Micro Exam 3

Vaccination

deliver foreign antigens into the body to stimulate an individual’s immune response

vaccine goal

develop long term immunological memory → T and B cell responses

feature common to all vaccines

the presence of an antigen designed to safely trigger an immune response

*Administered before exposure

antibiotic vs antiviral vs vaccine

antibiotic - target bacteria, post-exposure

antiviral - target viruses, post-exposure (PEP)

vaccine - does not treat infection

herd immunity

when a significant portion of a population is immune to a disease and that immunity subsequently protects the remaining portion that cannot become immune

lots of vaccines = herd immunity for all

critical threshold = decrease/slow of pathogen spread

polio virus mutations

(enters orally and replicates in the throat and intestines)

mutations in the 5’ end of the poliovirus genome result in a loss of tropism for neurons → attenuated strain could no longer induce paralytic polio

vaccine types

whole = use whole virus/bacterium

subunit = using parts that trigger the immune system (safe/weak)

nucleic acids = just the genetic material

live attenuated vaccine

whole

contains a living pathogen that is actively replicating and can stimulate an immune response but has lost its ability to cause disease

inactivated vaccine

whole

contains a pathogen that is inactivated using a physical or chemical procedure (heat, irradiation) → eliminates infectivity/pathogenicity

*safe for pregnant/immunocompromised

chimeric/vector

whole; both use a whole carrier organism to deliver genetic material

chimeric: a single hybrid virus combining genes from both → virus produces antigen, outside looks like the target

vector: a carrier virus housing genetic material from another virus → host produces the antigen, virus vector delivers the target

recombinant

subunit

antigen is made by genetic engineering

e coli

conjugate

subunit

a polysaccharide chemically linked (conjugated) to a carrier protein

HIV, works in infants

toxoid

subunit

a virulence or pathogenicity factor (protein) from a bacteria that best stimulates the immune response; toxins are inactivated by heat or chemicals to reduce rxns

clostridium tetani

genetic

nucleic acids

contains genetic material (dna/mrna) for a pathogen’s antigen instead of the antigen itself; body cells read and make antigen → b and t cells

fast/updated but short-lived immunity

adjuvant

improves the immune response to the vaccine, sometimes by keeping the vaccine at the site of injection for longer or by stimulating the local immune response

*produces a more robust immune response with less antigen

vaccine failure

1 failure to elicit the anticipated protective immune response (evasion, strains, mutations)

2 too many side effects

3 repeat vaccine does not produce long term immunity (mutations, people don’t want injections)

metabolism

biochemical reactions required for life

catabolic (energy-obtaining) - produce ATP

anabolic (energy-consuming - building) - use ATP

chemical work

synthesis of complex molecules

anabolic

transport work

uptake of nutrients, elimination of waste, and ion balance

mechanical work

cell motility and movement of structures within cells

ecosystems

MM (microbial metabolism) contributes nutrients and oxygen

cyanobacteria: primary producers, dissolved oxygen, eutrophication, nitrogen fixers

climate changes

microorganisms produce and utilize key greenhouse gases (carbon cycling)

respiration, fermentation, photo/chemoautotrophy, methanogens, methanotrophs

agriculture

effective agriculture is heavily impacted by microbial nutrient cycling

mineralization, assimilation, fermentation

food microbiology

food production often relies on fermentation for taste and preservation

infection (microbial metabolism)

determine sites of infection, targets of antibiotics/microbials, tissue damage, diagnostics

ATP

energy currency

ATP hydrolysis: exergonic, coupled with endergonic rxns

ATP has a high phosphate transfer potential → donates easily and more readily made

Substrate level vs oxidative phosphorylation

sub: ATP is produced by transferring phosphoryl group from another molecule

• glycolysis, TCA, fermentation

ox: transfer of electron from donor to recipient leading to proton gradient that is harnessed for ATP synthesis

• ETC, respiration

• * high ATP production

redox tower

[acceptor/donor]

[oxidized/reduced]

reduction potential and free energy

free energy: energy available to do work (delta G)

• high distance = high free energy produced

reduction potential: E’0

• negative = reduced substance has strong donation

• positive = oxidized substance has strong acceptance

good donor top, good acceptor bottom

half reactions

OIL RIG (oxidation is loss, reduction is gain)

oxidation: electron donated

reduction: electron accepted

enzymes

protein catalyst, reduce activation energy → increase rate of rxn

often interact with coenzymes and cofactors

regulated by competitive/noncompetitive inhibitors

photolithoautotroph

e - light

c - CO2

photoorganoheterotroph

e- light

c - organic carbon

chemolithoautotroph

e - inorganic chemicals

c - COs

chemolithoheterotroph

e - inorganic chemicals

c - organic carbon

chemoorganoheterotroph

e - organic chemicals

c - organic carbon

conserved (maintained over time) pathways

pathways are highly conserved because they provide the energy and raw materials needed for cells to grow, reproduce, maintain themselves, and survive

respiration

energy is captured from organic material by passing electrons through an ETC

*ETC generates PMF for oxidative phosphorylation of ATP

ETC

• plasma membrane (prok), inner membrane of mitochondria (euk)

• electrons from NADH and FADH2, not NADPH

• EMP and PPP are precursors to ETC

aerobic respiration, anaerobic respiration, and fermentation

respiration use NADH & NADPH from glycolysis for ETC

aerobic: oxygen final acceptor of ETC

anaerobic: something else is final acceptor of ETC

fermentation: energy is captured from organic material by oxidation of the organic energy source and substrate level phosphorylation of ADP

• final electron acceptor: pyruvate from glycolysis

glucose oxidation and TCA products → respiration

glucose oxidation = glycolysis (6C → 3C)

• produce: pyruvate (3C), NADH & NADPH

• located in cytoplasm of euk/prok

• oxygen not required

• 
  

TCA (no FAD+/NAD+ = inhibited)

• produce: NADH & FADH2 as a source of electrons for ETC

• located: cytoplasm (prok) and mitochondrial matrix (euk)

EMP (embden-meyerhof pathway)

most common (all microbes, plants, animals)

input: glucose, 2 ATP, 4 ADP, 2 NAD+

output: 2 NADH, 4 ATP, 2 ADP, 2 H2O, 2 pyruvate

• *net gain 2 ATP

substrate level phosphorylation

gluconeogenesis (pyruvate → glucose)

EDP (entner-dourdoroff pathway)

gram neg, no euk

input: glucose, 1 ATP, 1 NADP+

output: 1 NADPH, pyruvate, G3P (G3P → EMP → NADH, ATP, pyruvate)

• NADPH is important for anabolic rxns (lipid/na synthesis)

PPP (pentose phosphate pathway)

bacteria/euk, not archaea

input: 3 glucose 6-phosphate, 6 NADP+

output: 2 fructose 6-phosphate, G3P, 6 NADPH

• intermediate → EMP → ATP

  • erythrose 4-phosphate → aromatic amino acids

  • ribose 5-phosphate → nucleic acid synthesis

pyruvate oxidation/oxidative decarboxlyation

links glycolysis to TCA

TCA (acetyl-coa oxidation)

cycle turns twice per glucose

outputs: 2 CO2, 3 NADH, 1 FADH2, one GTP (ATP equivalent) per acetyl coa

amphibolic

amphibolic

intermediates are utilized in anabolic pathways

gluconeogenesis

builds glucose using smaller molecules (anabolic).

ETC carriers

electron and proton carriers: flavins, quinones

electron (only) carriers: cytochromes, Iron-sulfur proteins

proton motive force

chemiosmotic hypothesis: protons move across the membrane generating an electrochemical gradient → more protons outside

exergonic

ATP synthase: rotates and oxidatively phosphorylates ADP as protons flow to lower concentration

P/O ratio

phosphate/oxygen ratio: number of ATP molecules synthesized per pair of electrons donated by NADH/FADH2

*protons per electron pair

eukaryotes

• NADH 2.5, FADH2 1.5 (less ATP by oxid phosph), max 32 ATP yield

prokaryotes

• shower, lower P/O ratios, lower ATP yields

lower P/O for anaerobic respiration, shorter ETC

respiration detection

catalase: detects catalase produced by aerobic respiration

oxidase: detects specific cytochromes in aerobic respiration

nitrate: detects nitrate reduction/denitrification

four unifying themes of fermentation

1 O2 is not needed

2 electron acceptor is either pyruvate or a derivative

3 no ETC

4 far less efficient than respiration

fermentation re-oxidizes NADH removing the electrons from NADH onto pyruvate or some pyruvate derivative

fermentation

recycles electron carriers (NAD+ for glycolysis)

lacks ETC

lactic acid fermentation

reduction of pyruvate to lactate

*most common

homo vs heterolactive fermenters

homolactic: directly reduces pyruvate to lactate using lactate dehydrogenase

hetero: make other products (CO2, H2 gas)

mixed acid fermentation

production of lactate, formate, succinate, and ethanol due to glucose or other sugar fermentation

butanediol fermentation

production of butanediol (a neutral alcohol) to achieve redox balance by organisms that produce lower concentrations of acid products

detecting lactose catabolism

macconkey agar: neutral red changes pink due to accumulation of acid

phenol red lactose broth: phenol red changes yellow due to accumulation of acid

secondary fermentation

use a product of fermentation and ferment it further

propionic acid fermentation: cheese production (swiss)

fermentation gg and td

gas gangrene: gas products from fermentation lead to gas formation in soft tissue → direct tissue damage

• Clostridium perfringens - gram pos, spore forming, obligate anaerobe

tooth decay: biofilm formation, capsules → fermentation leads to high concentrations of secreted acidic waste products

• Streptococcus mutans: gram pos, acid-tolerant, facultative anaerobe

ocean microbiome, catabolism, oil spills

hydrocarbons from oil spill are degraded by naturally occurring bacteria

inorganic molecules

chemolithotrophy = eating rocks

these inorganic molecules have more positive redox potential → far less effiicient than glucose oxidation

• very high concentrations of electron source are required

important for nutrient cycling / primary producers

use ETC, oxidative phosphorylation

peptidoglycan synthesis

*anabolic

1 fructose 6-phosphate from EMB and PPP are used to synthesize UDP derivatives of NAM and NAG in the cytoplasm

2 animo acids are added to UDP-NAM to form the peptide side chains

3 NAM-NAG units are formed using the bactoprenol carrier molecule (conveyer belt)

4 flippase moves bactoprenol-NAM-NAG to the periplasmic space and is attached to growing peptidoglycan chain

5 cross-links form by transpeptidation

nitrogen fixation and nitrogenase

*anabolic

NF: reduction of nitrogen gas to ammonia

nitrogenase: enzyme that catalyzes nitrogen fixation

Frederick Griffith

studying avirulent and virulent strains of streptococcus pneumoniae to develop a vaccine

virulent: capsule, smooth colonies

• alive=killed host; heat killed = host lived

avirulent: no capsule, rough colonies

• alive=host lived; heat killed=host lived

transformation: DNA from heat-killed virulent was taken up and expressed by living avirulent, leading to host death (horizontal gene transfer)

Avery/MacLeod/McCarty

used Griffith’s observations of transformation to demonstrate the genetic info passed from heat-killed virulent to living avirulent

RNase and protease + heat killed virulent → transformation

DNase + heat-killed virulent → no transformation

Hershey/Chase

used bacteriophage infection to determine what was injected into bacterial host cells to induce production of viral proteins

radioactive labeling of DNA → pellet is radioactive

DNA vs RNA nucleotides

nucleotide: contain purine/pyrimidine base, ribose/deoxyribose sugar, and 1+ phosphates

dna: no OH at C2’

rna: has OH group on C2’

5’ and 3’ end

5’ has phosphate

3’ has OH group

phosphodiester bond

ester linkages between 3"‘ OH of one sugar and 5’ OH on the phosphate group

base pairing: AT (two H bonds), GC (three H bonds)

dna initiation

initiating proteins bind to oriC at AT rich regions

helicase unwinds by breaking H bonds (after topoisomerase)

SSBs stabilize strands

primase make short rna primers

dna polymerase attaches and adds nucleotides

dna elongation

dna pol adds in 5’→3’ direction

leading = continous / lagging = okazaki

rna primers removed and replaced with dna

dna ligase joins fragments

dna termination

replication forks meet at termination site

catenanes: interlocked chromosomes formed from topoisomerase breaking and joining strands to each super coiling

dimerized chromosomes: happens due to dna recombination between two daughter molecules

two identical dna molecules → each contain 1 old and 1 new strand

bacterial replisome

a complex of proteins at each replication fork involved in synthesizing the replicons

single stranded dna binding proteins (SSBs)

coat single stranded dna to protect from damage and to keep the two strand separated

topoisomerase

relieves supercoiling to prevent chromosome breaks

ahead of helicase

primase

RNA polymerase which synthesizes a short RNA primer providing a 3’ end for DNA polymerase to initiate DNA synthesis

helicase

six subunit protein powered by ATP hydrolysis, which separates DNA by breaking hydrogen bonds between the bases

dna polymerase

adds nucleotides by catalyzing the formation of phosphodiester bonds between the 3’ OH of a growing dna strand and the 5’ phosphate of an incoming nucleotide

DNA polymerase III

template DNA

3’ OH group of a primer of the growing DNA molecule

5’ phosphate group of the nucleotide to be added

*each replication fork as two DNA pol III

lagging and leading strand

lag: synthesized in okazaki fragments with many RNA primers because the 3’ end points away from the replication fork

• dna pol I removes rna primers and dna ligase joins fragments by phosphodiester bonds

DNA proofreading

dna poly III has 3’ to 5’ exonuclease activity → remove incorrect nucleotide and add correct one

DNA replication prok vs euk

prokaryotes

• cytoplasm, circular dna

• one replication bubble / oriC

• fast

eukaryotes

• nucleus, linear dna

• multiple replication bubbles / oriCs

• slow

mRNA, tRNA, rRNA

m: direct ribosomes to produce polypeptides

r: components of ribosome

t: carries amino acids to ribosomes for polypeptide synthesis

structural gene parts

operon

sigma factor

RNA polymerase core

transcription initiation

transcription elongation

transcription termination

transcription euk vs prok

open reading frames

silent mutation

universal genetic code

ribosome parts

translation initiation

translation elongation

translation termination

polycistronic RNA