive inhibitors

1. binds the ALLOSTERIC site - some OTHER place on the enzyme

2. causes active site to change shape

3. can be reversible or irreversible

4. feedback inhibition - an end product in a series of reactions inhibits the first enzyme in the series (keeps the cell from wasting resources

Energy production (oxidation-reduction reactions)

1. oxidation → lose an electron

2. reduction → gain an electron

3. these are coupled

4. dehydrogenation - movement of hydrogen atom and an electron

Energy production (generation of ATP)

1. substrate level phosphorylation - the high energy phosphate is transferred from a substrate to ADP to produce ATP

2. oxidative phosphorylation - electrons are transported down a chain of proteins and energy is produced by chemiosmosis (osmotic gradient drives it)

Energy production (energy is released in a series of steps)

1. too fast → heat → cell damage

2. energy produced by passing electrons through oxidation reactions

What is the substrate in reaction step 1?

A

What is the main product in reaction step 1?

B

What does the curved line mean in reaction step 1?

Coupled or joined reaction

Is coenzyme NAD+ reduced or oxidized in reaction step 1?

Reduced (gained an electron)

Is substrate A reduced or oxidized in reaction step 1?

Oxidized (gave electrons to NAD+)

What is also involved with this pathway (#1)?

Enzymes

In reaction step 2 changing B substrate coupled to get ATP (form it). Where did the phosphate come from?

Comes from B (enzyme is allowed B + ADP to exchange)

Where is a reversible reaction in reaction step 2?

Step 3

What is O2 at reaction step #4?

Reactant (for step 4 to work it needs O2)

What is the main end product (of the pathway)?

E

What are the by products or waste products?

• CO2

• H2O

Carbohydrates

The primary energy source for most microorganisms

Glucose

Most common carbohydrate energy source used by cells

Cellular respiration

• aerobic respiration: O2 as final electron acceptor in process (ends at O2)

• anaerobic respiration: another inorganic molecule as the final electron acceptor in process (ends at a inorganic molecule)

Fermentation

Organic molecule as the final electron acceptor in process (ends at organic molecule)

2 processes to get energy from glucose

• cellular respiration

• fermentation

3 pathways for aerobic respiration

1. glycolysis

2. Krebs cycle

3. Electron transport chain

Glycolysis

1. means splitting sugar

2. also called Embden-Meyerhoff pathway

3. most organisms use this pathway

4. glucose (starts with oxidation of this) → lots of reactions → 2 pyruvic acid (end product of pathway)

5. net gain of 2 NADH + 2 ATP for each glucose

6. step doesn’t require O2

7. occurs in the cytoplasm (prokaryotes and eukaryotes)

Krebs cycle

1. also called the tricarboxylic acid cycle (TCA cycle) or citric acid cycle

2. generally doesn’t occur in anaerobic organisms

3. 2 pyruvic acid (starts to with oxidation of this) → lots of steps → cycles

4. net gain of 8 NADH, 2 ATP, 2 FADH2, and 6 CO2

5. CO2 (by-product) lost in humans during exhaling

6. occurs in the cytoplasm of prokaryotes and the mitochondria matrix of eukaryotes

In Krebs cycle

ATP made from substrate level phosphorylation - the high energy phosphate is transferred from a substrate to ADP

In Glycolysis

ATP made from substrate level phosphorylation - the high energy phosphate is transferred from a substrate to ADP

What is NAD+ and FAD in Krebs cycle?

Electron acceptor (get electrons and take them somewhere and dump off)

What is NADH+ and FADH2 in Krebs cycle?

Electron carrier

Electron transport chain

1. reduced coenzymes/electron carriers are now oxidized (lose electrons) at this step → OR the pass electron to start of chain and then electrons are passed down a chain of carrier molecules

2. energy (ATP) produced by chemiosmosis via a proton gradient created by carrier molecules

3. after gradient made - protons rush back into the cell through the enzyme ATP synthase to produce TP

4. O2 as a final electron acceptor can produce oxygen radicals

5. inhibited by cyanide, carbon monoxide, or the antibiotic Antimycin A

6. occurs on the plasma membrane of prokaryotes and the mitochondria membrane of eukaryotes

7. net gain of 34 ATP molecules/glucose

8. anaerobic respiration

CHEMiosmosis

Chemical gradient due to pH

ChemiOSMOSIS

Osmotic gradient due to higher concentration outside the cell than inside

Anaerobic respiration in electron transport chain

-Use something other than O2 (final electron acceptor)

• NO3-

• SO4 2-

• CO3 2-

-Not as efficient as aerobic respiration

-Use parts of pathways

How many electron carriers from Krebs cycle?

10

How many electron carriers from Krebs cycle + glycolysis?

12

How many net ATP made during glycolysis/glucose molecule?

2

How many ATP made from aerobic respiration/glucose molecule (prokaryote)?

38

Eukaryotes total ATP?

36

Fermentation

• Free up coenzyme to continue glycolysis

• releases energy from organic molecules (glucose)

• steps:

1. glycolysis

2. some fermentation cycle (depends on organism as to which cycles they do)

• does not require O2 (can occur when around)

• use organic molecules as a final electron acceptor

• small amount of ATP produced (step 2/glycolysis)

• main end products (depends on the fermentation cycle)

1. acid

2. alcohol

3. gas (waste products)

The glycolysis step/pathway does produce ATP (but it is a very small amount). So why is fermentation (step) important?

O2 is usually not around and the organisms need some energy production → recycles coenzymes (oxidizes them) and can keep them doing glycolysis

How much ATP is produced in the fermentation step/pathway?

0

Latic acid fermentation

• first step is glycolysis

• 2 pyruvic acid → steps →

• fermentation step oxidizes the reduced coenzymes

Alcohol fermentation importance

1. food industry - bread and beer production

2. gasohol - part of NE’s economy (ethanol plants)

3. cheese (propionic acid and carbon dioxide)

Fats

1. good storage form of energy

2. break down to form fatty acids and glycerol

3. beta oxidation of fatty acids

• lipase → enzyme to breakdown

• feed into glycolysis and Krebs cycle

• huge amounts of energy released

Protein catabolism

• start with enzyme protease and peptidase

• amino acids transported across plasma membrane

• converted to other molecules to enter glycolysis and Krebs cycle

1. deamination - remove amino group off amino acid

2. decarboxylation - remove - COOH

3. Dehydrogenation - remove H

Coronavirus

• coronavirus is common animal virus

• many strains of coronavirus that circulate constantly

• for most species-specific coronaviruses, the disease process is mild

• when coronavirus from one species gain the ability to infect a new species (an emerging virus) the disease can be severe

Coronavirus in humans

• many strains that circulate constantly and cause the common cold (15-30% of colds)

• most of the rest are rhinovirus infections

• deadly strains have emerged previously - which did not persist or don’t spread well

SARS

• 2002-2004

• bats to masked palm civets (animals) to humans

• 8000 infected (600-800 dead)

MERS

• 2012

• bats to camels to humans

• 2575 cases (886 deaths)

COVID-19

Bats (horseshoe) to pangolins to humans

Why bats?

• common infectious disease reservoir

• internal nose shape of a horseshoe

• infects same cells

• high core body temperature with flight

• special immune systems (interferon always on)

SARS COVID-2 pathogenesis

• incubation period 2-14 days

• like other human coronaviruses, target cells are respiratory epithelial cells

• receptor is angiotensin converting enzyme 2 (ACE-2)

• ACE-2 is present in lower respiratory tract cells (deep in the lungs)

COVID-19 disease

• flu-like, loss of taste/smell

• destruction of lung tissue

• severe pneumonia-like symptoms

• shortness of breath

• acute respiratory syndrome

• heart attack-like symptoms

• acerbation of underlying disease-heart disease, lung problems

• secondary infection

Prevention

• mask, social distance, avoid poorly ventilated, wash hands, avoid sick folks

• vaccine - make artificial immunity to spike protein

• mRNA version (Pfizer, Moderna)

• viral vector version (J & J)

Why is COVID-19 such a problem?

• virus better adapted to human receptors

• 80% of cases have few or mild symptoms

• about 30-50% may have no symptoms

Cholera signs/symptoms, complications

• acute, diarrheal illness

• rice water stool

• 12-20 liters of water a day

• dehydration

• no fever

• vomiting

Death from cholera

• dehydration - loss of fluids

• electrolyte imbalance

• mortality: 50% left untreated

Cause of Vibrio cholerae

• found in brackish water (mix between fresh and salt)

• produce enterotoxin

• makes epithelial cells highly permeable to water

• serotype 01 or 0139

Infectious does of cholera

• 10^6 in water

• 10^4 in food

• a high dose to get past stomach acid (don’t like acidic environments)

Transmission of cholera

• fecal-oral

• water

• raw foods

• undercooked foods

• washing foods in contaminated water

• found in copepods

Treatment for cholera

• IV fluids

• ORT (oral rehydration therapy)

• vaccination (oral)

Trace minerals are needed in low amounts as enzyme cofactors for growing bacteria.

True

What is the order of pathways for alcohol fermentation?

Glycolysis, ethanol fermentation

Lactic acid fermentation is common in muscle cells that have run out of oxygen.

True

What category of bacteria based upon their optimal growth pH would inhabit the human blood (around pH 7.4)?

Neutrophiles

Which of the following is the reason jams and dried meats often do not require refrigeration to prevent spoilage?

Low water activity due to hypertonic conditions

What category of bacteria based upon their optimal growth temperatures would include most human pathogens?

Mesophile

Only eukaryotic cells can grow by anaerobic respiration using other inorganic molecules as final electron acceptors.

False

Fermentation can be used to preserve foods like dairy and fruits.

True

What category of bacteria would not contain any enzymes to inactivate oxygen radicals?

Obligate anaerobes

Epidemiology of cholera

• John Snow - mid 1800s

• London outbreaks

• disassembling water pumps in certain areas to clean up cholera

Some bacteria grow by fermentation because they are able to get more ATP/glucose than in aerobic respiration.

False

Public health measures put into place to prevent cholera

• cleaned up refuse

• contained animals

• instituted water treatment

Background of EHEC (Enterohemorrhagic Escherichia coli)

• caused by Escherichia coli O157:H7

• cause of illness in 1982 during outbreak of severe bloody diarrhea

• most strains of E. coli are harmless and live in the intestines of healthy humans and animals

• part of normal microbiota

Escherichia coli O157:H7

• gram negative

• rod shaped

• facultative anaerobe

• has flagella

• O refers to the antigen associated with the lipopolysaccharide

• H refers to the flagellar antigen

Toxin (E. coli)

• strain produces a “shiga-like” toxins

• toxin starts by destroying cells which line the large intestine

• escapes into the blood stream and kills erythrocytes

• damages endothelial cells lining the blood vessels

• only need about 100 organisms to cause disease - can resist acidic conditions of stomach

Symptoms/signs E. coli

• abdominal cramps and diarrhea

• progress to bloody diarrhea (hemorrhagic colitis)

• maybe fever and vomiting

• illness resolves in 5-10 days

• severe - heamolytic ureamic syndrome (HUS)

Haemolytic uraemic syndrome (HUS)

• 5-10% of all infections develop into this

• children under 5 years o age and the elderly

• red blood cells are destroyed and the kidneys fail

Diagnosis (E. coli)

• isolation of E. coli in stool sample grown with Sorbitol MacConkey (SMAC) agar

• Selective - inhibit the growth of the gram + microbial and flora

• Differential - cannot utilize sorbitol on plate, therefore, not red

Source (E. coli)

• reservoir of this pathogen appears to be mainly cattle - can be up to 50% of feedlot cattle

• organisms can be mixed into beef when it is ground

• dried cured salami

• unpasteurized fresh-pressed apple cider

• yogurt, cheese and unpasteurized milk

• fruits and vegetables (sprouts, lettuce, coleslaw, salad)

• swimming in or drinking sewage-contaminated water

• petting zoos