Microbiology Part 2 Second Exam

Front 1

Metabolic pathways hpic

Back 1

Metabolism

- all chemical reactions in a cell

Catabolism

- breakdown of complex molecules into smaller ones with the release of energy for

Anabolism

- the reactions that build microbial cells

Hint 1

ATP is the energy currency of cells

Front 2

Cells energy cycle (hpic)

Back 2

Aerobic respiration: Oxygen is used to make ATP

Anaerobic respiration: other than oxygen is used

Fermentation: substrate level phosphorylation, how fermentation makes ATP

Photosynthesis, using light energy to make ATP

Hint 2

Front 3

Energy provided in a cell

Back 3

we can predict the direction of a reaction using

a quantity - Gibbs free energy change ∆G

- ∆G for a reaction determines how much energy is available to do work such as:

–Rotate a flagellum

–Build a cell wall, or

–Store information in DNA

Front 4

Reaction mechanism and Keq

Back 4

Keq= [C][D]/[A][B]

∆G=-2.303RT log(Keq)

∆G units= Kcal/mol or Kjoules/mol

Front 5

Unbalanced reaction mech

Back 5

Left: Keq>1

∆G = is negatve, E is released

Right: Keq<1

∆G = is positive, E was required (endergonic)

Front 6

ATP input makes endergonic reactions more favorable

Back 6

Front 7

Oxidation-Reduction (Redox) Reactions

and Electron Carriers

Back 7

-electrons move from donor to acceptor

-utilize carriers (to move electrons)

-redox reactions can result in energy release, which can be used to form ATP

Front 8

Oxidation-Reduction Reactions

(hpic)

Back 8

-oxidation = loses electrons

-reduction = gain electrons

-oxidized substance is the donor, and the reduced substance was the acceptor (redox couple)

*easy stuff, you better not forget

Hint 8

Krebs cycle (TCA cycle)

malate gets oxidized, NAD is reduced, NADH is the cell reducing power

Front 9

Rhodoferax (hnotes)

Back 9

Rhodoferax metabolins is psychrophillic, strictly anaerobic bacterium that oxidizes Acetate with the reduction of Iron

(psychrophillic, survives in cold temps)

oxygen is toxic to it. survives where Atmosphere doesnt reach

Hint 9

-uses Acetate as electron donor (its being oxidized)

dumps electrons onto Iron.

-Malate dehydrogenase: the enzyme that oxidizes the Malate

Front 10

Enzymes (hpic)

Back 10

-proteins (mostly) that catalyze reactions( ribozymes = catalytic RNAs)

-Activation energy: the energy required to bring reacting molecues together, (enzymes lower this Act-Energy)

-Often named after the reactions they catalyze ex( phosphatase, kinase, cellulase)

Hint 10

-increase local concentrations of substrates

-orientation substrates for proper reaction

Front 11

Induced fit model of enzymes (hpic)

Back 11

1) ATP and glucose bind to active site of enzyme

2) enzyme undergoes conformational change that makes substrates fit the mold

3) substrates are converted into products

4) release products

Hint 11

Front 12

Reduction potential: (Eg) the ∆G equivalent (hnotes)

Back 12

- Equilibrium constant for redox rxns

(Eg is like ∆G)

-measure tendency of donor to lose electrons

more negative Eg = better donor

-more positive Eg = better acceptor

Hint 12

redox couples with more negative Eo, will donate electrons to more positive Eo

Front 13

The Electron tower: plot/chart of Eo values

Back 13

Front 14

2 classes of electron carriers in redox reactions (hnotes)

Back 14

-freely diffusible: ex_ NAD+ and NADP+

-membrane-bound: ex) flavoproteins, cytochromes, quinones

Hint 14

carriers function togheter moving electrons from donors to acceptors.

AKA electron transport systems

(most are found and formed in the plasma membrane, not the cytoplasm or cell wall)

Front 15

Types of diffusible electron carriers

Back 15

NAD+ and NADP+

(NADH and NADPH are the reducing power of the cell)

Hint 15

NAD+ = nicotinamide adenine dinucleotide

NADP+ = nicotinamide adenine dinucleotide phosphate

Front 16

Types of membrane bound electron carriers

Back 16

Quinones: ex) Coenzyme Q

Cytochromes: are proteins, use iron to transfer electrons (iron is part of a heme group)

Front 17

Mircrobes transfer energy by moving electrons from:

Back 17

reduced food molecules --> carriers in cytoplasm (NADH) --> membrane-bound carriers, (ex cytochromes, quinones) latch onto O2, metals, and oxidized Nitrogen and sulfur

Summary: gain energy from movement of electrons through redox rxns

Front 18

2 metabolic groups of the carbon cycle

Back 18

Heterotrophs

-reduced, preformed organic compounds as Carbon source

-convert large amounts of C --> CO2 (ex humans, many microbes)

Autotrophs: use CO2 as carbon source

-ex: plants and microbes

-produce organic compounds used by heterotrophs eg. sugar

Front 19

Organisms that require sources of energy and electrons for growth

Back 19

Phototrophs: use light for energy

Chemotropths, ozidize chemical compounds, often same as their carbon sources

Front 20

Microorganisms have 2 sources for electrons

Back 20

lithotrophs: inorganic molecules as electron donors

organotrophs: organic molecules as donors for electrons

ultimate electron acceptors:

-inorganic molecules in Respiration

-Organic molecules used in Fermentation

Front 21

Quiz: you identify a bacterium that utilizes compounds as a source of carbon, energy, and electrons, what is this bacterium?

Back 21

ANS: chemo-organo-heterotroph.

Hint 21

Table 11.2 in book. test yourself

Front 22

nutritional types of organisms

Back 22

Almost all Eukarya are either photoautotrophs (plants/algae) or heterotrophs( anmals, protozoa and fungi)

**Lithotrophy is unique to a few bacteria and archaea

Front 23

Fueling reactions

Back 23

Despite diversity of energy, electron and carbon sources used by organisms, all have same basic needs:

-ATP is energy supply

-reducing power to supply electons for rxns.

-precurser to biosynthesis

Front 24

Energy source (organotrophs)

hpic

Back 24

ATP by 2 means:

Substrate level phosphorylation

oxidative phosphorylation :ADP --> AGP

Hint 24

Front 25

two functions of organic energy sources hpic

Back 25

Amphibolic pathways: a pathway that works catabolic and anabolic.

oxidized to release energy

these are building blocks for anabolism.

Hint 25

Front 26

Aerobic respiration hnotes

Back 26

process that can completely catabolize an organic energy source to CO2 using:

-glycolytic pathways (glycolysis)

-tricarboxylic acid cycle

-electron transport chain with oxygen as final electron acceptor.

-produces ATP (indirectly by E-transport)

Hint 26

normally uses glucose as carbon source

Front 27

breakdown of glucose to pyruvate (3 pathways) know key rxns and products of each pathway

Back 27

embden-meyerhof (glycolysis)

pentose phosphate pathway

Entner-doudoroff pathway

Front 28

embden-meyerhof (glycolysis)

hpic

Back 28

most common form of glucose breakdown

occurs in cytoplasm

functiosn in presence or absence of O2

has 10 rxns in two stages (6 carbon stage and 3 carbon stage)

Hint 28

6C stage: glucose is phosphorylated twice, generating fructose 1,6-biphosphate

3C state: fructose 1,6 bisphosphate, spits into 3 glyceraldehyde then converted into pyruvate

Front 29

3-C stage indepths hpic

Back 29

key steps- oxidations come from NADH as reducing power

substate level phosphorylations generate ATP

net yield: 2 ATP, 2 NADH, and 2 pyruvate

Hint 29

G3p oxidized and phosphorylated generates high energy p-bond

Front 30

pentose phosphate: prokaryotes and eukaryotes do this

Back 30

starts by converting Glucose-6-phosphate into ribulose-5-P

generates sugars for biosynthesis (transketolases, transaldolases)

-1ATP and 6NADPH is the reducing power for biosynthesis

Front 31

Entner-doudoroff (found in a few prokaryotes, not in euqkaroytes)

Back 31

combines rxns of glycolysis and pentose phosphate

generates 1ATP, 1NADH, 1NADPH

Front 32

Tricarboxylic acd (krebs) cycle

Back 32

pyruvate is completely oxidized to CO2

happens in mitochondria of eukaryotes, and the cytoplasm of prokaryotes.

Generates: CO2, NADH, FADH2

Front 33

TCA cycle hpic

Back 33

-pyruvate first oxidizes CO2 and acetyl CoA,

-acetyl-CoA condensed with oxaloacetate

-oxidation and decarboxylation forming NADH and CO2

Hint 33

Front 34

Electron transport and oxidative phosphorylation

Back 34

only 4 ATP molecules synthesized directly from oxidation of glucose

most ATP is made when NADH and FADH oxidzed in electron transport chain

Front 35

Electron tranport chain hpic

Back 35

electrons from NADH and FADG are terminal acceptors

they will flow from carriers with more negative energy to more positive energy and the energy released is used to make ATP by Oxidative phosphorylation

-3ATP per NADH using O2

Hint 35

Front 36

Oxidative phosphorylation

Back 36

Chemiosmotic hypothesis:

- energy released during e-transport is used to establish proton gradient and charge difference across membrane

-AKA Proton Motive Force (PMF)

**memorize!!

Hint 36

PMF: drives ATP synthesis

e-flow causes protons to move outward across membrane, ATP is made when they move back in

ATP synthase (enzyme) uses proton movement to catalyze ATP synthesis

Front 37

ATP synthese (F0, F1) hpic

Back 37

we use proton flow down the gradient to make ATP

top half (F0) contains a proton channel, ring of C-subunits rotates

Bottom half (F1) sphere (gamma) shaft will rotate, causes conformational changes in alpha and beta subunits, this synthesizes ATP from ADP

Hint 37

Front 38

"Shewanella" electron transfer (an example of a microbial fuel cell) hpic

Back 38

this bacterium is aquatic, gram negative, and can transfer electrons to extracellular metals.

must be in a Anoxic environment (low O2, the anode)

it will connect to a chamber that is Oxic (high O2, the cathode) and a wire will connect the two chambers, circuit is generated. This is known as a microbial fuel cell

Hint 38

Front 39

methods and types of Electron acceptors microbes use (hpic)

Back 39

Organic electron donor:

-fermentation: endogenous electron acceptor (ex: pyruvate)

-Aerobic respiration: O2

-anaerobic respiration: NO3, SO4, CO4, fumerate

Inorganic E donor:

chemolithotrophy: O2, SO4, NO3

(exogenous electron acceptors, ex) O2, NO3, SO4, CO4, Fumerate))

Hint 39

Front 40

Anaerobic Respiration

Back 40

Less ATP is generated anaerobically (influenced by reduction potential)

"Paracoccus": uses NO3 as terminal e- acceptor

"geobacter" uses Fe3+ in anaerobic environments

Denitrification: dissimilatory nitrate reduction: nitrate as terminal e- acceptor, becomes reduced to N2 gas) causes loss of Nitrogen in soil

Front 41

Fermentation "the consequence of life without air" hpic

Back 41

completion of catabolism without the electron

transport system and a terminal electron acceptor.

-Occurs in the cytoplasm

-Hydrogen from NADH transfers onto pyruvate

-generates: fermentation products(lactic acid, ethanol)also NAD+,

-ATP by SLP (substrate level phosphorylation)

Hint 41

Front 42

Chemolithotrophs hnotes

Back 42

acquire e-from the oxidation of inorganic sources such as H2, NO2 or Fe2+

the e- are transferred to terminal (exogenous) acceptors (O2) by e- transport chains

Hint 42

2 examples: iron oxidizing and nitrifying

Front 43

Iron-oxidizing Bacteria

ex) acidithiobacillus ferroxidans

Back 43

-oxidizes ferrous Fe2+ to ferric Fe3+

-O2 as an e- acceptor

-forms insoluble ferric hydroxide

Front 44

Nitrifying Bacterium

Back 44

obligate aerobes

nitrification: oxidation of ammonia into nitrate

-2 genera (nitrosomonas - ammonia to nitrite) (nitrobacter - nitrite --> nitrate)

this is used to remove ammonia in waste water

denitrification: nitrate back into N2 gas

Front 45

Sulfolobus (domain archaea)

Back 45

H2S---> H2SO4

Front 46

Photosynthesis - phototrophs

hnotes

Back 46

Two parts

Light reactions: light every trapped, and converted into chemical

Dark reactions :(calvin cycle) used to reduce CO2 and synthesize cell material

(many phototrophs are also autotrophs)

Hint 46

oxygenic: oxidize H20 for electrons, form oxygen. this is also called oxygenic photosynthesis. done by eukaryotes and cyanobacteria

anoxygenic photosynthesis: electrons from other sources, done by all other types of bacteria

Front 47

Light reactions (trapping light) hpic

Back 47

Chlorophylls: Major light abosrbing pigments. eukaryotes, and cyanobacteria do this

bacteriochlorophylls: major light absorbing pigments, done by purple and green bacteria

Hint 47

gram negative: because outer membrane(lipid bilayer)

Front 48

Domain "bacteria"

Genus: prochlorococcus

hpic

Back 48

Habitat: tropical oceans

the rings in the picture are the thylakoids (where photosynthesis takes place)

>100,000 cells/ml

smallest known photosynthetic organism

small genome 2000 parts)

Hint 48

Front 49

Accessory pigments hpic

Back 49

-transfers light to chlorophylls

-absorb different wavelengths than chlorophylls

-quench toxic forms of oxygen (photoprotection, antioxidants)

increase the number of wavelengths that can be absorbed

-ex) carotenoids, phycobiliproteins

Hint 49

Front 50

Photosystems

Back 50

when chlorophylls and accessory pigments are assembled into light harvesting arrays. these are imbeded in the thylakoids.

2 types, photosystem 1 and photosystem 2

Front 51

Light reactions: green plants and cyanobacteria (hpic)

Back 51

photosystem 1 absorbs P700 (cyclic phosphorylation)

photosystem 2 absorbs P680 (non-cyclic phosphorylation)

electron from PS1 travels down a electron transport chain to PS2, then back to the beginning of PS1. the electron can also travel in a different direction to make NADP+ into NADPH (calvin cycle)

PS2 absorbs 2 protons, goes up in redox potential, and feeds electrons into PS1.

Hint 51

Front 52

Photosystem 1 & 2 diagrams

Back 52

Use the inward flow of protons to synthesize ATP.

Hint 52

Front 53

Light reactions: green and purple

hpic

Back 53

The reactions occur in the plasma membrane

-utilizes bacteriochlorophyll (not chlorophyll) to generate energy

-Anoxygen (doesnt use water as reactant)

-only one photosystem (restricted to only cyclic phosphorylation to make ATP)

-Reverse electron transport (pushes electron backwards) to make NADP into NADPH

-H2S or other organic donors are needed to replace the electrons

Hint 53

Front 54

Archaea and photosynthesis?

hpic of rhodopsim

Back 54

Some are photosynthetic, but lack both chlorophyll and bacteriochlorophyll.

instead they use a protein pigment called Rhodopsin

-there are 7 transmembrane helices.

-conformational changes in rhodopsin- this is a proton pump, makes a proton motive force, generates ATP (normally this proton motive force comes from electron transport chain)

Hint 54

Front 55

Metabolic reactions hpic

Back 55

metabolism

–total of all chemical reactions

catabolism

–breakdown of larger,molecules into simpler ones

–energy released used for work

anabolism

–synthesis of complex molecules from simpler

ones with the input of energy

Hint 55

Front 56

Anabolism hpic

Back 56

synthesis of complex molecules

from simpler ones with the input of energy

Hint 56

Front 57

Principles of Anabolism hpic

Back 57

-Large from small

-Many enzymes do double duty(catabolic), others function in only one direction (anabolic)

-Catabolic and anabolic pathways use different

cofactors

-Catabolism produces NADH, NADPH

-Anabolism uses NADPH as e donor

Hint 57

Front 58

Calvin Cycle - Anabolic Pathway for Fixing CO2 Into Carbohydrate

(hpic) AKA the dark reaction of photosynthesis

Back 58

AKA the dark reaction

-energy demanding

-occurs in chloroplasts in plants, and cytoplasm in bacteria

-crucial for life, provides the organic matter for heterotrophs.

-three phases: carboxylation, reduction, regeneration)

Hint 58

Front 59

Calvin cycle energy path hpic

Back 59

-Biosynthesis requires energy and raw material

-Materials are intermediates of central metabolic pathways

-precursor metabolites (circled red)

Hint 59

Front 60

Reversal of catabolic pathways hnotes

Back 60

Hint 60

arrows are in reverse, instead of generating CO2, and power driven NADPH, its fixing CO2 to be used up.

Front 61

Carboxylation phase of calvin cycle hpic

Back 61

makes 3 phosphoglycerates

this phase occurs in carboxysomes

Hint 61

Front 62

Reduction phase or calvin cycle hpic

Back 62

its the reverse of two key reactions in glycolysis. this step requires ATP

Hint 62

Front 63

Regeneration phase of calvin cycle hpic

Back 63

Numerous carbohydrates are produced in this phase

Hint 63

Front 64

Gluconeogenesis hpic

Back 64

glucose from noncarbohydrate substrates

-animals, plants, fungi, microbes use gluconeogenesis to maintain blood glucose levels

-requires ATP and GTP

-there are 6 enzymes also used in glycolysis, but also 4 that are unique to gluconeogenesis

Front 65

Gene Structure and Replication hpic

Back 65

-DNA and RNA

structure and roles

-3 processes of genetic

information flow:

1. DNA replication

2. Transcription

3. Translation

Hint 65

Front 66

DNA as Genetic Material: Microbes (and some luck)

Provided Proof, Griffith’s Transformation Experiments

hnotes

Back 66

Hint 66

bacterium used in experiment

streptococcus pneumoniae

Front 67

Terms for genomic vocab

Back 67

Gene

– functional unit of genetic information

– deoxyribonucleic acid (DNA)

–Genes: pilA, lacZ

– Proteins: PilA , LacZ

•Genome

– all genetic material in cell or virus

– bacterial genomes consist of one (usually) or more DNA chromosomes

genotype: specific set of genes carried in genome

phenotype: observable characteristics

Front 68

Organization of cystronic RNA hpicRibonucleic acid (RNA) Structure

Back 68

promotor: where RNA polymerase binds to begin transcription

operator: where repressor proteins bind to block transcription

Hint 68

Front 69

DNA Structure hpic

Back 69

polymer of nucleotides

•each nucleotide 3 parts:

–sugar-deoxyribose

– nitrogenous base

•adenine (A), guanine (G)-purines

•cytosine (C), thymine (T) -pyrimidines

•phosphate group

•base + sugar + P group = deoxynucleotide

Hint 69

Front 70

DNA structure part 2

Back 70

Double helix, 2 complementary strands

-each helix connected by phosphodiester bonds

-sequence of one strand determines the other

base pair rules ( A -->T 2H's & G --> C 3H's)

Front 71

DNA Size

Back 71

bacterial genomes vary greatly:

–one of smallest - Mycoplasma - 580 kb

encoding 480 proteins (lack a cell wall)

expressed as base pairs (bp) 1000 bp=1kb

-E. coli = 4,640kb or 4.64 Mb

Front 72

Ribonucleic acid (RNA) Structure

Back 72

3 types"

- tRNA: carries amino acids during protein synthesis

-rRNA: component of ribosomes (ribosomes + RNA)

-mRNA: template for protein synthesis

RNA: uses ribose vs deoxyribose, uracil replaces the thymine, usually singlestranded

Front 73

How is DNA Organized in Cells?

Back 73

Double helix in both prokaryotic and eukaryotic cells

• However, organization differs

Front 74

Prokaryotic DNA hpic

Back 74

usually closed circular, supercoiled molecule

-bacteria pack their DNA into loops or domains, collectively called the nucleoid

Hint 74

Front 75

Eukaryotic DNA hpic

Back 75

-Linear

-Eukaryotes wrap their DNA around proteins called histones, collectively called nucleosomes.

-genes in human genomes are interrupted by introns ( archaea have circular chromosomes with histones)

Hint 75

Front 76

DNA replication hpic

Back 76

Semiconservative:

-parental strands are conserved

strands separate as templates for synthesis of new strands

Hint 76

Front 77

DNA replication in eukaryotes hpic

Back 77

-bidirectional

- multiple origins of replication

Hint 77

Front 78

DNA replication in Prokaryotes hpic

Back 78

-bidirectional

-single ori

-2 forks

moves in opposite directions

How is ori selected? (DnaA is the DNA binding protein, initiates the opening at the site, begins the replication)

Hint 78

Front 79

DNA polymerase

Back 79

Catalyzes DNA synthesis in 5' -->3' direction

Needs these to function:

- a template

- deonxtnucleotide triphosphates (dNTP's)

- Primer with 3' OH group (an RNA that is complementary to the DNA)

- In bacteria, DNA Pol III is major DNA replication enzyme

Front 80

DNA polymerase pictures for both eukaryotes and bacteria

hpic (SSB = single stranded binding proteins)

part 1, then part 2

Back 80

Hint 80

Front 81

DNA gyrase (DNA topoisomerase II)

hnotes

Back 81

DNA gyrase is the underwinder that is ahead of the replication fork. helps faciliate unbinding. the target for quinolone antibiotics

Hint 81

DnaB Helicase: hydrogen bond breakers, is the thing that actually unwinds the DNA strand

DNA Primase: it lays down RNA primer.

Front 82

Transcription of Bacteria

Back 82

-RNA comes from DNA

-RNA polymerase composed of = core + sigma factors

-Sigma factors = proteins, that direct the core to the promotor, dictates where the RNA polymerase will bond

- antibiotic "rifampin" targets bacterial RNA polymerase, disabling it

-Generates 3 types of RNA: mRNA, tRNA, etc

Hint 82

template strands direct rNA synthesis

terminator is a DNA sequence that signals RNA pol to stop transcription

Front 83

What is a terminator? corresponds to picture in the next slide

Back 83

-it is a DNA sequence

-broken into two groups: one group encodes an RNA stem loop structure. causes RNA pol to release (come off the template)

protein Rho binds RNA, moves towards RNA pol and causes release

Front 84

RNA polymerase "3 steps" (hpic)

transcription

Back 84

Hint 84

Front 85

Bacteria use two-component signal transduction systems to control gene transcription in response to their environments

Back 85

Signal, enters sensor kinase, requires ATP to power the kinase domain. histodine will grab the phosphate group that got bumped off. the phosphate will phosphorylate Aspertate, and activate a protein(response regulator)

will interact with gene.

Front 86

Transcription - Eukarya hpic

Back 86

takes place in the nucleus

-has 3 RNA polymerases

-dont have sigma factors, but instead transcription factors

-TATA box, promoter element.

-RNA splicing to remove introns

-mRNA further modified, also has capping, Methylguanosine added at the 5' end,

polyadenylation: adenine nucleotides add (cap) to the 3' tail end

Front 87

Translation

Back 87

-synthesis of polypeptide directed by nRNA sequence

- requires ribosomes and energy in the form of ATP and GTP (guanosine triphosphate)

bacterial ribosomes: 2 subunits, 30S + 50S.

the 30S = 21 proteins + 16 rRNA

the 50S= 34 proteins + 23S and 5S rRNA

Front 88

rRNA's play important roles in translation hnotes

Back 88

-23S rRNA: peptidyltransferase, Ribozyme( participates in translation),

-16S rRNA: helps aligns mRNA with ribosomes so translation can proceed, has sequence complementary to Shine-Dalgarno sequence of the mRNA

Front 89

ribosomes: sites of protein synthesis

Back 89

-ribosomes "read" mRNA sequence as a code

Codons:

-3 nucleotides (triplet)

-there are 64 codons

- 61 specify amino acids (sense)

-3 are stop codons (nonsense)

-code is degenerate- multiple codons can encode the same amino acid.

Front 90

tRNA (transfer RNA) molecules hpic

Back 90

Convert and transfer aminoacids to ribosomes:

-convert of the language of RNA into that of proteins

-clover leaf shape

-two functional regions

Hint 90

Front 91

translation initiation hpic

Back 91

Hint 91

shinedalgarno mRNA sequence aligns with 165 rRNA of ribosome. shinodalgarno mRNA sequcne aligns with 16S rRNA of ribosome.

mRNA with ribosome aligns with 16S, attaches at psite, and begins initiation

Front 92

translation: elongation hpic

Back 92

Hint 92

1) tRNA+AA binds to A-site (requires GTP)

2) peptide bond joins amino acids, (catalyzed by 23S rRNA)

3) ribosome moves 1 codon along mRNA

4) empty tRNA moves from P to E

Front 93

Translation: Termination

Back 93

-initiation: methionine coupled together with formyl group engages ribosomes at P-site

-Elongation: bring another tRNA molecule with its corresponding Anti-codon, peptide bond formed tween two amino acids

-Termination: takes place at any 3 stop codons, -UAA, -UAG, -UGA, (no tRNAs that correspond to these)

-release factors, cleavage, release polypeptide

Front 94

Mutations and their chemical basis (hnotes)

Back 94

•Mutations

–stable, heritable changes in nucleotide

sequence relative to wild-type

–may or may not effect phenotype

•Wild-type strains possess the typical or

representative characteristics of a species

•Mutant strains have mutations - stable, heritable changes in nucleotide sequence relative to wild-type strain

Hint 94

Although mutations may or may not effect

phenotype, they are often classified in terms of their effect on phenotype ...

•Morphological - change colonial or cellular morphology

•Lethal - kill

•Conditional - expressed only under certain conditions (e.g., high temperature)

Front 95

How Mutations Arise hnotes

Back 95

•Spontaneously

–absence of added agents (errors in DNA replication)

–Transitions, Transversions

•However, DNA replication errors are rare

– DNA pol works at rate of 1000 nucleotides

per second

•but incorporates incorrect base one in a billion

•has proofreading activity

Hint 95

•Induced - after mutagen exposure – chemical or physical agents - damage, alter DNA

Examples:

-UV light(generates thymine dimers)

-Base analogs- resemble bases, cause mis-pairing to occur, ex) 5-bromouracil, T-analog that pairs with G

-intercalating agents wedge themselves between certain segments

Front 96

Mutations in protein-coding genes can affect

protein structure in a variety of ways. The most

common types of such mutations are:

Back 96

-missense: single base substitution, changes codon for one amino acid into codon for another amino acid

-nonsense: converts a sense codon to stop (disables it)

-frameshift: insertion or deletion of one or two base pairs in coding region of a gene

Front 97

More Types of Mutations (hnotes)

Back 97

-Auxotrophs: mutations in biosynthetic pathways. (auxo- refers to the mutant strain) cant make the product of the pathway. requires product in media. (ex: lysine auxotroph cannot make lysine) labeled lys-

-

Hint 97

replica plating is used to identify auxotrophs.

Front 98

Microbes are equipped with a variety of

molecular tools that repair DNA damage,

let’s consider two.

Back 98

-"light" repair: photoreactivation, light activated photolyase, binds and cuts thymine dimer, fixing it.

-"dark" repair: nucleotide excision repair. UvrABC endonuclease removes section of damaged nucleotides. DNA polymerase I fills, and ligase joins.

Front 99

Creating Genetic Variability

Back 99

•Mutation (all domains)

–new alleles, new phenotypes

•Vertical gene transfer (Eukarya)

–sexual reproduction

–new combinations of genes when gametes from parents fuse

•Horizontal gene transfer (Bacteria, Archaea)–transfer from one mature, independent

organism to another

Front 100

Gene Transfer by Transformation

Back 100

-discovered by Fred Griffith

-Uptake of free DNA from environment.

-Competent cell: a cell that can take naturally take up DNA from the environment via transformation

• Only a few bacteria are known to be naturally competent:

•Gram +Streptococcus, Bacillus

•Gram- Haemophilus, Neisseria

Front 101

Membrane-bound protein complexes

bring DNA into the cell. How is DNA

changed during this process?

Back 101

Fig16.26

gram positive vs gram negative

gram positive: no outer membrane, needs additional proteins to allow DNA to enter cell

gram negative:

note* as DNA comes through barrier membrane, becomes unwounded by nuclease that is in membrane converst DS into SS

Front 102

Artificial Transformation

Back 102

•In lab with bacteria not naturally competent (e.g.,E. coli)

•Critical step in cloning

•Two techniques:

-Calcium chloride: makes cells more permeable

-electroporation: pulses high voltage, temporary holes- cell wall, plasma membrane

Front 103

DNA Transformation

Back 103

Front 104

Gene transfer by conjugation hpic

Back 104

-DNA transfer by direct cell-to-cell contact

-requires pill, and plasmids is commonly what is transmitted

-major mode of spreading antibiotic resistance genes between bacteria

Hint 104

Front 105

Plasmids

Back 105

•double-stranded, circular DNA

•extrachromosomal

•carry genes that confer advantage

•can be transferred by conjugation

• are replicons - have their own ori

-episomes- plasmids that exist with or without integrating into the chromosome.

Front 106

Example of conjugative plasmid (hpic)

Back 106

The F (Fertility) Factor of

Escherichia coli is a well-studied example of a conjugative plasmid

Hint 106

Front 107

The F Factor is an Episome hpic

Back 107

-can integrate into chromosome

-Cell now designated Hfr (high frequency of recombination)

-Can transfer F factor and part of its own chromosome into F-cell

Hint 107

Front 108

Transposition

Back 108

•Pieces of DNA move, insert different sites in

genome

•Transposable elements - called“jumping genes”

–Transposons

–Insertion sequences

•Insert, turn genes on or off