Central Michigan Universitypeople.cst.cmich.edu/alm1ew/208 unit 2 metabolism.docx · Web viewAs a result of their fermentative metabolism on this bounty of glucose, E. coli will produce

1BIO 208 Unit 2 Patterns of Metabolism

This outline is intended to facilitate your preparation for lecture. This web outline will NOT substitute for regular lecture attendance.

While we will be covering the topics outlined in Chapter 5 of your text, we will be doing it in a very different manner from how it is presented in the text. Please be prepared to take careful notes in class.

III. Patterns of Metabolism in the Microbial World (a.k.a. how do microbes make a living – and who cares?)

A. The Basics: background info

Metabolism = sum of all chem. rxns occurring within a living organism

All cells need a source of energy for:

Catabolism- breaking bonds in molecules –

Ex. glucose to carbon dioxide and water

Anabolism – creating bonds -


The coupling of catabolism and anabolism is made possible through adenosine triphosphate (=ATP. ADP stands for adenosine diphosphate)

During catabolism – ATP ADP + Pi + energy Pi = inorganic phosphate

During anabolism – ADP + Pi + energy ATP

ATP can be formed in 3 ways:

1. by substrate level phosphorylation – the simplest, oldest and least-evolved way to make ATP - a high energy phosphate is removed from a substrate and is added to ADP to make ATP. Ex. C-C-C~P + ADP C-C-C + ATP

2. by oxidative phosphorylation aka electron transport phosphorylation – electrons are transferred from organic compounds to electron carrier molecules and then to final electron acceptor molecules. The transfer of electrons releases energy that is used to convert ADP ATP.

3. by photophosporylation – occurs in photosynthetic cells only. Light energy is converted to ATP.

We will see examples of #1 and #2 as we continue our discussion

adenine

ribose

Adenosine diphosphate (ADP)

Adenosine triphosphate (ATP)

unstable bond = high energy

Adenosine monophosphate (AMP)


Oxidation-Reduction Reactions

Electrons (e-) in molecules contain energy.

Oxidation – removal (loss) of e- -

Reduction – addition (gain) of e- -

Oxidation and reduction reactions are always coupled-

In biological molecules it is usually the entire H atom (electron and proton) that is lost or gained, but not always; sometimes the electrons are separated from the proton and only the electrons are lost or gained; and sometimes it may be one H entire atom plus 1 additional electron (from a second H atom) that are lost or gained.

In any pair of molecules you can distinguish which is in the oxidized state and which is reduced:

Oxidized state: Reduced state:Contains more oxygen atoms OR Contains fewer oxygen atoms ORfewer hydrogen atoms AND more hydrogen atoms ANDtherefore has fewer electrons and is therefore has more electrons and isless negative (or more positive) more negative (or less positive)

Glucose 2 PyruvateC6H12O6 2 C3H4O3

NAD+ NADH

Sulfate Hydrogen sulfide SO4 H2S_____ _____ ________________________


B. Patterns of Energy Production

1. Basic information

Patterns among Eukaryotes: alcohol fermentation (yeast) lactic acid fermentation (muscle cells, neutrophils) aerobic respiration (mold, protozoa, animals) oxygenic photosynthesis (algae, plants)

Prokaryotes do all the above plus: anaerobic respiration: uses inorganic molecules other than 02 as a final electron acceptor lithotrophy: use of inorganic substances as sources of energy photoheterotrophy: use of organic compounds as a carbon source during bacterial photosynthesis anoxygenic photosynthesis: photophosphorylation in the absence of O2 methanogenesis: uses H2 as an energy source and produces methane light-driven nonphotosynthetic photophosphorylation: converts light energy into chemical energy

There are 2 initial sources of usable energy:1. sunlight – 2. chemical bonds of molecules -

Heterotrophs - energy is created by breaking bonds in a molecule and harvesting the electrons released from the H atoms in:

a. Organic molecules -

b. Inorganic molecules –

The more electrons a molecule has, the more energy it is capable of releasing. This initial molecule is called the electron donor.

Ex. glucose (C6H12O6) has a lot of H atoms (12) and therefore a lot of electrons, its oxidation will release a lot of electrons. Glucose is a high energy electron donor.


The electrons have to go somewhere - they get passed from the initial donor of released electrons (electron donor) to intermediate electron carrier molecules.

NAD (Nicotinamide Adenine Dinucleotide) -

accepts 2 e- (and 1 proton) and becomes reduced toNAD+ NADH

PROBLEM: NAD+ is present in limited amounts in the cell and could be depleted; it must be regenerated if energy production in the cell is to continue.

Oxidized state

missing an H,

(which means

missing an e-)

Reduced state

(complete with e-)

NAD = Nicotinamide Adenine Dinucleotide a mobile, cytoplasm soluble electron carrier


2. Example – generation of energy via carbohydrate catabolism – specifically glucose

Glycolysis (= Embden-Meyerhof pathway) – occurs in the cytoplasm – electron donor is glucose

Glucose + 2 ATP 2 Pyruvate + 4 ATP net ATP production = 2 ATPC6H12O6 2 C3H4O3


End of glycolysis - 2 ATP and 2 Pyruvate (C3H4O3) and 2 NAD+ converted to 2 NADH

Need to regenerate NAD+

Fermentation – occurs in the cytoplasm - pass e- from NADH to an organic molecule, regenerates NAD+

Ex. 2 C3H4O3 2 NADH + 2H+

2 C3H6O3 2 NAD+

(lactic acid)

Inefficient –

End of fermentation - 2 ATP and some fermentation end products and 2 NAD+ regenerated


Alternative (to fermentation) strategy:

Respiration – pass e- along a series of electron carrier molecules, ultimately to a final (or terminal) electron acceptor molecule, regenerates NAD+

Step 1 – Tricarboxylic acid (TCA) cycle (also known as citric acid cycle or Krebs cycle) – occurs in the cytoplasm - harvests the energy stored in pyruvate but transfers an even larger number of e- to NAD+ (which converts it to NADH).

At this point following TCA:For each pyruvate (C3H4O3) 1 ATP + 3 CO2 + 4 NAD NADH and 1 FAD FADH2

For each glucose 2 ATP + 6 CO2 + 8 NADH+ 2 FADH2


Step 2 – Electron Transport Chain – the soluble NADH and FADH2 carry e- from the cytoplasm to the cytoplasmic membrane and pass them off to a series of membrane associated electron carriers (NAD+ is regenerated when NADH passes the e-), ending with the final or terminal electron acceptor.

This final electron acceptor may be oxygen –

Electron acceptor (oxidized state) becomes reduced to½ O2 H20 aerobic respiration

1 molecule of C6H12O6 oxidized completely to CO2 coupled to reduction of oxygen to water (aerobic respiration) can yield up to a max of 38 ATP.


OR

This final electron acceptor is something other than oxygen –

Examples of final e- acceptors for anaerobic respiration:

Electron acceptor (oxidized state) becomes reduced to:

Fe3+ Fe2+ Iron respirationferric iron

NO3- NO2-, N2O, N2 Nitrate respiration

nitrate

SO42- HS- Sulfate respiration

sulfate

CO2 CH4 Methanogenesiscarbon dioxide methane

S0 HS- Sulfur respiringsulfur

yield of ATP is greater than 2 but fewer than 38 ATP


C. Some Exciting Implications of Microbial Activity:

1. Metabolism of the Human Intestinal Community

a. Where does your gut microbial community come from?

At birth

Progression of your gut community if you were a breast-fed baby

Day 1 - First colonizer was Escherichia coli

facultative anaerobe chemoorgano heterotroph (both C and E from organic)

Where did E. coli come from?

What organic cmpd does E. coli use as a C and E source?

How does E. coli get C and E from lactose?


galactose

Entner-Douderoff

2-keto-3-deoxygluconic acid 6-phosphate

pyruvate glyceraldehyde 3-phosphate

lactose

lactose

glucose

glucose 6-phosphate

2 pyruvate

2 acetyl CoA

lactic acidformic acid CO2 + H2

succinic acid

acetic acidethanol

CO2

CO2

Embden-Meyerhoff-Parnas

fructose 6-phosphate

2 glyceraldehyde 3-phosphate

Krebs cycle (=TCA = CAC)Electron transport

26 ATP from each glucose

(52 ATP from each lactose)2 ATP from each glucose

(4 ATP from each lactose)

outsideinside

Lactose utilization by E. coli


Day 3 – added 2 more bacteria

Enterococcusobligate but aerotolerant anaerobeschemoorgano heterotrophsobligate fermentative metabolism

BifidobacteriumLactic acid bacteria (named from their final fermentation end product)

e-1 Glucose NAD+

glycolysis2 ATP e-

2 Pyruvate NADH

fermentation

2 Lactic acid NAD+ regenerated(excreted waste)

Soon after added:

Enterobacter facultative anaerobeClostridium obligate (aerotolerant) anaerobe

fermentative metabolism

Butanediol fermenters (named from their final fermentation end product)

e-1 Glucose NAD+

glycolysis2 ATP e-

2 Pyruvate NADH

fermentation

ethanol Acetoin CO2 + H2 NAD+ regenerated

acetic acid lactic acid succinic acid

2,3-Butanediol + CO2 all of these are excreted waste products


Community of breast-fed infant from 1 week to ~ 3.5 months:E. coliEnterococcusBifidobacteriumEnterobacterClostridium

3.5 months to weaning99% Bifidobacterium infantis –another lactic acid fermenter

When meat is introduced:

Gram-negative anaerobes:BifidobacteriumClostridiumFusobacteriumEubacteriumRuminococcusPeptococcusPeptostreptococcusBacteroides – 30% of total adult community

Bacteroides obligate anaerobeextremely oxygen sensitivefermenter


b. What are the benefits of a stable, mature gut community?1) Nutrition 2) Prevents colonization by pathogens3) Trains the immune system

1) NutritionComplex polysaccharides are converted to volatile fatty acids (vfa)

Bacteroides is the key player

Host and dietary carbohydrates – complex carbs, starch, cellulose

saccharolaseshydrolases

fermentation

volatile short-chain fatty acids*

acetic acid butyric acid propionic acid

reabsorbed through the large intestine

used by you as an energy sourceprovide a significant proportion of your daily energy requirement (540 kcal)

* These products in brown are good for your health metabolic by-products


Dietary fats

Liver

Bile acids

Absorbed by small intestine

----------------------------------------------------------------------------------------------------

If fats and bile acids are not reabsorbed by small intestine but make it to colon

deconjugated

deoxycholic acid intermediate productslithocholic acid

Bacteroides thetaiotomicron

ethyl ester

*These products in purple are mutagenic, carcinogenic products; they can induce cancer – bad for your health products


Dietary protein

Peptides

Amino acids

Absorbed by small intestine

----------------------------------------------------------------------------------------------------Combined activity of thecolonic microbial community

If peptides are not reabsorbed by small intestine but make it to colon

Amino acids

R

+H3N – C – C – O-

H – O

deamination decarboxylationaromatic sulfuramino acids amino acids

reduced tophenolic SO4 H2S gascompounds anaerobic respiration by

fermentation sulfate-reducing bacteria by many microbes

ammonia H2 branched chain volatile CO2

fatty acids fatty acids reduced to

CH4 gas anaerobic respiration by Methanogens (which are Archaea)

brown and purple as explained before. red are final electron accepts in anaerobic respiration


We’ve seen aerobic respiration, fermentation, and anaerobic respiration in chemoorgano heterotrophs. Now let’s bring in some chemolitho autotrophs and look at the effects of their metabolism!

All microbes need:a source of energy (electrons = ATP)a source of C to build macromolecules (-C-C-)

Heterotrophs that we have talked about already– get C to make –C-C- by recycling the C contained in organic molecules (like sugars or amino acids). (all heterotrophs get energy (electrons and therefore ATP) to make –C-C- from breaking chemical bonds)

**Autotrophs – get C to make –C-C- from CO2

But there are 2 sources of energy that can be used to turn CO2 -C-C- and the source defines 2 groups of autotrophs:

1. Photo autotrophs

energy from sunlight (C from CO2) (I’ll leave this for Botany, but lots of microbes do this too)

2. Chemolitho autotrophs

energy is generated from inorganic chemicals (C from CO2)

Many different inorganic chemicals can serve as electron donors to provide the energy for microorganisms via aerobic respiration (notice the presence of O2 as final electron acceptor in all the equations following – therefore all chemolitho autotrophs are obligate aerobes):

a. Hydrogen gas as an electron donor

e- donor e- acceptor reduced toH2 + 1/2 O2 H2O

Hydrogen bacteriaEx. Alcaligenes faecalis (from Lab 8)

b. Sulfur compounds as electron donors

e- donor e- acceptor reduced to2S + 2H2O + 3O2 2H2SO4

Ex. Sulfur bacteria like Thiomargareta namibiensis or the bacteria that form snot-tites in caves.


c. Nitrogen compounds as electron donorsNitrifying Bacteria

2 groups of Nitrifying Bacteria:

1) 2NH3 + 3O2 NO2 + 2H2O + 2H+

Ex. Nitrosomonas

2) 2NO2 + 2O2 2NO3 + 2H+

Ex. Nitrobacter

d. iron as an electron donor

Fe2+ + 1/2 O2 + 2H+ Fe3+ + H2O

Iron bacteria like Ferroplasma

Two scenarios where chemolitho autotrophs are very important:

Metabolism of Wastewater TreatmentHow do we go from toilet water to treated water? (stay tuned, we will discuss this in Unit 4 )


Metabolism of the Deep - What? No photosynthesis???

Deep sea hydrothermal vents – provide all the necessary chemicals Black smokers – vent hydrogen, sulfur, iron (electron donors for energy), and CO2 (for

carbon) from the Earth’s core Sea water contains dissolved oxygen (electron acceptor for aerobic respiration) Everything that is needed for chemolithoautotrophs to grow.

Chemolithoautotrophic metabolism turns CO2 and inorganic chemicals into bacterial biomass, with excess energy to spare!

Animals (chemoorganoheterotrophs)Giant tube worms

with endosymbiotic chemolithoautotrophsGiant mussels

Brittle starsLimpetsWormsCrabsVent fishSharks

AssignmentRead Chapter 5Review 4, 5b,c, 6,7,9MC 1,4,6CT 1,3,5


Supplemental Information – For your information (FYI) onlyHow can 2 people eat the same foods, 1 person gains weight and the other stays lean?

colonization of gut by microbes increases glucose uptake in the intestine

↓

microbial fermentation↓

resulting in substantial elevations in serum glucose and insulin

results in production of short-chain fatty acids

stimulate lipogenesis in the liver

↓

triglycerides into the circulation

↓

taken up by adipocytes (fat cells)

The composition and operation of your gut microbiota influences your energy balance.

Relatively high-efficiency gut microbial communities would promote energy storage (weight gain), whereas lower efficiency communities would promote leanness.

Small but long-term differences between energy intake and expenditure can, in principle, produce major changes in body composition.

Ex. if energy intake exceeds energy expenditure by +12 kcal/day, >1 lb of fat could be gained in a year; this is the average annual weight gain experienced by Americans between ages 25 and 55.

2). Prevents colonization by pathogens – pathogens like Salmonella, Shigella, Campylobacter, the pathogenic strains of E. coli, etc. that cause intestinal disease. If we have time…

a. competition for attachment sites – the gut epithelium is so densely colonized by normal microbiota, nowhere for pathogens to attach.

b. competition for nutrients – if pathogens do attach, they have to fight normal microbiota for a share of nutrients

both


c. antimicrobial chemicals – and then the normal microbiota secrete antimicrobial chemicals that kill pathogens.

Ex. E. coli – produces a chemical called colicin


3) Trains the immune system

The primary barrier between the outside world and you is a single layer (1 cell thick) of gut epithelium. This barrier is tight, but not impenetrable.

Microvilli – where adsorption takes place

Epithelium

Submucosa

Muscle

The surface of the intestinal epithelium is protected by your immune system – the antibody IgA, the white blood cells called T and B lymphocytes, and phagocytic macrophages.

The gut epithelium tests the contents of the gut lumen (open cavity) and can directly sense the antigens of microbes using “pattern recognition receptors”

(PRRs) – the epithelium recognizes conserved structures of bacteria and

viruses and then alerts the host to the potential of infection.

Normal microbiota of the gut and dietary antigens in food are tolerated (should not stimulate an immune response).


c. How does what you eat influence your gut community and in turn your health? –So very cool!!

1). Over stimulation of microbial growth and metabolismEx. Lactose intolerance

In babies, the enzyme human lactase is secreted by the small intestine and will break milk lactose into glucose and galactose. By the age of weaning, humans stop secreting human lactase.

After the age of weaning if lactose is consumed in dairy, it will pass undigested to the large intestine. In the large intestine E. coli will secrete the enzyme -galactosidase, which will now break lactose in to glucose and galactose. The E. coli will use the glucose as a carbon and energy source to support rapid population growth.

As a result of their fermentative metabolism on this bounty of glucose, E. coli will produce a lot of 3 carbon fermentation end products, and a lot of CO2 gas. The 3 C end products increase the osmotic pressure in the large intestine, which combined with the CO2 will results in the symptoms of bloating and diarrhea

Adult lactose intolerance is the normal state for humans. People who as adults can tolerate lactose had ancestors that acquired a mutation that allows them to continue to secrete human lactase in to adulthood.

2). Diet can upset immune system training – if we have time…

The gut immune system has the challenge of responding to disease-causing microbes but not responding to food antigens and the normal gut microbial community.

In developed countries like the U.S., this discriminatory ability appears to be breaking down.

High-fat, high-sugar, low-fiber diet changes gut community composition, which upsets immune training resulting in allergies and/or chronic inflammation

Ex.1. Allergies Children w/ allergies have a higher chance of having bad Clostridium difficile and Staphylococcus aureus and lower prevalence of good Bacteroides and Bifidobacteria in their gut.

Ex. 2. Chronic inflammationCrohn's disease and ulcerative colitis (UC)

? breakdown in tolerance to Bacteroides initiates an autoimmune reaction?

Experimental txt - whipworms


3). Diet can promote abnormal cell growth – i.e., cancer

Examples of suggested links between microbial metabolism and cancer:

1. High fat diet – go back and look at diagram of what happens to fat in the gut

conjugated secondary bile acids – are carcinogens

2. High protein diet – go back and look at protein diagram again

protein fermentations may be sources of systemic toxins Heterocyclic amines (HCA) are converted into carcinogens. phenolics from aromatic amino acids may enhance production of mutagens. reduced sulfur compounds (like H2S) may be toxic to the colonic epithelium.

3. Alcohol consumption

acetaldehyde toxicity

Look again at diagram of lactose utilization by E. coli. See where ethanol is produced by mixed acid fermentation? An intermediate molecule in the pathway Acetyl CoA ethanol is a toxin called acetaldehyde

Acetyl CoA acetaldehyde ethanol

Part of this pathway also runs in the reverse direction:

oxidation mitochondria in the liver cellsethanol acetaldehyde (bad) acetic acid (good)

alcohol dehydrogenase aldehyde dehydrogenase

If there is a lot of ethanol being converted to acetaldehyde, the hepatic mitochondrial enzyme aldehyde dehydrogenase cannot keep up, and acetalydehyde levels build in the liver and blood. This causes symptoms of hangover in the short term, in the long term the acetaldehyde causes mutations in DNA that can lead to cancer.

Pr e biotics are complex carbohydrates that you cannot digest, such as fructo oligosaccharides (FOS). They pass to the intestines where they stimulate the growth and activity of intestinal bacteria that secrete beneficial metabolic end products. Fruits and vegetables contain oligosaccharides; bananas and artichokes are especially high.

Pr o biotics are living bacteria from genera that produce favorable end products, such as Bifidobacterium and Lactobacillus.

Documents

Central Michigan Universitypeople.cst.cmich.edu/alm1ew/208 unit 2 metabolism.docx · Web viewAs a result of their fermentative metabolism on this bounty of glucose, E. coli will produce