Module 2

Metabolism

a complex series of breaking down larger molecules to obtain energy and using smaller precursor molecules to obtain energy to build molecular components

Energy

defined as the ability to do work

i.e. move flagella, build cell wall

Metabolic Pathway

series of reactions that make an end product

\-mediated by enzymes which catalyze biochemical reactions

enzymes

proteins that accelerate the rate of a reaction without being changed themselves

Activation energy

the input energy needed in order to generate a high energy transition state

Transition state

high energy state that is on the way from converting a product or converting substrate into a product

\-

(+ or -) ∆G is spontaneous and exergonic

\+

(+ or -) ∆G is non spontaneous and endergonic

Reaction Rate

Determined by a reactions activation energy, the input of energy needed to generate the high energy transition state…. nothing implied by the spontaneity of rxn

Catalytic site

The site of activity

\-competitive inhibitor binds here

Allosteric site

The site of the regulator that changes the enzyme conformation (to help or block)

ATP

primary energy currency of the cell

\-contains a base (adenine), ribose sugar, and 3 phosphates

\-always forms a complex with Mg2+ because it partially stabilizes negative charge

\-energy comes from oxygens repelling each other

\-”medium sizes” energy carrier

Electron Carriers

Molecular shuttles that repeatedly accept and release electrons and hydrogens to transfer redox energy

\-Most common is NAD+ (oxidized)

NAD+

\-the most common energy carrier

\-carries 2-3 xs more energy than ATP

\-accepts hydrogens and a pair of electrons

\-Reduces NADH and H+

\

Catabolism

breaking down molecules to produce energy

Anabolism

using building blocks (sugars, amino acids) to make large macromolecules (energy requiring)

Fermentation

all the electrons from organic substrates are put back onto the organic products

\-occurs without oxygen… can occur in presence (won’t use it)

\-No ETC

\-Organic molecule is final e- acceptor

\-Low ATP produced

Respiration

electrons removed are ultimately transferred to an inorganic electron acceptor

\-can occur with or without oxygen

\-Oxygen is final electron acceptor

\-High energy yield

EMP Pathway

Glucose catabolism pathway (glycolysis)

\-glucose 6-phosphate → 2 pyruvate

\-final product: 2 ATP and 2 NADH

\-used by bacteria, archaea, eukaryotes

\-most common form of glycolysis

\-occurs in cytoplasm

\-functions in the presence or absence of oxygen

ED Pathway

Glucose catabolism pathway used primarily by gut bacteria

\-Final Product: 1 ATP, 1 NADH, 1 NADPH

\-essential for bacteria to colonize intestinal epithelium

\-many gut flora use as primary glycolytic pathway

\-forms 2 pyruvate

NADH

\-molecule used to transfer its electrons to store energy

NADPH

\-molecule used for biosynthesis (biosynthetic pathways)

Pentose Phosphate Pathway

\-Glucose metabolism pathway

\-Key intermediate is ribulose 5-phosphate (5C)

\-Produces sugars of 3-7 Carbons

\-Serves as precursors for biosynthesis or are converted to pyruvate

\-Generate 1 ATP, 2 NADPH

\-Many different intermediates

Pyruvic Acid

In strict aerobes and some anaerobes, __ enters the Krebs cycle.

\-can be fermented anaerobically to multiple end products

\-can serve as a source of raw material for synthesizing amino acids and carbs

Oxidative phophorylation

the process where ATP is formed as a result of transfer of electrons from NADH to O2 by a series of electron carriers

\-doesn’t require O2

\-Eukaryotes: occurs in mitochondria

\-Prokaryotes: occurs in cell membrane

\-NADH+ is oxidized

\-indirect energy source (ETC)

Substrate level phosphorylation

Formation of ATP from ADP and phosphorylated intermediates

\-NAD is reduced

\-direct energy source

Respiration

Reasons:

\-Cells lack a sufficient amount of an inorganic final electron acceptor

\-Cells lack genes to make appropriate complexes and electron carriers in the ETC

\-Cells lack genes to make 1 or more enzymes in the Krebs cycle

Fermentation

The transfer of electrons from a reduced, organic electron acceptor to an organic molecule

\-Yields 2 ATP through substrate level phosphorylation

\-Anaerobic process

\-Doesn’t involve ETS

\-Doesn’t directly produce any additional ATP beyond that made during glycolysis

\-Little ATP is produced but bacteria can still grow rapidly due to increased rate of glycolysis

Lactic Acid Bacteria (LAB)

\-Ferment pyruvate by reducing it to lactic acid

\-Pyruvate + NADH   Lactic acid + NAD+

\-used by pathogens (streptococcus), normal gut flora (Bifidobacteria) microbes used in yogurt/sour cream (lactobacillus)

Homolactic fermentation

\-Lactic acid is the only fermentation product

\-used for cheeses/yogurts

Heterolactic fermentation

\-Mixture of lactic acid, ethanol, and/or acetic acid, CO2

\-Used to sour vegetables

Alcoholic fermemntation

\-occurs when pyruvic acid is converted to ethanol. CO2 is also a byproduct

Mixed Acid Fermentation

\-Produces a variety of products… acetic acid, lactic acid, succinic acid, formic acid, CO2, and Hydrogen gas

Redox Reactions

\-involve transferring electrons from donor to an acceptor

\-Can result in energy release

\-the more e- a molecule has, the more energy rich it is

\-2 half reactions

Standard redox potential

v

\-the tendency of a molecule to acquire electrons
\-equilibrium constant for a redox reaction
\-more negative = better e-donor
\-more positive = better e- acceptor
\-+ E = spontaneous

\- -E = nonspontaneous

Electron Transport System

\-includes proteins and small organic molecules that can be reduced and reoxidized cyclically

\-redox reaction stores energy as ion gradients across cell membrane

\-no ion gradient => no H+ available (alkaline)  or too many H+ (acidic)

\-Occurs in the cell membrane in bacteria

\-cytochrome=e- acceptor in aerobic respiration

\-reduction state of cytochrome involves metal ion (heme)

\-electrons transferred to a final electron acceptor -> product leaves cell

Lipid Metabolism

\-Requires more energy accepting carriers like NAD+ and NADPH

FADH2

Alternate electron carrier that enables more efficient use of food

Geobacter

\-bacteria that reduces metal and can have 100 different cytochromes in its envelope to allow for reduction of metal

Oxidoreductases

Electrons transport proteins that oxidize 1 substrate and reduce another

Proton Motive Force (PMF)

\-Transfer of electrons in electron transport chain yields energy to move ions

\-stores energy to drive cellular processes

\-Chemiosmotic theory couples electron transport to ATP synthesis

Drug efflux pumps

\-Pump antibiotic out of the cell using antiport/symport type of movement

\-use H+ ion energy

Electron Transport System

\-Requires 3 functional systems: substrate oxidoreductase, mobile e- carrier, Terminal oxidase

Substrate Oxidoreductase (Dehydrogenase)

Oxidation of NADH and reduction of quinone (energy source)

Quinone

-can receive 2 e- from the substrate, oxidoreductase, and the 2 H+ from solution

\-negative charge is balanced to quinol (mobile e- carrier) which transfers onto cytochromes

\-adds proton potential (outside of cell)

ATP synthase

proton driven synthesis of ATP that completes cycle of oxidative phosphorylation

\-embedded in membrane

\-reversible process/pump

\-ADP → ATP

Cyclic Photophosphorylation

\-Energy source: light energy creates high energy electron donor and low energy electron acceptor

\-one photosystem (e-get sent back to PS I)

\-Anoxygenic process

non-cyclic photophosphorylation

2 different photosystems

electrons lost by PSII go into PSI

\-produces NADPH

\-Oxygenic → Needs constant supply of water

\-Net products: ATP, NADPH, O2

cytokines

small proteins made by the immune system that are secreted by microbes

\-Voice of the immune system

Chemokines

small proteins made by the immune system that are secreted by microbes

\-Directional Movement

Bacteriodetes

A genus of gram negative, obligately anaerobic, non-endospore forming bacteria in the gut

\-25% of gut microbes

\-energy source: plant glycans

Short Fatty Acid Chains

carboxylic acids with aliphatic tails of 1-6 carbons

\-product from indigestible glycans in microbial fermentation

\-signal through G-protein coupled receptors to initiate immune signaling cascades

Obese

____ individuals have more Firmicutes and fewer Bacteriodetes than lean individuals

\-due to mutated leptin gene (controls hunger)

genome

all the genetic info that defines an individual

* (microbial) usually consist of 1 circular chromosome

vertical gene transfer

Transfer of genes from parent to offspring

Horizontal gene transfer

Transfer of genes from cell to cell

\-only in prokaryotes

\-ex. antibiotic resistance gene from cell → cell

Capsule

source of difference between smooth and rough strains

\-provides adhesion, defense against host immunity, protection against drying out (dessication)

\-S strain = pathogenic/deadly

Plasmids

virulence factors encoded for on plasmids

\-nonessential pieces of DNA

\-Double stranded circles of DNA

\-Can be duplicated and passed on during conjugation (horizontal)

\-often confer protective traits

\-replicated independent of chromosome

\-S strain coded for a capsule on ____

Transformation

the process by which R strains picked up genetic material from S cells in environment in Griffith Experiment

\-NOT HORIZONTAL/VERTICAL TRANSFER

monocistronic RNA

Genes can act independently of one another

\-1 promoter, 1 gene

Polycistronic RNA

Genes can exist in tandem with other genes

\-Operon region, one promoter, many genes

Operon

collection of genes and operons at different points on the chromosome with a unified biochemical purpose

\-SINGULAR

Regulon

collection of genes and operons together

\-MULTIPLE

molecular face

the surface of the grooves on DNA

\-provide a unique surface for binding sites for DNA binding proteins to recognize

Bacterial Nucleoid

Bacteria pack their DNA into a series of loops or domains

\-loops anchored by histone-like proteins

OriC

Contains 9 base pair repeats called DnaA boxes.

\-DnaA-ATP binds to the nonamers and then interacts with the 13 bp repeats

\-Causes DNA to open

SeqA

prevents initiation of another DNA replication event by binding to hemimethylated DNA and preventing binding

\-occurs once active levels of DnaA decrease

DnaB

helicase

DnaC

helicase loader

DnaG

(primase) lays down RNA primers and single stranded binding proteins bind to the DNA

DnaN

sliding clamp; increases processivity

\-helps keep polymerase onto strand for longer

DnaQ

episolon subunit of DNA polymerase III that contains proofreading activity/nucleotide excision repair

alpha subunit

Subunit of DNA polymerase III responsible for synthesis

Beta subunit

Subunit of DNA polymerase III that contains sliding clamp

Sigma subunit

Subunit containing RNA polymerases

Replisome

consists of 2 DNA polymerase III enzymes (1 for each strand), primase, and helicase

DNA polymerase I

The polymerase that excises the RNA primers and fills in the resulting gaps

\-DnaQ; exhibits proofreading

ligase

fills in phosphodiester bond (not adding new nucleotides)

Tus protein

protein that binds to the termination sequences

\-stops DnaB (helicase) activity → ending replication

Topoisomerse

Separates rings between chromosomes with help of proteins XerC and XerD

\-can cut the backbone of DNA

XerC and XerD

proteins that pull 1 chromosome through the other to get 2 individual pieces of DNA

MreB

a cytoskeletal protein that forms a spiral inside the periphery of the cell. It is the major shape determining factor in prokaryotes. If mutated, chromosomes do not separate.

Septation

the formation of cross wall between daughter cells that occurs in several steps:


1. Assembly and formation of Z ring (FtsZ protein)
2. Linkage of Z ring to plasma membrane
3. Constriction of cell and septum formation

SlmA

protein that prevents Z ring formation

Z ring formation

\-Step 1 in septation

\-Regulated by Min CDE system, limiting the Z ring to the cell center (where there’s no CDE)

Transformation

Uptake of DNA from outside the cell

Transduction

Transfer of a DNA fragment from 1 bacterium to another by a bacteriophage

Conjugation

transfer of DNA from a living donor bacterium to a living recipient bacterium by cell-cell contact

Sigma Factor

Part of DNA dependent RNA polymerase holoenzyme

\-Detects the promoter, which signals the beginning of the gene

\-”houskeeping” factors that recognize consensus sequences at the -10 and -35 positions

\-10 site

site of promoter and pribnow box (like TATA)

\+1 site

site of the start of transcription

Rho-dependent

Transcription termination signal that relies on a Rho protein and a strong pause site at the 3’ end of the gene

Rho-independent

Transcription termination signal that requires a GC-rich region of RNA, as well as 4-8 consecutive U residues

Rho protein

a 3’ C rich sequence that appears to be a pause site

\-slows down because more H bonds to break between C and G

\-Rho factor binds to a pause sequence forming a hexamer

\-Rho helicase activity unwinds RNA-DNA duplex, releasing polymerase

Rho independent termination

GC rich region and sequence of U’s at the terminus

\-GC region forms a stem loops structure that interacts with the RNA polymerase, causing a pause

\-Complex falls apart

Rifamycin B

Antibiotic that selectively binds to the bacterial RNA polymerase, inhibiting transcription initiation

Actinomycin D

Antibiotic that non-selectively binds to DNA to inhibit transcription elongation

ribosome

composed of 2 subunits, each of which includes rRNA and proteins

* in prokaryotes, the subunits are 30S and 50S which combine to form a 70S ribosome

Shine Dalgarno Sequence

ribosomal binding site used for recognition

\-Complementary to a sequence at the 3’ end of 16S rRNA of the 30S subunit

\-encoded in RNA

Streptomycin

antibiotic that inhibits 70S ribosome formation