Genetics of Microorganisms Test 2
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- Stage 1 Rolling-Circle Replication
- The Rep protein binds to DSO; SS break; Rep remains attached to 5' end; DNA-PolIII uses free 3' end as primer; Replication around the circle; Second break by Rep and ligation by Rep; Give replicated DS plasmid and SS plasmid
- Theta Replication
- Most common form of plasmid replication; Uni- or bidirectional; Gram negative plasmids
- Gel Electrophoresis
- Purified plasmid from other processes; DNA migrates according to size and shape; supercoiled moves furthest
- Separation on CsCl-EtBr gradient
- Centrifugation separates based on density; EtBr intercalates into DNA but not plasmid as it is circular; Plasmid is lower in density
- Amplification by Cloramphenicol
- Chloramphenicol inhibits chromosomal replication by protein synthesis inhibition; plasmid continues to replicate, building up inside the cell
- Alkaline lysis
- Treat bacteria with lysozyme and alkali which lyses the cell and denatures DNA; Add salt which precipitates DNA and cell wall material; centrifuge; Detect by running supernatant on agarose
- 4 techniques for plasmid isolation
- Alkaline lysis; Amplification with chloramphenicol; separation on ScCl-EtBr gradient; Gel electrophoresis
- Curing Experiment
- Grow bacteria in prescence of acridine orange or EtBr which causes loss of plasmids; Thest whether trait is lost when plasmid is lost
- What do you use to determine whether mutants act in cis or trans?
- Complementation Test
- Stage 2 Rolling-Circle Replication
- RNA polymerase makes a primer at SSO and this RNA primes replication around the circle by DNA Polymerase III;
- How is the second stage of rolling-circle replication regulated
- the SSO is located immediately before the DSO so the SSO will not appear until the first stage is almost complete
- What are relaxed Plasmids?
- high copy-number plasmids
- What is ColE1-derived Plasmid replication regulated by
- RNAI
- How does RNAI regulate ColE1-derived plasmid replication
- forms a kissing complex with RNAII. RNAII is then not available as a primer to start replication
- What is Rop?
- Stabilizes kissing complex of RNAI-RNAII complex
- What is Handcuffing or Coupling?
- RepA, which regulates plasmid replication, binds to iterons on two plasmids, linking the two and preventing both from replicating
- What are the parts of the par-system
-
parS - cis acting site; plasmids are pulled apart
parA - ATPase, binds to parS/parB complex
parB - binds to parS - How do kill-proteins work in relation to plasmids
- production of a protein that kills cells that do not contain the plasmid. The plasmid expresses an antidote. Both the toxin and antitoxin are present in cells with the plasmid. If the plasmid is lost, the toxin is activated by the instability of the antitoxin
- What is Inc
- An incompatibility group of different plasmids based on identical replication control and identical par system
- What is Conjugation?
- Plasmid mediated exchange of DNA between bacteria
- What is self-transmissible plasmid?
- both conjugative and mobilizable
- What is a Conjugative plasmid
- Can form pairs
- What is a Mobilizable plasmid
- Can be moved by self transmissible plasmid
- What is a donor in conjugation
- bacterium containing the conjugative plasmid initially
- What is a recipient in conjugation
- plasmid free bacterium
- What is a transconjugate in conjugation
- result of a mating. It is now a potential donor
- What is promiscuous in conjugation
- conjugative plasmid with a wide host range
- Most naturally occuring plasmids are what in relation to conjugation?
- transmissable or mobilizable
- How are promiscuous plasmids important?
- important in evolution and spread of antibiotic resistance
- What is the cis-acting site in genes of self-transmissable plasmids
- oriT
- What are trans-acting factors encoded by in self-transmissable plasmids
- tra
- What is a Dtr component
- DNA transfer and replication
- What is an Mpf component
- mating pair formation
- What does an mpf form
- large, membrane associated structures between mating pairs, holding them together
- How do coupling proteins communicate
- through dtr and mpf
- Are pili vehicles of DNA transfer?
- No
- In the F plasmid tranfer in E. coli, what does F+ make and which cell contains the plasmid (donor or recipient)?
-
makes sex pilus
donor - What two items are required for transfer in conjugation
- relaxase and oriT
- What does relaxase do
- creates a ss break at the nic site in oriT
- What does relaxase interact with during tranfer
- coupling protein of mpf-complex
- What are male specific phages
- bacteriophages that use sex pili as a receptor
- What is M13
- a filamentous, ss DNA, phage
- The expression of tra genes depends on what
- TraJ, a transcriptional activator
- What regulates traJ expression
- finO and finP
- FinO stabilizes what
- finP
- How does F always contain a pili
- F lacks finO, a fertility inhibitor, so the pili is always there
- What is required for mobilization
- self-transmissable plasmids
- What do mobilizable plasmids not encode for
- mpf gene functions
- what region encodes tra genes in mobilizable plasmids
- mob
- What does the mob region do in terms of host range
- expands host range
- Agrobacterium tumefaciens has what kind of plasmid
- Ti or tumor inducing
- Ti plasmid contains two DNA transfer machineries. What are they?
- bacterium to plant and bacterium to bacterium
- A pilus is not involved in what kind of conjugation
- conjugation in gram positives
- Pheremone plasmids are found in what bacteria
- Enterococcus faecalis
- What are pheremone plasmids
- plasmids that can carry antibiotic resistances and virulence factors giving E. faecalis a high rate of antibiotic resistance
- What activates plasmid transfer in inducible plasmid transfer
- Peptides secreted by recipient and used to activate plasmid in donor
- Which cell releases the pheremones
- recipient
- What is transformation
- uptake of naked DNA molecules
- What is compentent in reference to transformation?
- capability to take up DNA
- What is a transformant in transformation?
- Cells that successfully took up DNA
- What is natural competence in transformation?
- Bacteria that can take up DNA on their own
- What is artificial competence in transformation?
- chemical or physical manipulation to achieve competence in bacteria
- In transformation, do ds DNA or ss DNA enter into the cytoplasm of the cell
- One strand is degraded in a ds DNA and a ss enters into the cytoplasm
- Integration of naked DNA into the cell's DNA is done by what process
- homologous recombination
- What are the com genes in Bacillus subtilis
-
comEFG - operon structural genes
comEA - DNA binding
comF - DNA translocation
comG - channel formation - When does B. subtilis develop competence and sporulates
- under nutrient deprivation
- comEFG regulated by what
- comA and comK
- What is meant by induction density dependent in respect to regulation of competence in B. subtilis
- accumulation of peptide signals is required to induce competence
- comX pheremone and CSF are used in what process
- regulation of compentence in B. subtilis
- CSF is imported into the cell by what
- opp
- How do you test DNA uptake efficiency in respect to naked DNA uptake
- Label DNA, Add to cells, treat mix with DNase, Collect cells on filter
- How do yout test specificity in DNA uptake
- DNase treatment of the DNA outside of the cell,
- What is the mechanism of DNA uptake in S. pneumoniae
- dsDNA binds to receptors on cell surface, broken into smaller pieces by endonucleases, one strand degraded, one transported into cytoplasm, integration into chromosome by recombination
- What is the mchanism of DNA uptake in H. influenzae
- initial uptake of dsDNA in transformasome
- How do we know that ds DNA is broken down and ss DNA enters the cytoplasm
- DNase only breaks down dsDNA
- Plasmids have to be what in order to be taken up in naturally competent bacteria
- has to be dimeric so it can recyclize once inside the cell
- What are the possible roles for natural competence
-
DNA repair although compentence genes not induced in response to DNA damage
Nutrition is unlikely - What is the chemical treatment for artificially induced competence
- CaCl2. This is used for plasmid transformation
- What is electroporation
- an intense electric field used in artificially induced competence
- What are bacteriophages
- viruses that infect bacteria
- What are plaques
- holes in bacterial lawn from phage activity
- What is the host range of phages
- specific to bacterial strains
- What is T4
- One of the largest known viruses
- What are immediate-early and delayed-early genes transcribed from
- o70 promoters
- Middle genes require what as alternate promoters
- delayed-early gene products
- What do the true late genes code for
- Head, tail, and tail fibers
- What is the alternate promoter factor in true late genes
- gp55
- Some true late genes are only expressed if what occurs
- DNA replication
- What is required for gp55-RNA pol complex to start transcribing
- interaction between virus sliding clap and gp33
- Is T4 linear or circular
- linear
- What is a concatemer formation
- individual DNA linked end to end
- What are the two stages in DNA replication in T4
- replication from well-defined origins; ss ends invade daughter strands, form D-loops, prime replication to form branched concatemers. This is recombination dependent replication.
- What is the first stage of DNA replication in T4
- Primers invade DNA strand, form R-loop which primes the leading strand
- What is the second stage of replication in T4
- 3' end serves as primer, producing long concatemers, which are then ready for packaging
- What is required for pairing of invading strand in T4 DNA replication
- RecA
- What is cut from concatemers during packaging
- individual genome length DNA
- How is terminal redundancy created during packaging
- a larger than necessary phage head
- Are filamentous phages ss or ds
- ss
- When is the protein coat removed with filamentous phages
- removed on entry into the cell
- Do cells lyse with filamentous phages
- no
- What is the + strand in phage replication
- infecting DNA
- What is RF in phage replication
- replicative form
- What is primed by host RNA-Pol in replication of ssDNA phages in M13
- negative strand, ligation, and supercoiling
- How is the + strand replicated in phage replication
- rolling circle replication
- What is used to nick the ori in phage replication during RC replication
- endonuclease GpII
- What does GpV do in phage replication
- binds to ssDNA and prevents the negative strand formation
- Why can M13 be used as a cloning vehicle
- no fixed length
- What are permissive conditions
- phages can multiply
- What are non-permissive conditions
- phage cannot multiply
- What is MOI
-
multiplicity of infection
number of phage/number of bacteria - What does a high MOI signify
- phage numbers greatly exceed bacterial numbers
- What are crosses in reference to MOI
- infection of one host with two genetic distinct phages
- How do you find percentage of population infected in a cross
- take the percentage of infected population by one phage and square it.
- What type of Test uses the permissive host for both phages
- Recombination test
- What uses a non-permissive host for testing
- Complementation test
- In T4 analysis of rII how do you make a phenotypic distinction between the mutant and wild type
-
mutant has hard edges
WT has fuzzy edges - What type of phage has a stable relationship with the host
- lysogenic phage
- What is the phage called in lysogenic phages
- prophage
- What is the host in lysogenic phages
- lysogen
- When are lysogenic phages activated then leave the host
- cell damage
- Lysogenic phages prevent what from other phages of the same type
- superinfection
- What are the first genes expressed in phage lambda
- N and cro
- What is coded for in late gene expression in phage lambda
- head, tail, lytic enzymes
- What is cro in phage lambda
- inhibitor of repressor synthesis, which is required for lysogeny
- What is N in phage lambda
- antitermination factor
- What does N bind to and interact with
- binds to nut, interacts with RNA-Pol
- What is the general name for host proteins involved in antitermination
- nus
- What joins ends of linear lambda DNA and what are the cohesive ends called
-
cohesive ends called cos sites
DNA ligase joins ends - During packaging in lambda phage replication, where are the head proteins recognized and cut
- cos sites
- What are the two lambda gene requirements in lambda replication
- O and p, a primer at ori
- What does gam do in lambda replication
- shifts to rolling circle replication
- what does gam mutant do in lambda replication
- shifts to theta replication
- What are cII and cIII transcribed from
- respectively, pR and pL
- What is the function of cIII in Lysogeny
- inhibits host protease that degrades cII
- What is the function of cII in Lysogeny
- activates transcription of pI which integrates the phage
- cI shuts down what in the Lysogeny system
- pR and pL
- Integrase promotes what in Lambda integrating
- site specific recombination between attP(attachment site phage) and attB(bacteria attachment site)
- What is meant by the repressor in cI synthesis in Lambda integration acting as a homodimer
- repressor not active at low concentrations
- What is oR1 and what does it repress in relation to cI synthesis in lambda integration
- binding site for cI in low concentrations. represses pR.
- What does oR2 activate in relation to lambda integration
- in middle concentrations of cI, it binds to oR2 and activates transcription of cI from pRM
- What does oR3 repress in relation to lambda integration
- at high concentration of cI, it binds to oR3 and represses pRM and no transcription of cI
- What two things lead to the degradation of cI
- RecA + ssDNA
- During induction of lambda, Cro binds to what, preventing what
- binds to oR, preventing cI transcription
- What is Mu
- A mutator phage
- What does an Mu consist of
- transposon with phage coat
- What depends on the prescence of P2 as an example of parasitic growth on a virus
- P4
- What does Sid do in P2-P4 interaction
- causes P2 proteins to make smaller heads, thus only fitting P4 DNA into them
- What are pathogenicity islands
- large selections of bacterial genomes that are not common to all strains
- in packaging of host DNA into phage head in transduction, what is the phage unable to do
- generate progeny
- Transducing lambda are a defect in either one of two things. What are these
- head and tail formation or lysogen formation
- Generally, what are transposons
- DNA elements that can hop
- What is the responsible enzyme in transposons
- transposase
- What are the smallest transposons called
- Insertion sequence or IS elements
- Most times, what do two IS elements of the same type flanking other genes mean
- antibiotic resistance genes
- What are suicide vector systems
- replication defective phages or plasmids
- What is replicative transposition
- copy of transposon in donor and target DNA
- cut and paste transposition
- only one copy remains; donor DNA is destroyed