MIBO FINAL EXAM SPRING 2016

Learning Objectives

1. Flagella, Cilia, Pili

Contrast the difference between flagella, cilia and pili (both conjugative and non-conjugative). Also identify
which cells, prokaryotic and eukaryotic, possess these appendages.

Flagella Cilia Pili (conjgative) Pili (non-conjugative)=

FIMBRAE
Prok ( on the surface and Euk Prok Prok
have 3 parts) -gram Negative**
-proton driven

Euk (intracellular and

bigger)
-atp driven
Basal Body Hook and short, hair-like Pilin- short and rigid Pilin- short and rigid
Filament
-Flagellin – prok projections that OriT
extend from -- passing plasmids
9+2 arrangement of throughout the cell
microtubules made of
tubulin- euk surface; made of
--covered by cell tubulin
membrane extension

**movement *intracellular (lipid

bilayer)

9+2 orientation (9
couplets + 2 individual
tubulin filaments)

Chemotaxis helps locomotion, Transfers DNA between Attachment (type 4 pili)

Run- counter clockwise F+ and F- : conjugation *adhesion and BIOFILMS
Tumble-clockwise feeding circulation,
aeration
Prok: Shorter than Flagella
Monotrichous- polar
Lophotrichous
Amphitrichous-polar
Peritrichous

*endoflagella in spirochetes… in the space between the outer sheath and the cell wall peptidoglycan layer

2. Cell wall
Again, contrast the structure of the cell wall in bacteria, archaea and eukaryotic cells. What are the
components? What is the arrangement of subunits? Do all microorganisms have a cell wall? How does
staining pertain to the structure of the prokaryotic cell wall (gram staining vs. acid-fast)?

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 *mycoplasms are wall-less and are like “gram positive.” Instead they have sterols in their plasma membranes

Bacteria (Gram +)--- Pep
Almost all
Exception: mycoplasma
which are “like gram +”
Peptidoglycan (thick layer)
-long glycan chains
(glycosidic links) cross
linked by short peptide
fragments
NAG and NAM- Sugar**-
Cross bridge: D-Ala-D-Ala
*antibiotic attack
Gram stain: purple
Bacteria (Gram -)--- Pep Archaea---peseudomeurin Eukaryotes (carb)
Almost all Conditional… wall-less or Yes- Plants, Fungi, Algae
pseudomurein No-Animal
Peptidoglycan (thinner Pesudomurein (lack NAM Cellulose (glucan) and
layer) + D amino Acids) chitin (mannan)
-long glycan chains
(glycosidic links) cross “polysaccharide cell wall”
linked by short peptide
fragments
NAG and NAM- Sugars**
Cross bridge: D-Ala-D-Ala
*antibiotic attack
Nam connects
Gram stain: red

Acid-Fast for
mycobacterium cells
1. Teichoic Acid 4. LPS Glycocalyx
2. Lipoteichoic Acid 5. Lipoprotein -Carbs extending from the
3. Mycolic Acids 6. Porin Proteins plasma membrane of an
animal bonded to proteins
and lipids
S layer that does 7. Always
not protect competent
against
hypertonic
environment!

Only gram + can

produce
endospores!

*need high
density through
quorum sensing
to ebcoem
competent : use
transformase
 Name couple antibiotics that interfere with the integrity of the peptidoglycan? How do these
antibiotics work?
o Penicillin inhibits the enzyme transpeptidase which cross links the peptides (breaks cross
link)
o Vancomycin- prevents release of amino acid so that linkage cannot be made

Glycocalyx on Euk cells attachess to outside of ell wall:

o Gelatinous, sticky material on the outside of the cell

o composed of polysaccharides, polypeptides or both
o Two types:
 Capsule- highly organized, tightly attached
 Slime layer- loosely organized and attached
o Helps organisms attach to the cell surfaces
o Capsule can runaway from a phagocyte from an immune system
o Protective layer against dehydration and nutrient loss

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 o Inhibit killing by white blood cells by phagocytosis contributing to pathogenicity

3. Media
Look at the example question from Exam 1 regarding media. What makes this list of ingredients a complex
media?

Chemically Defined Media- Exact chemical definition of each ingredient that I could repeat this experiment… I know
exactly what the “agar” is or the “animal component”
---- can be used to grow specific heterotrophs

Complex Media- Must be rich in nutrients from extracts of plants or animals, but I do not know the exact chemical
composition
------will grow most heterotrophs

4. Gene structure and replication

Describe the arrangement of genetic material using the following terms: origin, regulon, operon, gene,
promoter

Origin : bacteria: replication begins at origin (oriC) … which after initation opens to make a bubble fork

-replicates bidirectionally until it terminates at the ter sites at the opposite end of the molecule.

-once started cell is committed

-A partially replicated chromosome can start new rounds of replication at the two daughter origins even before the first round is
complete

Promoter- -promoter site: sigma factor and rna pol recognizes… Sigma Recognizes consensus sequences at the –
10 and –35 positions, relative to the start of the RNA transcript (+1)

-- genes can have multiple promoters depending on environmental condition… one for normal growth versus heat
stress

 Mutations in the consensus sequence can affect the strength of the promoter

- Some mutations can cause decreased transcription (called “down mutations”), while others
cause increased transcription (“up mutations”).

 Origin- site on chromosome where replication begins (replisome machinery starts)

 Promoter- site before a gene or operon where transcription starts (RNA polymerase holoenzyme starts)

 Transcription: 1 start, 1 promoter, 1 transcription

 Translation can have multiple

 For each protein (Specific )

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 Regulon- collection of genes and operons at different positions on the chromosome. Regulated by same protein and have same
biochemical purpose ( aa biosynthesis) … transcribed together but translated at different times???

Operon-other genes in a unit acting in tandem… share the same promoter but each gene has its own start and stop
codons and is transcribed into one mRNA. polycistronic”

Gene :

Do we need to be able to explain every part on this?

Name the elements/enzymes involved in the replication machinery as well as how nucleotides are arranged
as they are added onto the growing chain.

Replication: 53

----Hydroxyl group on the 3’-C of the last sugar on the growing strand of DNA

Replication is : semiconservative and in bacteria also rolling circle

- DnaA: initiator protein : ratio of DNA to cell mass… when DnaA accumulates during growth it
triggers initation of replication… DnaA-ATP complex binds to 9-bp repeats upstream of origin
causing DNA to loop

- DnaB: helicase: opens the DNA

- DNA primase: synthesis of RNA primer:

- DNA Pol III: major replication enzyme: Sliding clamp (beta) tethers DNA Pol to DNA so it wont
fall off

- DNA Pol I: replaces RNA primer with DNA: synthesizes DNA using 3’ Oh end of preexisting DNA
as priming site in lagging okazaki strands

- DNA Ligase: repairs the phosphodiester nick using NAD (bacteria) and ATP (euk)

- Replisome: Two DNA Pol III with DNA primase and DNA helicase … make sure leading and
lagging are being made silumtaneously in 5 3

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 - DNA gyrase: relieves DNA supercoiling: Topoisomerase II that has multiple subuntis that
cleaves both strands to relieve tension… targeted by Quinolone antibiotics ..uses energy

- RNaseH: removes RNA primers

Dna Pol III exonuclease 3’—5’ ***

Single stranded binding protein

5. Carbon and energy sources; cardinal terms

Describe an environment where you would find a: chemoorganoheterotroph, chemolithoautotroph,

photolithoautotroph

Chemoorganoheteroph- bacteria and fish (euk, protists, fungi, animals) …

Chemolithoautotroph- caves where they use sulfur from the bedrock to fuel life… deep terrestrial subsurfaces

Photolithoautotroph- algae, bacteria, land plants

State what would be a possible carbon, energy and electron source.

Energy

Phototroph- light

Chemotroph- oxidation reactions (redox)

Electron

Lithotrophs- inorganic molecules (fe or h)

Organotrophs- organic molecules *fadh2 or nadh

Carbon

Hetero- preformed carbon

Auto- fix CO2

Describe environments where you would find barophiles, acidophiles, neutrophiles, halophiles, etc.

Psychrophiles : 0-20C: refridgerator, artic

Mesophiles: 14-45 C- our body

Thermophiles: 40-70 C: compost

Hyperthermophiles: 70-120 C: volcanic vents

Barophiles/pizeophiles: 110 MPA

Barotolerant: 1-50 mPA

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 Halophiles: 2-4M NaCl (10-20%).. high osmotic pressure

Acidophiles : 0-5 pH - pathogens

Neutrophiles: 5-8 pH- chemoautotrophs

Alkaphiles: 8-11 pH- soda lakes

6. Mutations
Categorize mutations according to their effect on transcription (whether or not mRNA is fully made) or translation
(whether or not a full and functional proteins is made): nonsense, silent and missense mutation, frameshift mutation,
transposition

Transcription (mRNA) Translation (protein)

Frameshift mutation.. adding or deleting nucleotide
causeing a who change in protein function
Silent Mutation… no change in AA but can affect protein
synthesis by slowing it down
Transposition… jumping genes that affect genome size
-A coding mutation affects translation NOT
transcription. ???
Point mutation (single base changes to affect mRNA)
Missense.. changes the AA sequence and if conservative
it does not change amino acid but if non-conservative it
changes AA sequence
Nonsense (stop codon so stop translation)… destroy
protein function
Conservative: same aa group
Nonconservative: different AA groups

Transition: purine – purine

Transversion: purine—pyrimidine

Transposition : Not autonomous like plasmids (plasmids can exist outside outside of larger DNA molecule)

-transposase catalyzes the transfer or copying of the elemtn form 1 DNA to another by binding to inverted
sequences

chromosomes and plasmids

Complex transposon (insertion sequence and transposon)--- these are what carry antibiotic resistance after
conjugation!

7. CRISPR
Using the diagram shown in the Exam 2 final exam topics, describe the function of CRISPR/cas in bacteria

Clustered Regularly Interspaced Short Palindromic Repeats

Needs: homing device and endonuclease (restriction enzymes)= CAS

Pros: Gene therapy, RNA targeting, drug target, vector control, pathogen gene disruption,

Cons: permanent changing evolution, ethical

1. adaptation 2. Transcription 3. Targeting 4. Destroy

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 2. CripsR rna is a hairprotein so that it binds to viral DNA so that cas can degrade it
3. Homing device is RNA

in bacteria and not used to move genetic material in-between species but as a defense
mechanism… capturing invading phage and inserting some of its genome into u …
—use RNA off of kept fragment DNA to couple that with CAS proteins to look for other
invading phages

8. Gene regulation (TRANSCRIPTION)… binds to DNA

Describe the difference between an operator and an activator; a repressor and an operon, constitutive,
induced and repressed expression. @ Transcription level

Operator- segment of DNA to which a transcription factor binds to regulate gene expression

-repressor binds

Operon: … transcription factor can be a repressor to stop or an inducer to start

--made into single RNA… all have 1 promoter and single start and stop codons

-- has to be next to each other (genes)

Promoter// regulatory sequences: rna transcription binds **

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 Regulon- single repressor or activator control with a sigma factor to control the regulon simultaneously of all the
genes … different positions at genome

--all genes have common mechanism they are controlled (same repressor or activator protein)****

Activator: bind to the regulatory sequence to stimulate transcription… needs ligands and touch the Stuck RNA pol @
promoter to initiation … induction inhibits repression

Repressor: Bind to operator sequence to prevent transcription… some ligands help repression (corepressors).
Repressor physically obstruct RNA POL

Induced –can displace a repressor protein from the operator DNA site to uninhibit operon

Constitutive- Glucose... always on!

Inducible-Lac operon when lactose is present in body

9. Respiration vs. Electron source

Explain the difference between aerobic and anaerobic respiration – how is this different from the electron
donor for ETC?

Chemo-organotrophy… aerobic vs anaerobic

Donator: organic when NADH

Inorganic: H2 or Fe2+

Aerobic Anaerobic
ETC final E- acceptor: O2 ETC final E- acceptor: S, No3-, So4 2-, Organic e-
acceptors
Ex: ETS transfers most commonly to O to make water Ex: salmonella uses tetrathioante as a terminal E
and reduce FADH2 and NADH acceptor
*prokaryotes

Fermentation: Nad+ make it **** so we use it when there is no terminal electron acceptor

10. Degradosomes vs proteases

Contrast the activity of degradosomes and proteases and their effect on the cell’s ability to adapt to
environmental change.

Both can remove the mRNA and proteins that are no longer needed in light of a new carbon source

-intracellular

-degradasomes- degrade mRNA

-proteases- Degrade protein

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 11. Prokaryotic carbon fixation
Explain the role of the carboxysome and how it enables the autotroph to use CO2 as a carbon source

---Calvin Cycle: Rubisco is within the polyhedral structure called Carboxyosomes.. Carboxysome take up Bicarbonate
to convert it to CO2 by carbonic anhydrase… The CO2 is fixed by Rubisco .. without it CO2 could pass out so need to
take it in as bicarbonate and fix it inside the carboxysomes

1. CO2 is converted to bicarbonate (HCO3-) which is trapped in cytoplasm and can’t leak out like CO2 could
a. Carbon –concentrating mechanism (CCM)
2. Rubisco w/I carboxysomes… that are in CO2-fixing lithotrophs, cyanobacteria, and chloroplasts. Polyhedral shell
of proteins surrounding tightly packed molecules of Rubisco
a. Inside the bicarbonate is converted to CO2 via carbonic anhydrase… this Rubisco is fixed to PGA… PGA
exits to complete the Calvin Cycle in the cytoplasm
b. When CO2 levels decline, genes induced to express trans membrane transporters of CO2 and
bicarbonate
c. CCM have low and high CO2 affinity gene, and a low and high bicarbonate gene.
d. High affinity transporters for low CO2 (starvation)
3. Two unlinked operons each encoding different set of CO2 fixation genes called Form I and form II
a. Have key enzymes like that of Rubisco… when CO2 is limiting form I works best

If the carboxysome was degraded by proteases, which of the following reactions may not be able to occur?

a. Condensation of CO2 with ribulose 1,5-bisphosphate

Ribulose 1, 5 is carboxylated *** condensed with CO2

*know NADPH and ATP steps

KNOW PGA

Explain how this done in cyanobacteria.

They have heterocysts to Fix N2 (energetically expensive)… must be anaerobic so photosynthesis is turned off. N2
 ammonium

---- others around them share energy and food sources and they fix the N2 for the other cells

Supposed you have discovered a mutant cyanobacterium that is incapable of producing heterocysts. Which
of the following observations would you expect to see?

Dependence on supplemental ammonia for growth

12. Biosynthesis
Describe how does the cell balances the cost of genomic material and energy for biosynthesis

Regulation- turn on or off via repressors or activators …. Catabolic or anabolic

---enyzmatic regulation

Genome degeneration- degrade genes you don’t need anymore

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 Secondary products- toxins, antimicrobials, virulence factors that help survive and compete better but are not
necessary… energy expensive

List the catabolic pathways that contribute intermediates to amino acid, purine and pyrimidine synthesis.

-Amino Acids

--from glucose metabolism and TCA cycle … linked by Acetyl-CoA

- some AA arise from key metabolic pathways where others can be made from other AA

-- AA can arise from 1+ source

Purine and Pyrimidine

- built onto a ribose 5-phosphate substrate… converted into 5-phosphoribosyl-1-PP (PrPP) via activation step
of ATP AMP
- First purine made is inosine then converted to AMP and GMP
- First pyrimidine is Uracil  CMP and TMP

Carbon fixation:

Strict autotroph- only way to get carbon to assimilate to aa, lipids, etc is through calvin cycle … not EMP, PPP, or ED

-generates just carbon not necessary energy

-energy can be photosynthesis or respiration with aerobic or anaerobic respiration

Heterotroph: use emp, ppp, or ed

--- have preformed organic sources

Devise a strategy to engineer an industrial strain to create an excess of the amino acid lysine.

--Lysine comes from pyruvate so increase glycolysis to pyruvate and keep pulling out lysine so it will make more.
Decrease pyruvate dehydrogenase ability so a build up in pyruvate will make more Lysine but also must pull lysine
out (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3026452/)

13. Viruses, viroids and prions

Contrast the structure and genetic makeup of viruses, viroids and prions.

• Differences/similarities between

• Viruses – protein, RNA/DNA, lipid (envelope).. non cellular

• Capsid head can be symmetrical (icosahedral ) or assymetrical

• Viroids – RNA only infecting plant and use host RNA dep RNA pol for its ssRNA

• Prions – protein only infecting animals/humans, no nucleic acid ***

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 Prions: Made entirely of protein


Linked to fatal human disease
 (Mad cow Disease0

Cause brain degeneration - sponge-like holes


Disease = transmissible spongiform encephalopathy


Symptoms may not appear for years after infection


Non-immunogenic

Categorize the activities of the T4 phage into early, middle and late stages of replication.

--Adsorption: entry by digestive enzyme eating peptidoglycan

--Penetration: only dsDNA is injected

-- Transcription of EARLY proteins.. DNA POL and DNase by using host RNA POL…

--Replication: rolling circle replication (linked in concatemer cleave)

--Late Genes: produce the capsid and tails and lysosome for lysis

---ATP driven motor packing DNA replication

--Assembly: on the membrane and is predetermined from genes encoded

--Lysis: via lysosome

As a +RNA virus, explain the steps in HCV virus synthesis after a single virion infects a host cell.

--Attachment: Lipoprotein finds LDLR at the liver, E2 binds to receptor and HCV virion attaches to tight junction

--Entry: endocytosis containg virion that casues a fusion to lysosome. Acidity uncoats the + RNA and releases to
cytoplasm

--Replication and translation: RNA genome uses host ribosomes (ER) for translation (IRES.. initates translation to
make polyprotein) and RNA DEP RNA POL for transcritption

--all in cytoplasm

--Assembly:

--Exit: Budding or move through tight junction to neighbor

*Don’t forget HVC can easily mutate through template switching to change all the alleles (quaispecies) whereas
influenza uses Antigenic shift to reassembly and make new virus

14. Virulence factors

Describe the difference between exotoxins and endotoxins, their composition and symptoms associated with
their release

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 Exotoxin Endotoxin
Kills host cell nutrients LPS on gram (-) outer membrane
Chemoorganoheterotophs Bacteria release this when they die (lipid A) which is toxic
alone causing a huge release in cytokines
Protein synthesis affected; plasma membrane affects; Hyperactivates immune system to cause shock and
superantigens (avoids antigen presenting process) death
ALPHA TOXIN(hemolytic )
Proteins
AB toxins
Superantigens ***

SaPI function and transmission

--Pathogenicity islands are inherited by horizontal transfer

-- PASSED ON BY TRANSDUCTION: mobile and shared along strains… need another phage to provide protein for
it to help it move… genome excises and goes to phage genome “coinfector phage” to help

15. Vaccines
Analyze the composition of an attenuated vaccine vs. an inactivated vaccine.

Attentuated Inactivates
Pros: Pros – long lasting immunity; exposure to non- Pros: does not replicate, few reactions
immunized individuals

Cons – exposure to non-immunize individuals; require Drawback: does not replicate, lower adaptive response ,
refrigeration; not applicable to pregnant women; could requires boosters and less antigens presented
mutate

Living virus or bacterial cell… so we see it longer in our Whole agents and toxoids or subunit agents
body and learn to respond stronger (recombinant, piece)
Can infect and replicate but with mild symptoms
--genetic engineering

Use figure 24.13 to explain how vaccines are used to generate a higher titer of antibodies when the natural
pathogen is acquired.

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 primary vaccination or natural infection leads to antibodies produced and stored but second exposure or “booster”
causes more T and B cells to be formed…. More exposure to epitope… rapid repsonse for IgG cells the second time

16. Antibiotics
Be able to identify the qualities of an antibiotic using the following terms: bactericidal or bacteriostatic;
broad or narrow; mechanism of action as discussed in the activity

-Bactericial- kill

Bacteriostatic- inhibit

-broad

-Narrow

Mechanism of action: targeting cell wall, DNA, RNA symthesis with examples *

Cell Membrane: disrupt the function /phospholipid bilayer

---make it more permeable to disrupt balance and cause linkage of ions  cell death

--- Ex:

Cell Wall:

--Bacterialcidal

Ex: Penicillin (beta Lactam), Cephlasporin (beta lactam), Vancomycin

DNA Synthesis

--Quinolenes – Dna Gyrase

--Nalidixic Acids

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 Rna Synthesis

-Rifamycin.. prevents this and thus protein synthesis

Protein Synthesis

Identify a mechanism of resistance

Acquired:

1. vertical gene transfer : bacterial replication !!! spontaneous mutation (rare)

2. horizontal gene transfer: conjugation, transduction, transformation

Gram – have resistance to penicillin because penicillin targets peptidoglycan layer proteins.. because it is thick and
outer most layer. Penicillin are not effective against gram – because of outer membrane layer and the layer is so thin

1.) Plasmids usually hold the resistant genes

---become antibiotic degrading enzyme

ex: beta lactamase which breaks beta lactam rings on penicillin ***

--- become an efflux pump that pumps out antimicrobial

--modify antibiotic target…

ex: penicillin targets penicillin binding proteins but if bacteria has gene to modify penicillin binding proteins, then
penicillin cannot bind ! found in MRSA!

To Remember:

Co factors can be inorganic or organic but coenzymes are only organic!

Heterotrophs- other feeding

Antigen presenting cell (macrophage, dendritic) picks up a microbe and diGEsts it to put antigen on MHC II Toll like
receptor which recognizes mamps/pamps  relays info to make cytokines !!! also will degade cells like it

Antigen presenting cell goes to lymph node to find a T0 which becomes a helper cell … helper cell finds B cell with
same antigen

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