1
Q
Metabolism [Definition] (10.1)
A
- All chemical reactions in a cell
- Requires the flow of energy (capacity to do work) and the participation of enzymes
2
Q
Catabolism [Definition] (10.1)
A
Breakdown of complex molecules into smaller ones with release of energy for anabolism
3
Q
Anabolism [Definition] (10.1)
A
- Reactions that build cells
- Synthesis of complex molecules from simpler ones with the input of energy
4
Q
What does ATP stand for? (10.2)
A
Adenosine Triphosphate
5
Q
What does the amount of Gibbs Free Energy (delta G) determine in a reaction? (10.2)
A
- It determines how much energy is available to do work such as:
- Rotate a flagellum
- Build a cell wall
- Store information in DNA
6
Q
What does Gibbs Free Energy measure? (10.2)
A
The change in free energy that can predict the direction of a reaction
7
Q
What do solutes (such as sugar / salt) do to the availability of water? (7.1)
A
- Solutes decrease the availability of water to microbes
- Availability of water affects growth of all cells
- Expressed as: a (sub w)
- Higher solute = Lower a (sub w)
8
Q
Hypotonic (7.3)
A
- Low extracellular solute concentration
- Water flowing into the cell
- Ex: Freshwater lakes & streams
9
Q
Isotonic (7.3)
A
-Same concentration of solute both in & out of the cell
10
Q
Hypertonic (7.3)
A
- High extracellular solute concentration
- Water flowing out of the cell
- Low a (sub w)
- Ex: Dead Sea, Great Salt Lake, Peanut butter
- Osmophiles live in these conditions
- Microbes living in these conditions have compatible solutes in an effort to increase the materials inside the cell
11
Q
Halobacterium [archaea] (7.3)
A
- A halophile
- Cause of pink coloration to Pink Lake in Australia
- –Yes, this is an archaea even though it has ‘bacterium’ in its name
12
Q
Staphylococcus [bacteria] (7.3)
A
- A halophille
- Found on human skin
- Isolated using Mannitol Salt Agar
13
Q
Compatible Solutes (7.3)
A
Help halophiles to survive under high salt concentrations
–Also help other osmophiles live in their highly concentrated environments
14
Q
What are the two types of extremophiles that can withstand strong pHs? (7.3)
A
- Alkaliphiles
- –Withstand high pH (basic conditions)
- Acidophiles
- –Withstand low pH (acidic conditions)
- –Ex: E. coli can withstand pH of 2 - 10 — very wide range, though not typically thought of as an extremophile
15
Q
What is a Biofilm? [overview] (7.4)
A
- Microbial community
- Attached to a surface
- Covered with a matrix of polysaccharide, DNA, & protein
- –“Protective Matrix”
- The cells + The Protective Matrix = Biofilm
16
Q
Four Stages of Biofilm Formation (7.4)
A
1) Attachment
2) Colonization
3) Maturation
4) Dispersal
17
Q
Attachment - Biofilm Formation Stage (7.4)
A
- First stage
- Use of pili & adherence proteins
18
Q
Colonization - Biofilm Formation Stage (7.4)
A
- Second stage
- Quorum sensing
- –Cell-cell signaling
- –Density dependent
- Activates gene expression
- –Genes that make the Protective Matrix are turned on
19
Q
Maturation - Biofilm Formation Stage (7.4)
A
- Third stage
- Forms a “mushroom” with:
- –Channels for nutrients
- –Oxygen gradients
20
Q
Dispersal - Biofilm Formation Stage (7.4)
A
- Fourth / Final stage
- Reactivation of motility
- Allows the bacteria to spread out again
21
Q
Dental plaque (7.4)
A
- A biofilm
- Bacterial film on tooth surface (over 300 microbial species)
22
Q
Caries (7.4)
A
- A biofilm
- Tooth decay
- Bacterial fermentation –> Acidic products –> Damage to enamel
- –Streptococcus mutans - fermentation
- –Poryphromonas - fermentation
23
Q
Periodontal disease (7.4)
A
- A biofilm
- Inflammation & tissue destruction
24
Q
How is ATP created in aerobic & anaerobic respiration? (10.3)
A
ATP is created via Oxidative Phosphorylation
25
How is ATP created in fermentation? (10.3)
ATP is created via Substrate-Level Phosphorylation
26
How is ATP created in photosynthesis? (10.3)
ATP is created via Photophosphorylation
27
Exergonic reaction (10.2)
- Favors products
- -- [A+B] -------> [C+D]
- K(eq) > 1
- Delta G Prime
28
Endergonic reaction (10.2)
- Favors reactants
- -- {A+B] 1
- Energy required
- -Fig. 10.2
29
Oxidation - Reduction Rxns [general] (10.3)
- Electrons move from donor to acceptor
- Utilize carriers
- Redox rxns can result in energy release, which can be used to form ATP
30
O.I.L.R.I.G. (10.3)
```
Oxidation
Is
Loss
Reduction
Is
Gain
```
- -Oxidation: Removal of e-
- --Substance that is oxidized in the donor
- -Reduction: Addition of e-
- --Substance that is reduced is the acceptor
31
In the following reaction, what is oxidized? Reduced? What enzyme is required to catalyze the reaction? (10.3)
[Malate + NAD+] -----> [Oxaloacetate + NADH + H+]
Oxidized: Malate
---Oxidized to oxaloacetate
Reduced: NAD+
---Reduced to NADH
Enzyme: Malate Dehydrogenase
32
Rhodoferax metabolins [bacteria] (10.3)
- Psychrophilic, obligate anaerobe that oxidizes acetate w/ the reduction of iron
- --Habitat: Cold, no oxygen
- --Donor:Acetate
- --Acceptor: Iron
33
Enzymes (10.6)
- Proteins (usually) that catalyze reactions
- --Ribozymes: catalytic RNAs
- Act on substrates & convert to products
- Require activation energy to bring reacting molecules together
- Increase the rate of reaction by lowering the activation energy
- Often named for the reactions that they catalyze
- --Ex: Phophotase, Kinase, Cellulase
34
How do Enzymes Lower Activation Energy? (10.6)
- Increase local concentrations of substrates
| - Orient substrates properly for reactions to proceed
35
Reduction Potential [E(0)] (10.3)
- Equillibirum constant for redox rxns
- Measure the tendency of the donor to lose electrons
- More negative E(0) is a better donor
- More positive E(0) is a better acceptor
36
What does a redox reaction accomplish? (10.3)
It pairs molecules with a negative E(0) to molecules with a positive E(0)
37
Electron Tower (10.3)
- Reference fig. 10.6
- Negative delta G' - Better e- donors
- Positive delta G' - Better e- acceptors
38
How do microbes transfer energy [4 steps]? (10.4)
- Microbes transfer energy by moving electrons from:
- --Reduced food molecules (glucose) -->
- --Diffusable carriers in the cytoplasm -->
- --Membrane-bound carriers -->
- --O2, Metals, or oxidized forms of N & S
Overall: From food to O2, Metals, or oxidized N & S
39
What are the two types of electron carriers? (10.4)
- Freely diffusable
- --Ex: NAD+ & NADP+
- Membrane-bound
- --Ex: Flavoproteins, cytochromes, quinones
40
What does NAD stand for? (10.4)
Nicotinamide Adenine Dinucleotide
41
What does NADP+ stand for? (10.4)
Nicotinamide Adenine Dinucleotide Phosphate
42
What do the reduced forms of NAD & NADP look like? What do they do for the cell? (10.4)
- Reduced forms:
- --NAD: NADH
- --NADP: NADPH
- These reduced forms are the "reducing power" of the cell
43
Quinones (10.4)
- A membrane-bound carrier
- Made of organic compounds
- Ex: Coenzyme Q
44
Cytochromes (10.4)
- A membrane-bound carrier
- Made of proteins
- Use iron to transfer electrons
- --Iron is part of a heme group
45
What are the two types of metabolic groups in the carbon cycle? (11.1)
- Heterotrophs
| - Autotrophs
46
Heterotrophs (11.1)
- Use reduced, preformed organic compounds as their source of carbon
- Convert huge amounts of C --> CO2
- Ex: Animals, many kinds of microbes
47
Autotrophs (11.1)
- Use CO2 as their source of carbon
- Synthesize organic compounds that are used by heterotrophs
- Also called Primary Producers
- Ex: Plants, many kinds of microbes
48
Phototrophs (11.1)
-Use light as a source of energy
49
Chemotrophs (11.1)
- Oxidize chemical compounds as source of energy
| - Often the same chemicals that are used for the carbon source
50
Lithotrophs (11.1)
- Use inorganic molecules as their source of electron donors
- Use respiration to accept electrons
- Table 11.1 & 11.2
51
Organotrophs [basic] (11.1)
- Use organic molecules as their source of electron donors
- Use fermentation to accept electrons
- Table 11.1 & 11.2
52
What would a photolithoautotroph use for a source of energy? Electrons? Carbon? (11.1)
- Energy : Light (photo)
- Electrons: Inorganic compounds (litho)
- Carbon: CO2 (auto)
53
What kinds of organisms are lithotrophs? (11.1)
- Microbes (prokaryotes) exclusively
| - --Eukaryotes are either photoautotrophs or heterotrophs
54
What are the three basic needs that fulfill all sources of energy, carbon, and electrons? (11.1)
1) ATP as energy currency
2) Reducing power to supply electrons for chemical reactions
3) Precursor metabolites for biosynthesis
55
Organotrophs [complex] (11.1)
- Many different energy sources are funneled into common degradive pathways
- Most pathways generate glucose or intermediates of the pathways used in glucose metabolism
- Substrate Level Phosphorylation (high energy)
- Oxidative phosphorylation
56
What are the two functions of organic energy sources? (11.1)
1) Oxidized to release energy
2) Provide building blocks for anabolism
- Amphibolic pathways
- --Catabolic & anabolic
- --Ex: Glycolysis
57
Aerobic Respiration (11.2)
- Process that can completely catabolize an organic energy source to CO2 using:
i) Glycolytic pathways (glycolysis)
ii) Tricarboxylic Acid cycle (TCA cycle / citric acid cycle)
iii) Electron transport chain with oxygen as final electron acceptor
- Produces ATP (mostly indirectly, via electron transport)
58
What are the three different paths in the breakdown of glucose to pyruvate? (11.4)
i) Embden-Meyerhof (glycolysis)
ii) Pentose phosphate
iii) Entner-Dourdoroff
59
Glycolysis / Embden-Meyerhof [general] (11.4)
- Most common form of glucose breakdown
- Occurs in the cytoplasm
- Functions in the presence or absence of CO2
- Ten reactions in two stages
60
Glycolosis [in-depth] (11.4)
- 6C Stage: Glucose is phosphorylated twice
- --Requires ATP
- --Generates fructose 1,6 biphosphate
- 3C Stage: Fructose 1,6 biphosphate split into two glyceraldehyde 3-P, then converted to pyruvate
- --Key Steps in 3C Stage:
- -----i) Oxidations --> NADH
- -----ii) Substrate-Level Phosphorylation --> ATP
- Big Picture: Glucose --> Pyruvate
61
What is the net yield of glycolysis? (11.4)
2 ATP, 2 NADH, 2 pyruvate
62
In glycolysis' 3C stage, how are NADH & ATP generated? (11.4)
- G3P is oxidized and phosphorylated
- --Generates high-energy phosphate bond
- --Uses G3P hydrogenase to do this
- NAD+ is reduced to NADH
- Phosphorylation of ADP by high energy metabolic substrate
- --Generates ATP
- --3PG Kinase (phosphoryglycerase)
63
Pentose phosphate pathway (11.4)
- Occurs in both prokaryotes & eukaryotes
- Starts by converting Glucose-6-P to Ribulose-S-Phosphate (pentase)
- Many sugars for biosynthesis
- --Transketolases & transaldolases
- Yields 6 NADPH (the reducing power of biosynthesis)
- Net yield of 1 ATP (indirectly)
64
Entner-Doudoroff pathway (11.4)
- Occurs in a few prokaryotes, does NOT occur in eukaryotes
- Combines the reactions of glycolysis & pentose phosphate
- Net yield: 1 ATP, 1 NADH, 1 NAHPD
65
Tricarboxylic Acid Cycle (TCA) / Citric Acid Cycle / Krebs Cycle (11.5)
- Pyruvate is completely oxidized to CO2
- Eukaryotes - Occurs in mitochondria
- Prokaryotes - Occurs in cytoplasm
- Generates:
- --CO2
- --Numerous NADH & FADH(2) (another type of diffusable electron carrier)
- --Precursors for biosynthesis
66
Describe the steps of the TCA / Citric Acid / Krebs Cycle [5 steps] (11.5)
- Fig 11.8
i) Pyruvate is oxidized to CO2 & Acetyl CoA
- --Acetyl CoA - high-energy molecule (thioester bond)
ii) Acetyl CoA condensed with oxaloacetate
iii) Oxidation & decarboxylation forming NADH & CO2
iv) Succinyl CoA --> Succinate
- --Generates high-energy guanosine triphosphate (GTP) via substrate-level phosphorylation
v) More oxidations form NADH & FADH(2)
67
How many ATP molecules are synthesized directly from the oxidation of glucose? (11.6)
- Four
| - Most ATP in cells is made when NADH & FADH are oxidized in the electron transport chain
68
Explain how the electron transport chain creates ATP (11.6)
- Electrons flow from the NADH & FADH2 --> Terminal acceptor
- Flow from carriers with more negative electron potential (Eo) to more positive Eo
- --Energy is released by doing this
- Used to make ATP by oxidative phosphorylation
- ---3 ATP per NADH using O2 as the acceptor
69
Where are electron transport chains located in the cell? (11.6)
- Eukaryotes: In the mitochondrial membrane
- Prokaryotes: In the plasma membrane
- Ex: Paracoccus denitrificans
- --Aerobic conditions
70
Oxidative phosphorylation (11.6)
- Chemiosmotic Hypothesis
- --Energy released during electron transport use to establish proton gradient & charge difference across membrane
- -----Proton motive force (PMF)
71
Explain how the proton motive force (PMF) drives ATP synthesis (11.6)
- Electron flow causes protons to move outward across membrane, ATP made when they come back in
- ATP synthase (F1Fo ATPase)
- --Enzyme
- --Uses proton movement to catalyze ATP synthesis
72
Bacterial ATP synthase structure (11.6)
- Fig. 11.16
- Fo
- --Proton channel
- --The part in the plasma membrane
- --Ring of C subunits rotates
- F1
- --The part in the cytoplasm
- --Gamma shaft rotates
- --Conformational changes in sphere of alpha & beta subunits
- --ATP synthesis
73
Shewanella [bacteria] (11.6)
- Aquatic, gram-negative bacterium
- Capable of extracellular electron transport
- --Transfers electrons to extracellular metals
- Facultative anaerobe
- --Prefers oxygen, but can live without it
74
Microbial Fuel Cell (11.6)
- Anoxic (low O2) chamber
- --Anode
- Oxic (high O2) chamber
- --Cathode
- Harnesses microbes' extracellular electron transfer to create electricity by connecting the two chambers
- ex: Using Shewanella
75
Organic electron donor [3 kinds] (11.6)
i) Fermentation
- --Endogenous organic electron acceptor
- --Ex: Pyruvate
ii) Aerobic respiration
- --O2 as acceptor
iii) Anaerobic respiration
- --NO3-, SO4(2-), CO2, fumarate as electron acceptor
76
Inorganic electron donor (11.6)
- Chemolithortophy
| - --O2, SO4(2-), NO3- as electron acceptor
77
Anaerobic respiration (11.7)
- Table 11.3
- ---Don't need to know all of these, he said he will point out which we need to know
- Produces less ATP than aerobic respiration
- Ex: Paracoccus
- Ex: Geobacter
- Use of anaerobic chamber to study these microbes
78
Dissimilatory Nitrate Reduction (11.7)
- Also known as nitrification
- ex: Paracoccus denitrificans
- Uses nitrate (NO3-) as terminal electron acceptor
- Reduced to nitrogen gas (N2)
- Major loss of nitrogen in soil
- --This is why farmers till the land-- in an effort to kill these anaerobic bacteria by exposing them to air, because they take nitrogen out of the soil
79
Fermentation (11.8)
- Completion of catabolism without the electron transport system & a terminal electron acceptor
- Occurs in the cytoplasm
- Hydrogens from NADH transferred onto pyruvate
- Generates:
- --Fermentation products
- -----Ex: Lactic acid, ethanol
- --NAD+ (oxidized form of NAD)
- ATP by substrate level phosphorylation
80
Sulfolobus [archaea] (11.10)
- Thermoacidophile
- --Lives in sulfur hot springs
- Oxidizes H2S --> H2SO4
- Chemolithotroph
81
Chemolithotrophs (11.10)
- Acquire electrons from the oxidation of inorganic sources such as H2, NO2, or Fe(2+)
- --Unlike most organisms which acquire electrons from the catabolism of an organic molecule such as glucose
- The electrons are transferred to terminal acceptors (usually O2) by electron transport chains
- Tables 11.5 & 11.6
82
Acidithiobacillus ferroxidans [bacteria] (11.10)
- An iron-oxidizing bacteria
- Oxidizes ferrous (Fe(2+)) --> ferric (Fe(3+))
- Uses O2 as electron acceptor
- Forms insoluble ferric hydroxide
83
Iron-oxidizing bacteria (11.10)
- Ex: Acidithiobacillus ferroxidans
- Very low reduction potential - Small amount of energy created
- --Look at the electron tower - Fe --> O is very small
84
Nitrifying bacteria (11.10)
- Obligate aerobes
- Nitrification: ammonia oxidized to nitrate
- Requires 2 genera to do this
- --i) Nitrosomonas - Reduces ammonia --> nitrite
- --ii) Nitrobacter - Reduces nitrite --> nitrate
- Used to reduce ammonia in wastewater
- Often followed by denitrification
85
Phototrophs & Photosynthesis (11.11)
- Two parts
- -- i) Light energy trapped & converted to chemical light (light reactions)
- -- ii) Chemical used to reduce CO2 & synthesize cell material (dark reactions)
- Many phototrophs are also autotrophs
86
Oxygenic photosynthesis (11.11)
- Provides all of the O2 for the Earth by oxidixing H2O --> O2
- A lot comes from microbes in the ocean
- Eukaryotic:
- --Higher plants
- --Green, brown, & red algae
- --Unicellular algae
- -----Ex: Euglenoids, dinoflagelates, diatoms
- Prokaryotic:
- --Cyanobacteria (gram negative)
87
Anoxygenic photosynhesis (11.11)
- Photosynthesis that does not oxidize water & therefore does not provide oxygen
- Prokaryotic only
- --Green sulfur bacteria
- --Purple sulfur bacteria
- --Green nonsulfur bacteria
- --Purple nonsulfur bacteria
- --Prochloron (bacteria)
88
Light reactions (11.11)
- Chlorophylls (Oxygenic)
- --Major light-absorbing pigments -- found in eukaryotic organisms & cyanobacteria
- Bacteriochlorophylls (Anoxygenic)
- --Major light-absorbing pigments -- found in purple & green bacteria
89
Prochlorococcus [bacteria] (11.11)
- Habitat: Tropical oceans
- >100,000 cells / 1 mL of seawater
- Smallest known photosynthetic organism (1 um)
- Oxygenic photosnthesis
- Uses chlorophyll
- Small genome: ~ 2000
- Thylakoids in tree-ring like formation
90
Accessory Pigments (11.11)
- Transfer light energy to chlorophylls
- Absorb different wavelengths than chlorophyll
- Quench toxic forms of oxygen (photoprotection, antioxidants)
- Ex: Carotenoids (lycopene, beta-carotene), Phycobiliproteins
91
Photosystems (11.11)
- A light-harvesting arrays composed of chlorophylls & accessory pigments
- Two types:
- --Photosystem I (PSI)
- --Photosystem II (PSII)
- Embedded into the thylakoid
- Occur in cyanobacteria & plants
92
Thylakoid (11.11)
Membranes that contain photosystems
93
What basic thing does biosynthesis require? (12.1)
- Requires energy & raw material
- These materials come from intermediates of central metabolic pathways
- Precursor metabolites examples: pyruvate, fructose-6-P, oxaloacetate
94
Cyclic photophosphorylation (11.11)
- Occurs in Photosystem Stage I (PSI)
- Has a reaction center of P*(700) - this is the wavelength of light that it absorbs at
- Makes ATP
- H2O is electron source
- Generates Proton Motive Force (Fig. 11.32)
- Fig 11.31
95
Non-cyclic photophosphorylation (11.11)
- Occurs in Photosystem Stage II (PSII)
- Makes ATP & NADHP - dark reactions
- H2O is electron source
- Generates Proton Motive Force (Fig 11.32)
- Fig 11.31
96
Describe light reactions in green & purple bacteria (11.11)
- Only 1 photosystem
- Can only make ATP (not NADPH)
- --Uses reverse electron transport to create NADPH
- Uses bacteriochlorophyll (Bchl)
- Anoxygenic
- Uses H2S or an organic donor to replace electrons lost
- Fig. 11.34
97
Photosynthesis in Archaea (11.11)
- Some archaea do perform photosynthesis, however, they do not contain chlorophylls or bacteriochlorophyll
- Use Rhodopsin
- --A protein
- --Mostly in archaea, but some bacteria have this
98
Rhodopsin (11.11)
- Light-driven proton pump
- Contains seven trans-membrane helices
- Pigment protein
- Retinal - the pigment in rhodopsin
- --Absorbs light
- --Induces conformational changes in rhodopsin
- --Pumps proton out
- -----Generates proton movement gradient
99
Calvin Cycle (12.3)
- Anabolic pathway for fixing CO2 into carbohydrate
- Dark reactions of photosynthesis
- Energy demanding
- Plants: Occurs in chloroplasts
- Bacteria: Occurs in cytoplasm
- Crucial to life, provides organic matter for heterotrophs
100
What are the 3 key steps in the Calvin Cycle? (12.3)
- Fig. 12.4
i) Carboxylation phase
ii) Reduction phase
iii) Regeneration phase
101
Carboxylation stage in Calvin Cycle (12.3)
- Use of rubisco (very important)
- Often occurs in carboxysomes
- --Proteinaceous shell containing large concentrations of rubisco
102
Reduction phase in Calvin Cycle (12.3)
- Reverse of two key reactions in glycolysis
| - Requires NADPH
103
Regeneration phase in Calvin Cycle (12.3)
- Numerous carbohydrates produced
| - Requires 18 ATP to make glucose
104
Gluconeogenesis (11.2)
- Functional reversal of glycolysis
- Glucose synthesis
- Occurs in animals, plants, fungi, bacteria
- --Humans use this to maintain blood glucose levels
- Requires ATP & GTP
- 6 enzymes also used in glycolysis, but 4 unique to gluconeogenesis
105
What are the three processes of genetic information flow (central dogma)? (13.1)
i) DNA Replication
ii) Transcription
iii) Translation
106
Griffith's Transformation [experiment] (13.2)
- Proved that DNA is the genetic material
- Used Streptococcus pneumoniae on mice
- Fig 13.1
- Found that the capsulated smooth strain killed mice, non-capsuled rough strain did not kill mice, killed smooth strain did not kill mice
- Found that when killed smooth strain and living rough strain were put together, it killed the mice
- Translation: When a microbe takes up free DNA from the environment and incorporates it into its own genome
- --The rough Streptococcus took up the genes for the capsule from the dead smooth strain
107
Gene (13.2)
- Functional unit of genetic information
- Deoxyribonucleic acid (DNA)
- Genes: (italicized) pilA, lacA
- Proteins: PilA, LacA (not italicized)
108
Genome (13.2)
- All the genetic material in a cell or virus
| - Bacterial genomes consist of one (usually) or more DNA chromosomes
109
Genotype (13.2)
-Specific set of genes carried in the genome
110
Phenotype (13.2)
-Set of observable characteristics (ex: motile bacteria, shape, etc)
111
Promoter (13.3)
- Place on DNA where the RNA polymerase binds to begin transcription
- Found upstream of the DNA fragment that is going to be transcribed
112
Transcription start site (13.3)
-Called +1 when in monocistronic
113
Operator (13.3)
-Where repressor proteins bind to block transcription
114
Operon (13.3)
-Cluster of genes regulated by one promoter (ex: Lac operon)
115
DNA Structure (13.3)
- Polymer of nucleotides
- --Each nucleotide is three parts -- sugar, nitrogenous base, & phosphate group: deoxynucleotide
- Double helix, 2 complementary strands
- --Each helix: Deoxynucleotides connected by phosphodiester bonds
- Sequence of one strand determines the other
- --Adenine (A) pairs with Thymine (T) - 2 hydrogen bonds
- --Guanine (G) pairs with Cytosine (C) - 3 hydrogen bonds
- ------Purines: G & A
- -----Pyrimidines: C & T
116
Name two bacteria that perform fermentation in the human mouth (7.4)
- Streptococcus mutans
| - Poryphromonas gingivalis
117
How is DNA organized in prokaryotes? (13.3)
- Double helical
- Closed, circular, supercoiled molecule
- Bacteria pack their DNA into loops, collectively called the nucleoid
- Archaea have circular chromosome that contain histones
118
How is DNA organized in eukaryotes? (13.3)
- Double helical
- Linear
- Wrapped around histone proteins, collectively called a nucleosome
- Genes in the human genome are interrupted by introns
119
What does DNA being 'semiconservative' mean? (13.3)
- The two strands separate in order to replicate
- Each of the two separated strands serves as a template for synthesis for a new strand
- Ends up with two parents strands each paired with a new daughter strand
120
In eukaryotes, is replication unidirectional or bidirectional? Is there one origin of replication or multiple? (13.3)
- Bidirectional synthesis
| - Multiple origins of replication (ori)
121
In prokaryotes, is replication unidirectional or bidirectional? Is there one origin of replication or multiple? (13.3)
- Bidirectional synthesis
| - One origin of replication (ori)
122
Which three things does DNA polymerase need in order to function? (13.3)
i) Template
ii) Deoxynucleotide triphosphates (dNTP)
iii) Primer (usually RNA) with a 3' OH group
123
What is the major replication enzyme in bacteria? (13.3)
DNA polymerase III
124
In what direction does synthesis occur? (13.3)
ALWAYS in the 5' -> 3' direction
125
DNA gyrase (13.3)
- Type II Topoisomerase
- Unwinds the DNA (releases tension from overcoiling)
- Cuts one DNA, passes through gap
- Seals the gap
- A target for quinoline antibiotics
126
What do quinoline antibiotics target? (13.3)
- Topoisomerase
| - If the DNA becomes too overcoiled, it can't continue synthesis, so this effectively kills the infection
127
DnaB Helicase (13.3)
-Breaks the hydrogen bonds between the base pairs so that translation can occur
128
DNA Primase (13.3)
-Primes the DNA for replication using an RNA primer
129
What does SSB stand for? (13.3)
Single-stranded Binding Proteins
130
Single Stranded Binding Proteins (13.3)
- A coating on the lagging strand of DNA
| - Keeps the DNA from rejoining together before replication is done
131
Okazaki Fragments (13.3)
- Occur on the lagging strand (which is discontinuous)
- The sections of DNA that are replicated on the lagging strand
- In the 5' -> 3' direction still
132
DNA Polymerase I (13.3)
-Removes the RNA primers after replication has been started, replaces the RNA with DNA
133
Ligase (13.3)
-Seals the gaps together between sections of replicated DNA (seals together okazaki fragments)
134
What does a DNA sequence of a gene correspond to? (13.4)
-It corresponds to the amino acid sequence of the protein encoded
135
What two processes are required to get DNA --> protein? (13.4)
i) Transcription
| ii) Translation
136
What does the antibiotic Rifampin target? (13.4)
- RNA polymerase
| - Targets this in order to prevent transcription
137
What dictates where transcription should begin and end in bacteria (not the promoters--what controls the promoters)? (13.4)
- A combination of core and sigma factors
| - Sigma factors: Proteins that direct the core promoters
138
Terminator (13.4)
-The sequence that signals RNA polymerase to stop translating
139
Start codon (13.4)
- In RNA
| - Signals the start of translation
140
Describe Rho-dependent termination (13.4)
- A DNA sequence that tells the Rho protein that it is time to bind
- Protein Rho binds to RNA
- This causes the RNA polymerase to pause
- The Rho protein moves towards polymerase quickly, and it boots the polymerase off the strand
- Ends translation
141
Describe rho-independent termination (13.4)
- A DNA sequence that encodes an RNA stem-loop structure
| - Causes RNA polymerase to release
142
Two-component signal transduction systems (13.4)
- Bacteria use these to control gene transcription in response to their environment
- Slide 13, Lecture 11
143
How is mRNA modified in eukarya? (13.4)
- Capping: Methylguanosine added to the 5' end of RNA
| - Polyadenylation - Adenine nucleotides added to the 3' end -- Poly-A Tail
144
Five features of transcription in Eukarya (13.4)
- Occurs in the nucleus
- Uses 3 RNA polymerases
- Only contains transcription factors (no sigma factors)
- TATA Box - Promoter element
- Uses RNA splicing to remove introns
145
What does translation require to synthesize protein? (13.7)
-It requires ribosomes & energy in the form of ATP & GTP
146
What does GTP stand for?
Guanosine triphosphate
147
What are the two subunits in a bacterial ribosome? (13.7)
i) 30S
- --21 proteins + 16S rRNA
ii) 50S
- --34 proteins + 23S and 5S rRNA
148
Translation definition (13.7)
The synthesis of polypeptide directed by mRNA sequence
149
What two rRNAs are used in translation? (13.7)
- 23S rRNA
| - 16S rRNA
150
23S rRNA (13.7)
- Peptidyltransferase
| - Ribozyme
151
16S rRNA (13.7)
- Aligns mRNA with the ribosome
| - Has sequence complementary to the Shine-Dalgarno sequence of the mRNA
152
Ribosomes (13.7)
-Read the mRNA sequence as a code
153
Codons (13.7)
- Triplets (3 nucleotides)
- 64 difference codons
- 61 codons encode protein
- --"Sense codon"
- 3 codons do not encode a protein
- --"Nonsense codon"
- --Stop codons
- The code is degenerate -- multiple codons can encode the same amino acid
154
Name the three stop codons (13.7)
UAG, UGA, UAA
155
Name the start codon (13.7)
AUG - codes for Methionine (Met)
156
What function does the tRNA serve? (13.6)
- It converts the language of RNA into that of proteins
- Collects amino acids and then gives them to the mRNA to assemble
- Clover-leaf shape
157
What does the 3' end of tRNA do? (13.6)
- Synthetase (an enzyme) attaches an amino acid to this end
| - ATP is required to do this
158
What does the anticodon of tRNA do? (13.6)
- It is complementary to a codon in mRNA
| - Tells the synthetase which amino acid it should attach to the 3' end
159
How does translation begin (Initiation) (13.7)
-Formylmethionine (f-Met) on a tRNA binds to the start codon in mRNA at the P-site
160
What does the Shine-Dalgarno sequence do? (13.7)
It aligns the mRNA with the 16S rRNA of the ribosome
161
A-Site (13.7)
-Aminoacyl / Acceptor Site
162
P-Site (13.7)
-Peptide Site
163
E-Site (13.7)
-Exit Site
164
Elongation [translation phase] (13.7)
- The tRNA with the proper amino acid attaches to the A-Site
- --GTP required to do this
- A peptide bond joins this new amino acid to the f-Met
- Ribosome moves 1 codon along the mRNA
- The empty tRNA (used to have f-Met) moves from P to E and exits the ribosome
165
Termination [translation phase] (13.7)
- Ribosome comes along a stop codon
- --There are no corresponding tRNAs for stop codons
- Release factors (RF) cleave & release the polypeptide
166
Translation & Transcription in Prokaryotes - Summary (13.7)
- Translation and transcription can (and often do) take place simultaneously in the cytoplasm in prokaryotes
- Fig. 13.30
167
How is genetic variation created? (16.1, 16.4)
- Mutations
- --Gives rise to new alleles & new phenotypes
- Vertical gene transfer
- Horizontal gene transfer
168
Vertical gene transfer (16.4)
- Sexual reproduction
- Occurs in Eukarya only
- New combinations of genes when gametes from parents fuse
169
Horizontal gene transfer (16.4)
- Occurs in Bacteria & Archaea
- Transfer from one independent organism to another
- 3 mechanisms: Conjugation, Transformation, & Transduction
170
Conjugation overview (16.6)
- DNA transfer by direct cell-to-cell contact
- Requires pili & plasmids
- Major mode of spreading antibiotic resistance genes among a bacterial population
171
Plasmids (16.6)
- Double-stranded, circular DNA
- Extrachromosomal
- Carry genes that confer an advantage
- Can be transferred by conjugation
- Are replicons
- Can be episomes
172
Replicons (16.6)
A gene having its own origin of replication
173
Episome (16.6)
Plasmids that can exist with or without integrating into the chromosome
174
What does F-factor stand for? (16.6)
- Fertilization factor
| - Fig. 16.16
175
Name the Four Steps in F(+) x F(-) Conjugative Mating (16.6)
1) Pilus connects the cells
2) F-factor begins replication & transfer
3) F-factor is replicated & transferred
- --Rolling circle replication
4) Now both cells are F(+)
- --Both contain the F-factor
176
In F(+) x F(-) Conjugative Mating, what does each F stand for? (16.6)
F(+) : Bacterial cell which contains the gene (F-factor) which is going to be transferred
F(-) : Bacterial cell which is going to receive the gene from the F(+)
177
Rolling Circle Replication (16.6)
- How plasmids replicate
| - Allows both cells at the end of the F(+) x F(-) mating to be F(+) instead of just transfer from one to the other
178
Hfr Cell (16.6)
- Fig 16.22)
- Can transfer the integrated F-factor AND part of the original bacterial chromosomal F-cell
- Leads to a high frequency of recombination
- --May accidentally give a bit of its regular chromosome when transferring its F-factor
179
Agrobacterium tumefariens [bacteria] (16.6)
-Causes crown gall disease in plants
Has a tumor-inducing (Ti) plasmid
-Piece of the Ti is transferred by conjugation into the plant cell
---Then that piece integrates into the plant genome
180
Transduction [definition] (16.8)
-The transfer of bacterial genes via phages
181
Phages (16.8)
- Cause of transduction gene transfer
- Abundant & diverse -- Over 10 billion per liter of seawater
- Impact the composition & behavior of microbial communities
182
What are the Two Major Types of Phages? (16.8)
i) Virulent
- --Lytic cycles (cause the host cell to lyse)
ii) Temporate
- --Lysogenic cycles (host cell remains intact)
183
Lytic Cycle (16.8)
- Fig. 16.27
- Phage attaches to host cell & injects its DNA into the cytoplasm
- Phage DNA undergoes replication & synthesizes new phages
- --Host genome becomes degraded during this process
- Host cell lyses to release the new phages
184
Lysogenic Cycle (16.8)
- Fig. 16.27
- Phage attaches to host cell & injects its DNA into the cytoplasm
- Phage DNA integrates into the host chromosome
- --Becomes a prophage
- -----Prophage: Phage genome that is integrated into the bacterial chromosome
- Prophage DNA replicates alongside the bacterial cell's replication
- Exposure to stress (such as UV light) can trigger excision from the host chromosome
- --General idea: GTFO before the host cell dies
185
Generalized Transduction (16.8)
- Occurs during the LYTIC cycle
- Any random part of the bacterial genome could be transferred
- During viral assembly, pieces of degraded host DNA can be mistakenly packed into the phage
- --The phage then goes on to put this DNA into a new bacterial cell
186
Specialized Transduction (16.8)
- Occurs during the LYSOGENIC cycle
- Specific part of the genome is transferred
- If the prophage incorrectly excises as it leaves the cell, it takes part of the bacterial genome with it
187
CRISPR-Cas System overview
- The prokaryotic "immune system"
- CRISPR is a cluster of genes, Cas is a protein
- Bacteria & archaea have RNA-based defense programs to destroy invading DNA from phage infection & sometimes conjugation
188
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
189
Describe the CRISPR-Cas System
- When a phage attacks, bacteria incorporate sequences of the viral DNA into their own genetic material, placing it between repeats
- Next time the bacteria encounter the phage, they use the DNA in the clusters to make RNAs that recognize the matching viral sequences
- The RNA guides the Cas proteins to viral DNA, where the Cas protein cuts the invading DNA
190
Spacer segments
- Part of the CRISPR-Cas system
| - Bits of phage DNA in the bacteria's genome from a previous infection
191
What does the Cas protein do?
- Processes the RNA
| - Cuts the DNA of invading phages
192
Transformation [definition] (16.7)
-Uptake of free DNA from the enivronment
193
Transformation [overview] (16.7)
- Discovered by Fred Griffith (did the experiments with the pneumonia in mice)
- Only a few genera of bacteria can do this naturally
194
Name two Gram + Genera of Bacteria that are Naturally Competent (16.7)
i) Streptococcus
| ii) Bacillus
195
Name two Gram - Genera of Bacteria that are Naturally Competent (16.7)
i) Haemophilus
| ii) Neisseria
196
Competent Cell (16.7)
- A cell which can undergo transformation naturally
| - --Can naturally take up free DNA from the environment
197
Artificial Transformation (16.7)
- A way to make genera which are not naturally competent undergo transformation
- --Ex: E. coli
- A critical step in cloning
198
What are the two techniques of Artificial Transformation? (16.7)
i) Calcium Chloride - Makes cell more permeable
ii) Electroporation - Pulses high-voltage, temporary holes through the cell wall & cell membrane to allow DNA through
199
Bacterial RecA Protein (16.7)
- Integrates DNA by homologous recombination
- Makes a stable transformation
- Fig. 16.34
200
Membrane-Bound Complexes (16.7)
- Bring DNA into the cell
- DNA is changed during this process
- --Fig. 16.26
- --Proteins labeled "com" mean "competence"
- --Nucleases
201
Nucleases in Transformation (16.7)
- Convert double-stranded DNA into single-stranded DNA as it enters the cell during transformation
- Does this because single-stranded DNA is much easier to incorporate into their genome, and also because single-stranded is easier to break down for nutrients if the cell decides to do that instead
202
Mobile DNA (16.7)
Segments of DNA that can move from one site to another within other DNA molecules
-Made up of transposable elements
Moves via the process of transposition
203
Transposable Elements (16.7)
- Fig. 16.13
- Two components: Transposons & Insertion Sequences
- --Insert & turn genes on or off
- IR - Inverted Repeats
- --Help the transposons move
- Transposase Enzyme
- --Cuts & Pastes
204
Mutations [definition] (16.1)
Stable, heritable changes in nucleotide sequence relative to the wild-type
---May or may not affect the phenotype
205
Wild-Type Strains (16.1)
-Possess the typical or representative characteristics of a species, whereas mutant strains contain mutations
206
Forward Mutation (16.1)
-A mutation from a wild-type to a mutant type
207
Reversion Mutation (16.1)
- A mutation reversing the initial mutation done to the wild type
- A mutation from mutant type to wild-type
208
Morphological mutation (16.1)
-Change colonial or cellular morphology
209
Lethal mutation (16.1)
Kill the cell
210
Conditional mutation (16.1)
Expressed only under certain conditions
| ---Ex: High temperature
211
Tomich et al 2004
-Showed a bacterial cell with pili vs. a pili mutant which contained no pili
212
Hirota et al (1968)
- Showed a rod-shaped E. coli (30 degrees C) vs. a mutant filamentous E. coli (42 degrees C)
- Found the existance of ftsZ
213
Spontaneous mutation (16.1)
- Absence of added agents
- Errors in DNA replication
i) Transitions
ii) Translations
214
Transitional Mutation (16.1)
- Type of spontaneous mutation
- Changes a purine --> purine OR Pyrimidine --> pyrimidine
- Ex: A-->G
215
Translational Mutation (16.1)
- Type of spontaneous mutation
- Changes a purine --> pyrimidine or vice versa
- Ex: A-->T OR C-->A
216
Rarity of DNA Replication Errors (16.1)
- DNA polymerase works at a rate of 1000 base pairs / second
- Only makes a mistake 1 / billion base pairs
- --Has a proofreading activity
217
Induced Mutation (16.1)
- Occurs after mutagen exposure
| - --Chemical or physical agents
218
How does UV Light cause a Mutation? (16.1)
- Generates thymine dimers
- Fig. 16.5
- Weak covalent bond between two adjacent thymines
- Causes the DNA polymerase to not realize that there are two thymines
219
Base Analogs (16.1)
- A type of induced mutation
- Resemble bases & cause mispairing
- Ex: 5-bromouracil
- --It is a T analog, but it base-pairs with G
220
Intercalating Agents (16.1)
- A type of induced mutation
| - Ex: Ethidium bromide
221
Missense Mutation (16.1)
- Single base substitution
- Changes codon for one amino acid for a codon into another
- May not affect the expression of the nucleotide (because the genetic code is redundant)
222
Nonsense Mutation (16.1)
-Converts a sense codon into a stop codon
223
Frameshift Mutation (16.1)
-Insertion or deletion of one or two base pairs in the coding region of a gene
224
Auxotrophs (16.2)
- Have mutations in biosynthetic pathways
- Can't make the product of that pathway
- Require that product to be in the media
- Ex: Lysine auxotroph - lys-
- --Bacterial cell that cannot make the amino acid lysine
225
Replica Plating (16.2)
- Fig. 16.6
- Use a piece of sterile velvet to stamp a grown plate and then press that onto a changed media to determine if any colonies have a mutation that doesn't allow them to grow on this new media (usually a chemical component is taken out of the altered media)
226
Light Repair (16.3)
- Also called Photoreactivation
- Light-activated photolyase (enzyme)
- Binds to and cuts the bond holding together thymine dimers
- Fig. 16.5
227
Dark Repair (16.3)
- Also called Nucleotide Excision Repair
- Fig. 16.9
- UvrABC endonuclease removes a section of damaged nucleotides (generally a thymine dimer)
- DNA polymerase I fills in the gap
- Ligase joins the segment back to the rest of the DNA
228
Ames Test (16.2)
- Method of identifying mutagenic substances
- Uses bacteria as guinea pigs instead of using actual guinea pigs
- Uses Salmonella mutant that cannot grow on media lacking histadine b/c it lacks the hisG gene
- If, when grown on a plate with the suspected mutagen, a reversion mutation occurs to allow the Salmonella to grow on a plate without histadine, then the substance is a mutagen
- In essence, if a substance can induce a reversion mutation, then it is a mutagen
MICB 3301
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