Week 12 - Revision (2018 ) (High Res) PDF

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Surveys & Prizes

https://www.sfs.uts.edu.au/index.php

5 movie vouchers up for grabs

https://goo.gl/forms/5vgS0DnaTILMW6c72 UTS…think-change-do UTS…think-change-do UTS…think-change-do

protozoa/helminth/parasite/algae/bacteria/mould/virus/mycoplasma

REVISION AND Q&A WEEK 12 A/PROF. MAURIZIO LABBATE UNIVERSITY OF TECHNOLOGY SYDNEY (UTS)

protozoa/helminth/parasite/algae/bacteria/mould/virus/mycoplasma

FINAL EXAM

Final Exam 75 Multiple Choice Questions (MCQ)! Pearson Quizzes can be used for practice ! 2 hours + 10 minutes reading time! 40% of total subject assessment! You must achieve at least 40% in final exam to pass subject otherwise you risk an X grade

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Where Do The Questions Come From? BASE KNOWLEDGE

Lectures

APPLICATION OF KNOWLEDGE

Laboratory Classes

YouTube Movies

In Class Learning Activities

Won’t include questions from the practical skills test UTS…think-change-do UTS…think-change-do UTS…think-change-do

Study Pearson Quiz available for study from Monday 9am 302 questions

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protozoa/helminth/parasite/algae/bacteria/mould/virus/mycoplasma

WINNING ASSIGNMENTS

Revision Topics: 1. Nutrition and Culture Media 2. Summary of metabolism 3. Transcriptional and RNA-based regulation 4. Lateral gene transfer 5. Antibiotics - modes of action 6. Fungi - Features 7. Parasites - Features

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protozoa/helminth/parasite/algae/bacteria/mould/virus/mycoplasma

NUTRITION AND CULTURE MEDIA

1. Essential Macronutrients

Composition of microbial cells (composing >95% dry weight): ! 1. Essential macronutrients - C, H, O, N, S, and P.! These are required in relatively large amounts! (Components of carbohydrates, lipids, proteins and nucleic acids) !

Amino acids - protein

Sugars - polysaccharide

Phospholipids & lipids DNA & RNA UTS…think-change-do UTS…think-change-do UTS…think-change-do

Nitrogen, Phosphorus And Sulfur All organic matter has C, H and O but can also contain Nitrogen, Phosphorus, and Sulfur.! These can be supplied from the same nutrients that supply carbon, or from inorganic sources.! Nitrogen • needed for synthesis of amino acids, nucleotides, some carbohydrates and lipids and as # enzyme cofactors! • supplied in numerous ways: ! • Many microorganisms can use nitrogen from amino acids and organic molecules! • Some incorporate ammonia directly and some reduce nitrite to ammonia ! • Some bacteria “fix nitrogen” (assimilate atmospheric nitrogen & reduce it to NH4+) Phosphorus! • needed for nucleotides (including ATP), phospholipids, cofactors and some proteins and cell components! • All microorganisms use inorganic phosphate • Most incorporate phosphorus directly! • Low phosphorus can limit growth Sulfur! • needed for the synthesis of amino acids cysteine and methionine, some carbohydrates, biotin and thiamine! • usually supplied as sulfate or via organic sulfur compounds! UTS…think-change-do UTS…think-change-do UTS…think-change-do

2. Co-Factors - Macronutrients 2. Co-Factors - Macronutrients - K and Mg. Ca and Na for some,! (Co-factors for enzymes, complexes with ATP (energy), stabilise ribosomes and components of cytochrome and electron-carrying proteins).!

Cations and enzyme activity

Fatty acids Phosphate Ethanolamine

Fatty acids

Glycerophosphates Fatty acids

Stabilisation of membrane and DNA and function of ribosomes (Mg & K)

Structure and function of ribosomes (Mg & K)

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3. Trace Elements

3. Micronutrients or Trace elements - Fe, Mn, Zn, Co, Mo, Ni, and Cu.! • These are required in trace amounts for certain enzymatic functions or protein stabilisation.! • In culture, these are provided in water or media components, or even by contaminants (water, on glassware etc).! • In nature, these are ubiquitous and are rarely growth limiting

e.g. Zinc finger proteins – DNA binding

Cations and cytochromes (e- transfer) Fe & heme, Fe and iron-sufur proteins

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4. Growth Factors 4. Growth Factors! Some microbes are unable to synthesise their certain molecules and these must be obtained from the environment or provided in growth medium.! Three categories:! • Amino acids (synthesis of proteins) e.g. Lactobacillus spp. and requirement for amino acids! • Purines and pyrimidines (synthesis of DNA & RNA)! • Vitamins (co-enzymes and functional groups of certain enzymes)! Microorganisms also have specific requirements that reflect their specific morphology and environment. For example, diatoms need sialic acid to construct their cell walls of silica.!

http://www.ucmp.berkeley.edu/chromista/ diatoms/diatomdiverse.jpg

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Nutritional Types

• All organisms require an energy source and a carbon source.

Carbon sources

Heterotrophic

Mostly Autotrophic

Preformed organic molecules

CO2

Heterotrophs

Autotrophs

Mostly Autotrophic

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e.g. Photoautotrophs

Bloom of Cyanobacteria • Energy = light! • Carbon = CO2!

Purple sulfur bacteria • Energy = light! • Carbon = CO2!

• Splits H2O for electrons in photosynthesis producing oxygen !

• Splits H2S for source of electrons in photosynthesis producing sulfur !

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e.g. Chemolithotrophs (rock eaters) Nitrobacter winogradsky • Energy = Inorganic chemical (NO2-)! • Carbon = CO2! Therefore, an autotroph or a chemolithoautotroph!

Beggiatoa alba • Energy = Inorganic chemical (H2S)! • Carbon = organic molecules! Therefore, a heterotroph or a chemolithoheterotroph!

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Culture Media Culture media must contain all the nutrients required by the organism to grow: Media are classified based on: 1. Chemical constituents (defined or complex) 2. Physical nature (liquid, semi-solid or solid)! 3. Function (supportive, enriched, selective or differential). 1. Chemical constituents Defined: All components and constituents (and concentrations) are known! Complex: Contain some ingredients that are of unknown composition and/or concentration! UTS…think-change-do UTS…think-change-do UTS…think-change-do

Culture Media - Defined

• Essential Macronutrients: C, H, O, N, S, P! • Co-factor Macronutrients: K, Mg UTS…think-change-do UTS…think-change-do UTS…think-change-do

Culture Media Defined • Essential Macronutrients: C, H, O, N, S, P! • Co-factor Macronutrients: K, Mg! • and a ton of growth factors!

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Culture Media - Complex

Peptone (protein hydrolysates prepared by partial digestion of various protein sources)! ! Extracts (usually yeast or beef extracts)! Provide carbon, nitrogen, sulfur, vitamins and minerals in the form of organic material such as amino acids, nucleotides, peptides, organic acids etc ! UTS…think-change-do UTS…think-change-do UTS…think-change-do

Culture Media - Function 3. Media function 3. Selective - allows growth for particular microorganisms while inhibiting the growth of others e.g. MacConkey agar – selects for enterics (i.e. bile salts)

Selective agent(s): Two dyes – eosin Y and methylene blue • Inhibits growth of Grampositive bacteria

Selective agent(s): Bile salts and crystal violet. • Inhibits growth of Grampositive bacteria

Selective agent(s): 7.5% NaCl • Selects for Staphylococcus spp.

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Culture Media - Function 3. Media function 4. Differential - distinguished on different groups of microorganisms based on their biological characteristics • e.g. Blood agar - haemolytic versus non-haemolytic bacteria • e.g. MacConkey Agar - lactose fermenter (red) versus non-fermenter (pale)

MacConkey agar Pink – lactose fermenter (E. coli) Yellow – non-lactose fermenter (Salmonella spp)

Blood agar α-haemolysis: Streptococcus haemolysi β-haemolysis: Streptococcus viridians γ-haemolysis: Enterococcus faecalis

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Summary • MacConkey and Mannitol Salt agar! • Blood agar ! • Know how they are used and how the selective and differential ingredients in these media work! • Both were covered in practical class revision

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protozoa/helminth/parasite/algae/bacteria/mould/virus/mycoplasma

METABOLISM

ATP Synthesis Intermediates Energy-rich Pi intermediates ADP A

B

B~P

ATP

C ~P

D

Substrate-level phosphorylation

Energized membrane Dissipation of proton motive force coupled to ATP synthesis

ADP + Pi

Less energized membrane

Oxidative phosphorylation UTS…think-change-do UTS…think-change-do UTS…think-change-do

ATP

Overview of chemoorganotrophs catabolism of organic energy sources Overview of aerobic catabolism:! 1. Large molecules into small molecules.! 2. Initial oxidation and degradation to pyruvate (glycolysis).! 3. Oxidation and degradation of pyruvate by TCA cycle.!

Note: ! Many different energy sources are funnelled into common degradative pathways.! ATP is made by:! 1.Substrate-level Phosphorylation 2.Primarily by oxidative phosphorylation! Source of intermediates for anabolism! UTS…think-change-do UTS…think-change-do UTS…think-change-do

The Electron Transport Chain

• Composed of a series of electron carriers that transfer electrons from donors (e.g. NADH) to terminal electron acceptor – O2 for aerobic microbes.! • Electron transfer from carriers with more negative reduction potential to those with more positive potential.! • Cytoplasmic membrane for prokaryotes and mitochondria for eukaryotes. UTS…think-change-do UTS…think-change-do UTS…think-change-do

The Electron Transport Chain δ

α ADP + Pi α

β

β

α

ATP

F1 In

b2

γ ε a

Membrane

c12

F0 Out

Proton motive force fuels: ATP synthesis - Flagella movement - Active transport Chemiosmotic hypothesis: as electrons move down the chain, protons move outward across the membrane.! • Formation of a concentration gradient – PROTON MOTIVE FORCE (PMF) • The PMF is used to perform work when protons flow back across the membrane down the concentration gradient. UTS…think-change-do UTS…think-change-do UTS…think-change-do

Anaerobic Respiration

Overview of anaerobic respiration 1. Terminal electron acceptor other than oxygen 2. Generally yields less energy (because the E0 of the electron acceptor is less than the E0 of oxygen)! 3. Final electron acceptor may be nitrate (NO2-), sulfate (SO42- ), CO2, but also iron (Fe3+) etc.! 4. Electron transport chain is/can be modified!

Electron acceptor

Related products

Aerobic

O2

H 2O

Anaerobic

NO3-

NO2-

NO3-

NO2-, N2O, N2

CO2

CH4

S0

H 2S

Fe3+

Fe2+

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Redox Tower Electron donor - higher up the redox tower Electron acceptor - lower on the redox tower

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2. Fermentation NO OXIDATIVE PHOSPHORYLATION Energy production from substratelevel phosphorylation. • In this process NAD+ is reduced to NADH ! • No electron transport chain.! • How to recycle NADH to NAD+??! FERMENTATION PATHWAYS • Pyruvate or derivative is electron acceptor! • Substrate is only partially oxidised! • Low levels of ATP compared to respiration! UTS…think-change-do UTS…think-change-do UTS…think-change-do

2. Fermentation • The electron from the organic compound is extracted by NADH and then delivered to the fermentation product to complete the oxidation and reduction reaction.

Fermentation is used: • In microbes that lack no ETC! • In microbes that find themselves in an environment that lack O2 or an alternative electron acceptor yoghurt

alcoholic! fermentation

yoghurt,! sauerkraut,! pickles, etc

alcoholic! fermentation

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protozoa/helminth/parasite/algae/bacteria/mould/virus/mycoplasma

TRANSCRIPTIONAL AND RAN-BASED REGULATION

Protein Coding Genes –35

Regulatory sites R: Operator A: ABS

–10

+1

Promoter RBS Structural gene Terminator 5′ DNA 3′ Activation Repression

3′ 5′

Transcription (making RNA)

RBS

RNA

RNA-based regulation

Start codon

Stop codon

5′

3′ 5′-UTR

3′-UTR Translation (making protein)

Feedback inhibition

Protein–protein interactions Mechanisms of controlling enzyme activity

Protein Degradation

Covalent modifications

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Protein Coding Genes arg Promoter RNA polymerase

arg Operator

argC

argB

argH Transcription proceeds

Operons and polygenic mRNA - unique to prokaryotes

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How Genes/Operons Are Regulated Induction

Repression

Total protein

Cell number

Total protein

Arginine added

Cell number

β-Galactosidase Lactose added

Arginine biosynthesis enzymes

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Negative Control Repression arg Promoter

arg Operator

argC

argB

RNA polymerase

argH Transcription proceeds

Repressor

arg Promoter RNA polymerase

arg Operator

argC Corepressor (arginine)

argB

argH

Transcription blocked

Repressor

The inverse can also occur where the repressor normally binds and is released when the effector is present. UTS…think-change-do UTS…think-change-do UTS…think-change-do

Negative Control Induction lac Promoter

lac Operator

lacZ

lacY

RNA polymerase

lacA Transcription blocked

Repressor

lac Promoter

lac Operator

lacZ

RNA polymerase

lacY

lacA Transcription proceeds

Repressor Inducer (allolactose)

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Positive Control - Induction Activatorbinding site

mal Promoter

malE

malF

malG

No transcription

RNA polymerase Maltose activator protein

Activatorbinding site mal Promoter RNA polymerase

malE

malF

malG

Transcription proceeds

Maltose activator protein Inducer (maltose)

The inverse can also occur where the regulatory protein always binds and is released when the effector is present. UTS…think-change-do UTS…think-change-do UTS…think-change-do

Lac Operon (Bonus Question) Glucose exhausted

Growth on lactose

Growth on glucose

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lac Operon - Positive and Negative Control CRP protein

lac promoter has a CAP binding site which activates transcription! Low levels of glucose = high cAMP! High levels of glucose = low cAMP!

cAMP RNA polymerase

Binding of CRP recruits RNA polymerase DNA

lacI

Transcription

lacI mRNA

CRP binds the CAP site when bound to effector cAMP!

lac Structural genes C

P

O

lacZ

Active repressor binds to operator and blocks transcription.

lacY

lacA

Transcription

mRNA

lacZ

lacY

Translation Translation LacI Active repressor

Inducer LacZ LacY

LacA

Lactose catabolism Inactive repressor

lacA

A. Lactose but no Glucose! High cAMP = activation via CAP! High lactose = No repression by LacI! =TRANSCRIPTION B. Lactose and Glucose! low cAMP = no activation ! High lactose = No repression by LacI! =NO TRANSCRIPTION C. No Lactose and No Glucose! High cAMP = activation via CAP! No lactose = Repression by LacI! =NO TRANSCRIPTION D. Glucose but no Lactose! low cAMP = no activation via CAP! No lactose = Repression by LacI! =NO TRANSCRIPTION

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Non-coding RNA non-coding RNA (ncRNA) • rRNA! • tRNA! • small RNAs (sRNA), 40-400 nt - regulatory 3′ 5′

Alanine tRNA Anticodon

CGG Key bases in codon: anticodon pairing 5′

tRNA!

GC U

Wobble position; base pairing more flexible here 3′ mRNA

Codon

16S rRNA! UTS…think-change-do UTS…think-change-do UTS…think-change-do

Regulatory RNA: Small RNAs • Antisense RNA ‣ transcribed from non-template strand Translation inhibition/stimulation 1.

3′

mRNA

5′

3′

RNA degradation/protection 1.

sRNA 5′

5′

3′

RBS

mRNA

5′

RBS

3′

3′

sRNA 5′

5′

RBS

3′ RBS Ribonuclease

Translation

No translation

Translation

No translation

3′

2.

2.

5′

3′

5′ 3′ RBS

5′

3′ RBS

3′

5′ RBS

5′ RBS

Ribonuclease No translation

Translation

1a - sRNA blocks mRNA RBS = no translation! 1b - sRNA releases mRNA secondary structure = translation

No translation

Translation

2a - sRNA encourages ribonuclease degradation of mRNA = no translation 2b - sRNA protects mRNA from ribonuclease degradation = translation

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5′ 3′

Regulatory RNA: Small RNAs • trans-sRNA ‣ Transcribed from regions separate from mRNA region! ‣ Limited complementarity to target mRNA! ‣ Binding often depends on small protein Small regulatory RNA