Micro410_Final

Front 1

Regulation (cellular control mechanisms) can act at various levels of metabolism: (5)

Back 1

1. Act by change gene structure like phase variation (S.typhi inverts DNA segments to switch flagella types)
2. Modulating frequency of transcription of genes
3. Modulating freq of translation like protein components of ribosomes or like antisense RNA or like a Riboswitch.
4. Posttranslational modification like adenylation of glutamine synthase
5. Modify protein activity. Allostery.
6. Product modification, allostery... effector molecules or ligands

Front 2

Regulation of gene expression can come from diff. mechanisms (more specific).. like

Back 2

1. New theta, new RNA pol
2. Blocking transcription initiation b binding of active repressor to operator
3. Enhancement of initiation by binding of active activator to Activator
4. Control of translation initiation
5. Attenuation of translation
6. Control of transcript stability
7. Methylation of DNA affecting it's activity
8. Degree of supercoiling of DNA affecting it's activity.

Front 3

Negative control vs Positive control in inducible and repressible genes...

Back 3

Neg control - binds to operator to stop translation.
Pos control - binds to activator to initiate translation.
Inducible genes need allosteric inducer to initiate translation both for neg.(release from operator) or pos. (bind to activator).
Repressible genes need to be free of allosteric corepressor/ inhibior in order to neg. (bind to operator) or pos. (release from activator)

Front 4

Lac operon natural inducer is ?
What does it do?
Other inducers are: TMG, IPTG, ONPG

Back 4

allolactose, it inactivates the Lac repressor that is supposed to bind to operator site.

Front 5

Lac Z gene encodes for ?

Back 5

Encodes for B-galactosidase, the enzyme that breaks lactose to glucose and galactose.

Front 6

Lac operon CAP is?
Exists in what forms?
What is it's key repressor?

Back 6

Catabolite Activator Protein also called cyclic amp receptor protein exists in two forms:
-active form when 3',5' cAMP is bound and
-inactive form when it's free of cAMP

Front 7

c-AMP synthesized from what by what? ... degraded into what by what?

Back 7

c-AMP is synthesized from ATP by Adenylcyclase and degraded into AMP by phosphodiesterase

Front 8

High/Low glucose levels do what for cAMP.. that in turn does what?

Back 8

High levels of glucose (don't need B-gal), cAMP is low and CAP is in inactive form.
Low levels of glucose (need to use lactose), cAMP is high and CAP is active.

Front 9

Glucose is gone and Enzyme II GlcB subunit levels are ___ because phoshphorulation system is plugged up, that activates ______ to be made and then more cAMP is made that binds to ___ and it goes to _______ site on Lux operon

Back 9

Glucose is gone and Enzyme II GlcB subunit levels are high because phoshphorulation system is plugged up, that activates adenylcyclase to be made and then more cAMP is made that binds to CAP and it goes to CAP ACTIVATOR site on Lux operon

Front 10

Expression of Lac Operon from most favorable glucose/lactose conditions to least favorable conditions

Back 10

1. Most favorible: Lactose but no glucose. (allolactose is repressing repressor and cAMP and CAP are bound to activator site)
2. Less Favorable Lactose and Glucose. (allolactose but no cAMP)
3. Less favorable No glucose or Lactose (no allolactose but cAMP and CAP)
4. Least favorable Glucose but no lactose.

Front 11

Inducer exclusion for Lac operon, explain.

Back 11

Since lactose can't get inside cell, it needs the Lac permease... but Transport of glucose inhibits lac permease and inhibits entry into cell.

Front 12

In the Maltose operon (regulation), the _____ needs to bind to the Maltose activator protein to bind to ______ so RNA pol will have higher affinity for mal promoter... so expression is controlled by an activator protein, not a _____ protein like in the Lac system

Back 12

In the Maltose operon (regulation), the inducer needs to bind to the Maltose activator protein to bind to activator site so RNA pol will have higher affinity for mal promoter... so expression is controlled by an activator protein, not a repressor protein.

Front 13

Tryptophan Operon and Biosynthetic pathway has several different types of regulation: (3)

Back 13

1. Feedback by L-tryptophan to Anthranilate synthase (enzyme that changes Chorismic acid to intermediate before tryp)
2. Repression, DNA binding protein represses transcription
3. Attenuation

Front 14

Isofunctional enzyme inhibition:

Back 14

Endpoint products allow for differential feedback effects by the two pathway end products. Each isozyme is sensitive to it's own allosteric effector. They catalyze same reaction but are subject to diff effector molecules. ie. Substrate to DAHP to Chorismate to Trp Phe and Tyr.

Front 15

Cumulative feedback

Back 15

Cumulative feedback involves multiple allosteric sites on the regulated enzyme assuring that thee will be some activity unless all of the end products are in excess.

Front 16

Sequential feedback

Back 16

Sequential feedback inhibition, different end products operate seperately on various branches of the pathway.

Front 17

Concerted feedback inhibition

Back 17

Where a change in one is a change to all.

Front 18

Glutamine synthetase as an example of ______ feedback inhibition.
GS is ___ by what to become less active?
GS is used by what for what?
How does it obtain cumulative feedback?
What triggers it to be produced?
What is GS used as a barometer for?

Back 18

Cumulative feedback inhibition . Glutamine synthase is adenylated with AMP by ATP.
Glutamine synthtase used to incorporate ammonia into glutamine or glutamic acid.
To have cum. feedback inhibition each of 12 subunits must be covalently modified (8 allosteric effector sites each).
Want GS in more active state when you are low on Glutamine and /a-ketoglutarate.
GS is used as barometer of nitrogen availability.`

Front 19

Phase variation example

Back 19

S.typhi Flagella phase variation. Flagella is major determinate and is recognized by immune sytem. Involves 2 diff genes that encode for flagellum subunit. Enzyme called Recombinase flips promoters (two inverted repeats of high homology)

Front 20

Antisense RNA used for _____.

Blocks _____. size = ____.

Back 20

Antisense RNA is an example of regulation where it blocks translation by being bound to homologous site. they are encoded by a gene on the chromosome.

Front 21

Riboswitch is what does what?
What's an example?

Back 21

a Riboswitch is a specific metabolite that blocks translation when it causes formation of hairpin structures on mRNA... like Thymine which is itself a riboswitch.

Front 22

Regulation at the level of gene structure

Back 22

Flagella phase variation, methylation of DNA, degree of supercoiling

Front 23

Regulatory mechanisms control at the transcriptional level

Back 23

Sigma subunit, negative/positive regulatory proteins

Front 24

(Glutamine synthetase) How does cell avoid futile recycling of ATP when AMP-adenyl transferase also removes adenyl groups?

Back 24

Because of the protein PII that promotes A.transferase to remove AMPs.

Front 25

(Glutamine synthase) What controls the protein PII modification? How is it modified? How does this impact the activity of GS?

Back 25

PII modification by GTP is controlled by low levels of Glutamine... that increases the activity of GS by making it more active and less sensitive to feedback inhibition.

Front 26

Functional elements of an environmental sensing/response system: (5)

Back 26

Stimulus
Sensor (His that can phosphorulate itself)
Transducer
Response regulator
Feedback mechanism

Front 27

Two component environmental sensing system : (2)

Back 27

The 2 component enviromental sensing system links external events to regulation of gene expression.
1. Sensor-kinase ("histidine kinase") proteins
2. Response regulators and activation by "phosphorelay"
ie EnvZ and OmpR

Front 28

Phospho-relay Env. sensing example. 1. What is the stimulus? 2. What is the sensor? 3. What is the transducer domain? 4. What is the response regulator/Receiver domain? 5. What is the feedback mechanism? 6. What are the key residues in this system?

Back 28

1. Stimulus is something from environment.
2. Sensor is usually an autophosphorulating Histadine on protein.
3. Space between sensor and reciever.
4. RR or receiver domain is usually a conserved Aspartate Asp that takes phosphate group.
5. depends.
6. Key residues in these proteins are conserved His sensor protein and conserved Asp as response regulator

Front 29

EnvZ/OmpR system:
1. Is an example of what?
2. OmpF is a ____ expressed when? Repressed when?
3. OmpC is a ____, expressed when?
4. What is EnvZ? Can detect _____ in _____ and auto________ using ______ and gives it to ______.

Back 29

1. E.coli's osmotic environmental sensing system to regulate gene expression for porin size.
2. OmpF is a Large porin used in low osmolarity expressed when there are low levels of OmpR -P (binds to high affinity binding site) or repressed when antisense RNA micF is expressed.
3. OmpC is a Small porin used in (the gut) high osmolarity, is expressed when there are high levels of OmpR-P where OmpF is repressed and OmpC is activated.
4. EnvZ is a sensor protein in innermembrane has a periplasm and cytoplasm domain and some how can sense osmotic environment in periplasm, there it autophosphorulates itself with ATP and gives it to OmpR.

Front 30

AHL signalling.
1. from _____ family of synthases.
2. Signal percieved by _____.
3. Signal made by ______.

Back 30

Acyl Homoserine Lactone.
1. Lux I family of AHL synthases diff species have diff length C side chains.
2. If have high enough concentration of signal it's percieved by reciever LuxR
3. AHL signal made by LuxI

Front 31

AI-2 based signaling.
1. Characterized by ___ gene that makes___ .
2. AI-2 used for _____?

Back 31

Extremely widespread, different from AHL signalling.
1. characterized by luxS gene that makes DPD released into environment and undergoes spontaneous arrangement (diff final for diff species)... makes AI-2.
2. AI-2 May be important for interspecies signaling in environments rich in bacterial species.

Front 32

V. harveyi AI-2 based signaling.
1. LuxS does what?
2. _____ detects AI-2 specific molecules and signals ____ that induces _____ .
3. ____ makes AI-1 signalsl and those are detected by membrane sensor called _____ which also regulates Lux__.

Back 32

V. harveyi AI-2 based signaling.
1. LuxS is involved in producing AI-2 signal that's released into environment.
2. LuxP detects AI-2 specific molecules and signals LuxQ that regulates LuxR (involved with initiation of LuxS .
3. LuxM makes AI-1 signals and those are detected by membrane sensor called LuxN which also regulates LuxR.

Front 33

Gram + quorum sensing involves?
1. _____ serves dual functions, what are they?

Back 33

Most Gram-positive quorum sensing involves peptide signal molecules that differ.
1. Peptide signal serves 2 functions - as signaling process to make peptide concentration stable in extracellular environment and as an antibiotic (Naicin).

Front 34

S.aureus agr AIP system is what?
1. when cell-density reaches critical threshold it upregulates ______ and down-regulates _______.
2. ArgA decides ___ . ArgB is _____ ArgD _______, ArgC is a _____, and ArgA again is a ______.

Back 34

S.aureus AIP system is an example of gram + sensing peptide.
1. When cell density reaches critical threshold, extracellular virulence factors are up-regulated and Surface virulence factors are down regulated.
2. ArgA decides which promoter to align to... RNAIII that is regulatory or induce ArgBCDA. ArgB is (involved with peptide processing/export "protease?"),D (makes AIP), C (histidine kinase), and A (Response regulator)

Front 35

Ralstonia solanacearum's volatile signalling molecule is primarily used to ____.

Back 35

Ralstonia solanacearum's volatile signalling molecule is primarily used to Get in Xylem of plant, clog up pores via the production of clogging polysaccharide causes plant to wilt... acts in gas phase, acts to signal as quorum sensing.

Front 36

In the lux system of Vibrio fischeri:
1. Structural genes:
2. Regulatory genes:
3. Transcriptional Units:
4. Mechanism of regulation for
- luxICDABEG transcription ___
- luxR
- luxICDABEG activation ___

Back 36

Lux gene organization in Vibrio fischeri.
1. Structural genes include luxCDABEG
2. Regulatory genes luxR and luxI
3.Transcriptional units luxR and lux ICDABEG
4. - luxICDABEG transcription that's leaky yields minimal AHL synthesis.
- luxR is membrane-associated to detect extracellular AHL.
- luxICDABEG activation results in bright luminescence and positive feedback on AHL synthesis.

Front 37

Pseudomonas aeruginosahas two signal generators are ____ and ____ and two response elements ___ and ____.
1. LasR-AHL activates the ____
2. Sequential activation of specific target genes is due to delayed synthesis of ____ relative to ___.

Back 37

P. aerugeinosahas two signal generatosrs LasI and RhlI and two response elements LasR and RhlR
1. LasR-AHL activates the rhlR gene
2. Delayed synthesis of RhlR relative to LasR results in sequential activation of specific target genes.

Front 38

Eavesdroppers:
Burkholderia and Ecoli.
Signal jammers:
Exoenzymes that degrade ____.
Structural analogs that _____.

Back 38

Eavesdroppers:
Burkholderia activates virulence genes in response to Pseudomonas signals.
E.coli and S.typhi have LuxR-like response elements, but no LuxI-like signal generators.
Signal jammers:
Exoenzymes that degrade AHLs so no LuxR to response to make light.
Structural anlogs (eg halogenated furanones) that block AHL function... no light is produced.

Front 39

Zobell model:

Back 39

Zobell studied stalked bacteria and their adhesion to surfaces in the great Salt Lake and was the first to carefully study surface-ssociated bacteria, noted the importance of the sorption of bacteria.

Front 40

In nature it's estimated that ____ of bacteria reside within biofilms and ____ of infections involve biofilm environments

Back 40

In nature it's estimated that 90% of bacteria reside within biofilms and 65% of infections involve biofilm environments

Front 41

Example of gradients in biofilms is Nitrification where 1st the _____ change NH4 to NO2 and 2nd the _____ change NO2 to NO3. Who needs who?

Back 41

Nitrification where 1st the aerobic ammonia oxidizers change NH4 to NO2 and 2nd the nitrite oxidizers change NO2 to NO3.
Nitrite oxidizing bacteria circle around Ammonia oxidizing bacteria.

Front 42

3 Reasons why biofilms are more resistant to antimicrobial agents than planktonic cells...

Back 42

1. The extracellular polymeric substance or exopolysaccharide matrix serves as a diffusion barrier.
2. Slow growing subpopulations. (most antibiotics attack fast growing organisms).
3. The "biofilm" phenotype

Front 43

Why form a biofilm?

Back 43

1. Promote metabolic interactions between community members.
2. Can facilitate horizontal gene transfer.
3. Increased resistance to antimicrobial stress (sometimes up to 1000x more resistant)
4. Can allow access to nutrients.

Front 44

One dispersal pattern starts in the center of biofilms...

Back 44

Center is empty (11day old biofilm) like a beehive with freeswimming cells in middle. Probably eating eachother.

Front 45

Clinically important biofilms
1. Native valve endocarditis
2. Cystic fibrosis pneumonia

Back 45

1. Viridans group streptococci: Staphylococci
2. P.aeruginosa and Burkolderia cepacia:

Front 46

Adaptations that occur in biofilm/CFibrosis lung environment (4)

Can detect Cfibrosis by ____ signal production because it indicates biofilm

Back 46

P. aeruginosa adapts to lung environment by:
1. Loss of motility functions.
2. Los of LPS O-antigens
3. Alginate overproduction (polysaccharide)
4. Amino acid auxotrophy

Can detect CF by quorum sensing signals of bacterium

Front 47

Inherited Cfibrosis?
1. Defect due to _____
2. _____ results in impaired _______
3. _____ colonizes environment initiating biofilm formation and persistant mucus hypersecretion by _______

Back 47

Inherited Cfibrosis?
1. Defect due to CFTR mutation
2. Dehydration results in impaired mucociliary clearance
3. P.aeruginosa colonizes environment initiating biofilm formation and persistant mucus hypersecretion by goblet cells

Front 48

A positive ssRNA virus such as ______ acts as _______ immediately after entering the cytoplasm of the infected cell.

Back 48

MS2, mRNA

Front 49

Morphology of viral capsids (3) and capsids of eukaryotes vs bacteriophages

Back 49

Morphology of capsids: 1. Filamentous
2. icosahedral
3. Prolate icosahedral with helical tails.
Eukaryote capsids can be either naked or enveloped...most bacterophages are naked (without membrane envelopes) Eukaryote replication cycle is also longer hrs vs min. Eukaryote, capsid often enters cell and voral rep. ccle often leads to prolonged association with host, cells often lyse but many viruses bud from cell.

Front 50

MS2
Describe -
Has ____ genes, they are and they do...

Back 50

Small icosahedral ssRNA phage genome size of 3569bp synthesis of 4 phage proteins during infefction is altered by elaborate translational control. 4 genes:
1. Coat gene has the most readily accessible RBS on phage RNA molecule which acquires complex secondary structure in the cell.
2. The 'A' maturation gene can only be translated during the replication of viral + strands.
3. Replicase protein associates with 3 other host proteins to form a viral RNA-specific RNA polymerase.
4. Lysis protein

Front 51

MS2 (second card)
1. Polymerase is viral origin,
High _______ because of lack of proofreading on _____.
2. MS2 controls translation by secondary structure of +RNA
3. Initial binding to ______.

Back 51

MS2 ssRNA (all RNA viruses require specific RNA polymerase BTW..since host pol only recognizes DNA)
1. MS2 polymerase has a high mutation rate because of lack of proofreading on it's own RNA pol.
2. During the initiation of translation for the capsid protein, the secondary structure that blocks the replicase translation is broken and then the ribosome can bind to translate replicase... then finally, transcription of viral + strand allowing for maturation proteins.
3. Initial binding to side of pilus. (F plasmid-deficient e.coli strains are resistant to both M13 and MS2.

Front 52

Overall viral particle size is proportional to

Back 52

Size of genome. Genomic material of phage is 25-50% of viral mass. 90% viral genome codes for protein

Front 53

Enzymes that get packaged along with viral nucleic acid

Back 53

Involved with production of RNA/DNA material. Other enzymes included in viral particle include Lysozyme.

Front 54

Several viruses infect Archaea:
1. Many known archeal viruses have ________.
2. Not well characterized.
3. Common ss and ds

Back 54

1. Spindle shape morphology
3. DNA/RNA
4. Icosohedral in symmetry
5. Most motifs have already been characterized by bacterial phages.

Front 55

Infection cycle (5)

Back 55

1. Attachment specific surface receptor. (protein or carbo)
2. Reaction triggered and Viral DNA/RNA enters via penetration or injection into host cell cytoplasm (During this process, the protein shell will remain outside of host bacterial cell).
3. Synthesis of nucleic acid and protein Early viral proteins inhibit host functions often leading to production of many copies of viral mRNA and then translate. 4. Later viral proteins are involved in capsid assebly and genome packaging.
5. Release (Lysis)

Front 56

Small genome viruses

Back 56

viruses with small genomes largely rely on host cell producing RNA and DNA

Front 57

Bacteriophage M13
1. What kind of genetic material?
2. What kind of structure?
3. What about the capsid?
4. How does it exit host?
5. No synthesis of viral proteins until after _____?

Back 57

1. + strand circular ssDNA, 6500 bases and 9-10 genes.
2. DNA does not form significantt 2ndary structure inside cell.
3. Assymetric capsid; 2700 proteins with distinct binding protein at one end
4. Virus buds from host without lysis/killing... complex phage assembly (or export)apparatus that assembles capsid proteins and DNA and allows trasit through cytoplasmic and outer membrane of cell.
5. No synthesis of viral proteins until after synthesis of viral negative strand.

Front 58

icosahedral ssDNA phage fX174:
(E.coli/enteric)
1. DNA enters cell and is converted to _______.
2. CisA is what?
3. How does it replicate early on?
4. Late infection products are?

Back 58

1. DNA enters the cell and is converted into a ds circular molecule by the host DNA synthesis machinery. Host topoisomerases supercoil the dsDNA creating the replicative form I(RFI) molecule that is active for replication of viral + strands.
2. CisA is an early protein that nicks the + strand of viral RFI (at origin of viral replication) and attaches to the 5' end of viral DNA forming an RFII.
3. 3'OH end of + strand is open for rolling circle replication. After 1 complete strand has been copied, CisA re-ligates DNA creating a circular ssDNA + strand and a dsDNA.
4. Late in infection the only prodct of replication is progeny + strands that associate wth other viral proteins nda re packaged into viral capsids.

Front 59

Rolling circle replication, where is - strand located? Which phages do it?

Back 59

Neg strand is always the circle/template for rolling circle replication.
fX174 and M13... both have similar replication techniques

Front 60

fX174 has overlapping genes, this means that:

Back 60

One secion of genomic material can specify the production of multiple translational products by:
1. Translation of 1 gene from 2 diff initiation sites ie CisA and CisA*.
2. Translation of 2 diff genes from the same section of mRNA in 2 different translational reading frames (ie CisA and CisB).

Front 61

Lytic "Beast" T4 169kd genome
1. Is 85% similar to ____ with the differences being ____.
2. It overwhelms host cell synthesis by: A,B,C

Back 61

1. Is 85% similar to T2/6 differences relating to receptor binding sites.
2. A. Inhibiting host RNA synthesis bia ADP ribosylation of sigma factor...makes own sigma factor that asociates with core RNA pol for T4 specific transcription. (T7 makes own RNA pol).
B. Nucleases (it's genes encode for) degrade host cell DNA.
C. T4 DNA is modified so that the T4DNA is resistant to the nucleases on the cytosine base in the form of 5-hmC.

Front 62

T4 DNA and Lambda/T7 DNA concatamers
1. Phage DNA undegoes recombination forming long ____.
2. These ___ are processed into _____ representing one "headful" of DNA
3. Individual pieces are ____.
4. Every piece contains _______.

Back 62

1. Phage DNA undergoes recombination forming long concatamers (linear molecules of several genomes attached to one another)
2. These concatamers are processed into 170kb pieces in length.
3. Individual pieces are packaged into phage heads (T4 has different terminal repeats, lambda has identical because of cos sites inbetween)
4. Every piece has complete genome (plus duplicated seuence at ends for T4 beast)(same pieces for lambda and T7)

Front 63

T4 Beast part II
What specific enzymes encoded by genome?
2. How does it protect DNA

Back 63

Amazing virus that encodes a lot of enzymes in it's genome that helps with proliferation of the phage

-T4 specific DNA polymerase
-T4 specific DNA ligase
-T4 specific Recombinase
2. Protects own DNA during lytic rep cycle so during degredation of host DNA - DNA is chewed up to bits down to the deoxynucleotide monophosphate compounds then normal host enzymes convert dNTP back to normal except for Cytosine, glucosylation (added post replication) this endows phage to complete resistance to OWN T4 nucleases eating host DNA.

Front 64

Phage Lambda
1. Size?
2. Structure?
3. Binds to ?
4. Temperate phage?

Back 64

1. dsDNA, 48.5kb
2. Icosohedral head with tail.
3. Binds to maltoporin in Out.Memb of E.coli
4. Temperate capable of 2 distinct fates upon infection.

Front 65

Prophage DNA means?

Back 65

Means the Viral DNA is integrated into host DNA

Front 66

Phage conversion is?
Examples: (4)

Back 66

Expression of prophage genes can give the host cell new characteristics. Is DNA encapsidated in a virus particle.
1. C. diptherium - Bphage -> dyptheria
2. V.cholera - Prophage CTX toxin -> Cholera
3. Streptococcus - Prophage -> Necrotiting Fascitis
4. E.coli - Lambda bacteria phage from shigella -> E.coli H0157 Jack in Box.

Front 67

Transduction

Back 67

Transfer of chromosomal DNA from one bacterial cell to another mediated by bacteriohages.
Specialized transuction and Generalized transduction

Front 68

Specialized Transduction:
1. How many kb of host genes can be replicated along with prophage?
2. What happens if phage infects new host?
... what happens to phage DNA?... what happens to host genes adjacent to prophage?

Back 68

In specialized transducing phage, only chromosomal DNA adjacent to the original prophage attachment site can be transduced.
1. As a result of an incorrect excision of prophage A few Kb of host genes adjacent to prophage can be replicated with partial (defective) phage genome.
2. If phage infects new host, bacterial DNA might recombine and alter host genome... bacterialDNA in he phage can recombine with host genome.. but Phage DNA is lost and only host genes adjacent to prophage (fewKb) can be copied with partial phage genome.

Front 69

Generalized Transduction.
1. Is what?
2. Depends on what?
3. What mechanism allows packaging?
4. How much host DNA?

Back 69

In generalized Transduction, error in packaging phage DNA into capsid structures. Any portion of chromosome can be packaged into phage capsids as long as it's of the correct size (ie S.typhi phage P22)
2. Depends on innate ability of capsid to inject DNA into infected cell.
3. "headful" mechanism without recognizing specific DNA sequence is what allows for this.
4. Can carry up to 50-100kb of host DNA

Front 70

Natural Transformation:

Back 70

1. Devopment of competence
2. Binding of DNA
3. Processinga nd uptake of DNA
4. Integration of DNA into chromosome by recombinationand expression.

Front 71

Gram-Positive Transsformation steps:
1. Before uptake into cell, what happens to DNA?
2. What do external nucleases do?
3. Is there DNA sequence that affects ability of DNA to bind?
4. B.subtillis

Back 71

1. Before uptake in gram+ cells, DNA fragments ~15kb in length are bound and processed ~8-10 kb in length.
2. On external surface of cell nuclease converts dsDNA to ssDNA during DNA entry into cell.
3. There is not any DNA sequence that affects the ability of DNA to bind to cell surface prior to transformation but in some ie B.subtilis, the acquisition of competence is regulated by quorum sensing peptide by a 2 component sstem, the activated response regulator induces expression of competence genes.

Front 72

Gram negative Transformation:
1. What structure forms?
2. How large of dsDNA can specific receptor bind for?
3. What's required for DNA binding and uptake?
4. H.influenzae

Back 72

1. Membraneous transformasome forms that contains..
2. a specific receptor for binding dsDNA up to 40kb in length.
3. specific recognition sequences within the DNA are required for DNA binding and uptake.
4. In H. influenzae, the DNA binding sequence is 11bp in length... find that this sequence occurs 1400 times in genome:).

Front 73

Lambda Phage:
1. Porin involved in passage of sugars is target =
2. 1st thing that happens to dsDNA after entering phage?
3. Early replication?
4. Late replication?

Back 73

1. LamD porin
2. Upon entering cell, Lambda DNA circularizes. (ss tails are of appropriate sequence and directionality that they can cirularize themselves 12bp overhang)
3. Early replication is in theta form.
4. Late replication for production of progeny genomes is symmetric rolling circle... both strands end up being replicated unike FX174.

Front 74

Repression of activator of lytic gene expresson is largely dependent on ratio of what?

Back 74

c1 (lytic repressor) and Cro (lytic activator)

Front 75

1. Lambda encoded _____ catalyzes sitespecific recombinationwith E.coli chromosome between ___ and _____ .
2. Site specific recombination on phage is called ____
3. Host sequence similar to phage is called _____?

Back 75

1. Lambda encoded integrase catalyzes site-specific recombination with e.coli chromosome between gal and bio.
2. att
3. Similar host sequence is called bacterial attachment site... integration occurs primarily at one location on genome.

Front 76

Induction of prophage....
1. What happens?
2. Why does it happen?
3. What can happen with incorrect excision?

Back 76

1. Induction of prophage pops out of genome
2. due to variety of environmental stresses such as uv starvation etc.. Inactivationof c1 repressor and off to the races for lytic function.
3. Durin induction, incorrect prophage excision can lead to specific host genes in replicating DNA molecule and in capsid

Front 77

F plasmid.
Size/Copy number?
Host range?
Traits?

Back 77

"Fertility" plasmid.
100Kb. One copy.
E.coli
Conjugal with phage sensitivity to MS2.... It contains genes that constituatively express for F pili (because of location of IS3) so it's always capable of conjugation and exchanging DNA.

Front 78

R 100 plasmid.
Size/Copy number?
Host range?
Traits?

Back 78

"Resistance" plasmid
89Kb with one copy.
Enterics
It is conjugal with phage sensitivity and drug and Hg resistance.

Front 79

Ti plasmid.
Size/Copy number?
Host range?
Traits?

Back 79

200Kb with one copy.
Agro/Rhizo
Causes plant tumor

Front 80

RP4 plasmid.
Size/Copy number?
Host range?
Traits?

Back 80

60Kb with 4 copies /cell
Broad host range Gram neg. E.coli and Pseudo
Conjugal, drug resistance and phage sensitivity

Front 81

ColE1 plasmid.
Size/Copy number?
Host range?
Traits?

Back 81

ColE1 the Hitchhiker (can only mobilize when Conjugal plasmid is also present)
9Kb with about 30 copies/cell.
Narrow host range
Mobilizable with colicins -growth advantage (genes coded toxins that act against other cells in environment as well as immunity to same toxins)

Front 82

F plasmid
1. What are some similarities and differences with R100?
2. Describe plasmid transfer via F conjugation.
3. What are some elements in plasmid? What region is responsible for conjugal ability?
4. What virus likes F pilus?
5. What protein nicks ss at oriT and acts as helicase in F plasmid... and also religates nicked DNA?

Back 82

1. Fertility plasmid doesn't have drug and Hg resistance like R100, but other than that they're pretty similar and even are compatible with eachother.
2. Pilus is extended and retracts pulling cells together,
- F plasmid oriT is nicked (by plasmid specified relaxase also acts as helicase) 5'-3' ssDNA transfer
- DNA pol III replicates (leading strand in donor)
- Religation of oriT at end.
3. 99Kb plasmid contains several IS/Tn elements and Rep/Par functions. Tra genes responsible for conjugal ability
4. MS2 covers F pilus.
5. TraI

Front 83

F plasmid chromosome integration.
1. What segments of plasmid allow it to recombine with host chromosomes?
2. What is frequency of popping back out?

Back 83

1. IS/Tns (specifically IS3) allow plasmids to recombine with chromosomes, formation of co-integrant, express conjugation in context of Hfr, cell can initiate transfer.

Front 84

Gated pore in DNA transfer allows for?

Back 84

Passage of DNA without exchange of nutrient contents

Front 85

What's the difference between
1. Plasmid transfer conjugation
2. Chromosome transf. conjugation?

Back 85

1. autonomously replicating DNA element.
2. Hfr mediated chromosomal transfer along with few plasmid base sequences... but MUST recombine with recipient.

Front 86

1. What genes on plasmid promote transfer of plasmid? What do they make?
2. Replication, partitioning and incompatibility functions are in what region of F plasmid? What do they contain?

Back 86

1. Tra genes. (one or two big blocks of transfer genes) specify syntesis of conjugal pilus, enzymes enable processing of DNA, and proteins that make up the cell envelope based DNA transfer pore.
2. Rep, par and inc genes.

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F+ means?
Hfr means?
F' is?

Back 87

F+ denotes cells that have the unintegrated form of F plasmid.

Hfr denotes cells with incorporated F plasmid (into genome) stands for High frequency recombination.

F' is a form of F plasmid that has inaccurately excised from chromosome and contains a portion of host chromosome too.

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Outcomes from exchange of genetic info. (2)

Back 88

1. Gene substitution:
-transduction,
-Natural transformation
-Hfr conjugation
2. Gene addition:
-phages forming lysogens,
-plasmid conjugation and mobilization
-artificial transformation with plasmids.

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Bacteria combat the flow of DNA into a cell via:

Back 89

Restriction-Modification (R-M) systems. Strain specific nucleases. Strain specific modification enzymes that protected against nucleases.

Front 90

Who gets to foreign DNA first, Host cell nucleases, or Host cell modification (methylases) enzymes?

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Nucleases are ~10000x's faster than modification enzymes.

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Hfr cells.
1. Cannot take up a second ___.
2. Hfr donating cells lead to what in recipient?
3. Direction of oriT during integration determines what?

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High frequency recombination.
1. Hfr (with full F copy)Cannot take up a second copy of the F plasmid because the alteration of surface receptors mean the cell is no longer able to behave as a recipient in conjugation. But partial Hfr F plasmid cells don't count.
2. Hfr donor leads to transfer of genes from the host chromosome and that of the recipient. Via homologous recombination and the RecA protein results in substitution.
3. Direction of oriT integration determines which part of F plasmid will be transferred since only part of the chromosome is usually transferred (entire plasmid is rarely transferred)