Plasmids and conjugative plasmids Flashcards
1
Q
Do plasmids cause disease?
A
Plasmids may not cause disease but can alter gene expression, including genes for antibiotic resistance.
2
Q
What type of DNA molecule are plasmids?
A
Plasmids are small, circular, double-stranded DNA molecules.
3
Q
How do plasmids exist in relation to host chromosomes?
A
They can exist independently (autonomous) or be integrated into the host chromosome.
4
Q
Do all bacteria have plasmids?
A
Most bacteria contain at least one type of plasmid and may also carry other extrachromosomal elements.
5
Q
Give an example of plasmids in E.coli.
A
E.coli can carry the F factor plasmid, lysogenic bacteriophage lambda, and a transposon.
6
Q
Are plasmids found in eukaryotic organisms?
A
They are rare but do exist in fungi, such as the 2μ plasmid in Saccharomyces cerevisiae.
7
Q
How large can plasmids be?
A
Plasmid sizes can vary from 2 to over 100 kilobases (kB).
8
Q
How do plasmid sizes compare to bacterial genomes?
A
Mycoplasma genome is ~580 kB, E. coli genome ~4600 kB, and viroids are 0.2–0.4 kB.
9
Q
What are engineered plasmids?
A
Engineered plasmids are artificial constructs used in recombinant DNA cloning.
10
Q
What is a plasmid vector?
A
A plasmid used to transfer, replicate, or express foreign DNA.
11
Q
How do plasmid vectors differ from viral vectors?
A
Plasmid vectors differ in origin and mechanism of DNA delivery compared to viral vectors.
12
Q
Are all cloning vectors natural?
A
No, cloning vectors are constructed and do not occur in nature.
13
Q
In which organisms can plasmid cloning vectors be used?
A
They are used in bacterial, fungal, plant, and animal host cells.
14
Q
Where are natural plasmids commonly found?
A
They are found primarily in bacteria and fungi.
15
Q
Are plasmids species-specific?
A
Yes, many natural plasmids are species-specific.
16
Q
How are transmissible plasmids transferred?
A
Via bacterial conjugation, requiring direct cell-to-cell contact.
17
Q
Are all plasmids transmissible?
A
No, some are non-transmissible and require artificial transformation to enter cells.
18
Q
What is the transfer of nontransmissble plasmids called
A
Bacterial transformation
19
Q
How does the bacteria decide which plasmid to keep and which to not keep
A
If the plasmid received gives the bacteria a competotive advnatage -> keep. If its not advantages -> cells will lose that plasmid
20
Q
How are non-transmissible plasmids introduced into cells?
A
Through protocols that weaken the host membrane or wall to allow DNA entry.
21
Q
How many origins of replication do bacteria have? Eukaryotes?
A
1, one, multiple
22
Q
What does the plasmid give the host cell
A
Specific abilities not normally essential to the host cell that contains the plasmids
23
Q
What does the deg plasmid in Pseudomonas do?
A
It encodes enzymes that degrade hydrocarbons, aiding in bioremediation.
24
Q
What does the nif-nod plasmid in Rhizobium do?
A
It encodes enzymes necessary for nitrogen fixation in plants. Rhizobium forms symbiotic relationship with legume plants. Ability of Rhibizobium is on its. Palsmits not in its genes
25
What is the R factor in bacteria? Where is it found?
In E. Coli, Salmonella. Contain genes that encode enzymes that inactivate certain antibiotics and allow the organism to survive in the presence of antibiotics.
26
What are R-factors or RTFs?
R-factors (resistance factor) or RTFs (resistance transfer factor) are transmissible plasmids carrying antibiotic resistance genes.
27
What genes do R-factors contain?
They contain antibiotic resistance genes (to tetracycline, streptomycin, chloraphenicol, ori (to replicated, like the chromonsones), oriT (to transfer as its replciated. Bacterial chromosoal does not have rolling replication), and tra genes for replication and transfer.
28
Where are antibiotic resistance genes often found?
Within transposons on the plasmid.
29
Why are plasmid-borne resistance genes a concern?
The transmission of plasmids carrying antibiotic resistance genes -> allowed many bacterial populations to become resistant to antibiotics contribute to antibiotic resistance in bacterial populations, reducing treatment options.
30
What happens in the presence of antibiotics?
Cells without plasmids die, giving an advantage to plasmid-containing cells.
31
What happens in the absence of antibiotics?
Cells without plasmids grow faster, outcompeting those with plasmids.
32
How can antibiotic resistance be mitigated?
By reducing antibiotic use to lessen selective pressure on bacteria.
33
Do plasmids have the same origin of replication?
No, different plasmids can have different origins (ori).
34
How does the affinity of the host replication machinery for a particular ori determine..?
How fast a particular plasmid will replicate, and the average number of copies of the plasmid per cell.
35
Give examples of ori and copy numbers.
F plasmid (oriV) in E. coli = 1 copy; ColE (oriC) = 10–20 copies per cell.
36
How do plasmids affect host cell growth?
Larger or multiple plasmids reduce growth rate due to competition for replication machinery.
37
how does size and number of plasmids affect growth
Smaller the plasmid -> faster growth rate. Cells with no plasmids growt quickets
38
What are episomes?
Plasmids that can exist both as autonomous entities and as integrated parts of the chromosome.
39
Most plasmids are… replicating
Autonomosuly - replicating independtly of the host chromosome.
40
What about integrated plasmids?
They become a physical part of the bacterial host cell chormoone. Only replicaate when the host replicates. But some retain ability to be autonomously replicating
41
How do integrated plasmids replicate?
Only when the host chromosome replicates.
42
Can integrated plasmids become autonomous?
Yes, episomes can excise and replicate independently again.
43
Give examples of episomes.
E.coli F factor, bacteriophage lambda, HIV.
44
What is the E. Coli F factor
Large plasmid that plays a major role in the conjugation of E. Coli
45
What is the size of the plasmid like compared to bacteria and viriod?
larger than viriod, smaller than bacteria
46
How does a bacterium acquire entirely new characteristics?
By acquiring genetic information from another bacterium.
47
What can be transferred in a single DNA transfer?
Entire biochemical pathways can be passed from one bacterial species to another.
48
What are some methods of horizonal transfer
1. Bacterial transformation: donor cell dies, recipient cell picks up the DNA. 2. Bacterial transducition. Phage infected donor cell -> picks up some of the genes, is released, infects another cell/ 3. Bacterial cojugation: transfer of plasmids between cells.
49
What is horizontal gene transfer?
The process by which bacteria acquire genes from other bacteria rather than inheriting them.
50
What happens when the donor cell dies?
It releases DNA that can be picked up by a recipient cell.
51
Why is horizontal gene transfer important for bacteria?
It allows bacteria to acquire genes that provide new traits or functions, aiding in survival and adaptation.
52
What happens to horizontally acquired genes over generations?
They can become integrated into the host's chromosomal DNA.
53
What is natural transformation?
The uptake of plasmid DNA by natural transformation compared to conjugation. Streptococcus makes use of competence factor that allows transformation of mid log cells. DNA fragements can be transfported in this manner. DNA is passed through a pore structure often through a pilin complex
54
Name bacteria capable of natural transformation.
Streptococcus and Bacillus.
55
How does Streptococcus enable transformation?
It secretes a competence factor that allows mid-log phase cells to transform.
56
What structure helps pass DNA during transformation?
A pilin complex in the cell membrane.
57
What did the Griffith experiment show?
That non-virulent bacteria can be transformed into virulent forms by absorbing genetic material from heat-killed virulent strains. Live S cells (with capsule) kill the bacteria. Heat killed S cells do not kill the bacteria. Live R cells (without capsule) do not kill the bacteria. But if you put the live R cells and the heat killed S cells together -> makes a S cell (with capsule) and kills the bacteira
58
What is essential for S. pneumonia virulence?
The capsule surrounding the bacteria.
59
How is a fragment of DNA incorporated into the genome of Streptoccous pneumonia
External DNA binds to the DNA binding protein. Competence-specific, ss DNA binding protein helps stabilize teh DNA. RecA protein allows for the uptake of sss DNA and RecA mediated homoglogous recbomibation
60
What is the role of competence proteins like ComE and ComC?
They help maintain and incorporate incoming DNA during transformation.
61
How do Gram-negative bacteria manage DNA uptake?
Through complex structures that allow DNA to bypass both outer and inner membranes.
62
Why is DNA uptake simpler in Gram-positive bacteria?
They lack an outer membrane, making their cell walls easier to penetrate.
63
What are some classes of conjugative plasmids
R factors or RTF, F factors, Col factors
64
Difference between transmissible and non transmissible plasmids
Tramssible = conjugative. Plasmids can transfer DNA from host cell to host cell. Process is plasmid encoded and requires donor and recipient cell. Non conjugative plasmids cannot transfer DNA from cell to cell because they do not have tra genes
65
Is conjugation bi/unidirectional
Unidirectlional
66
What is conjugation encoded by?
The plasmid
67
Most naturally occuring plasmids are
Non conjugative
68
Whats found on the normal flora of skin
Flora of skin can secrete bacteriocins that act as a line of defense againstt other bacteria. Some can work against host cell -> increase virulence
69
Who can bacterioins act on
Similar species or against another species
70
What are colicins
Kills E. Coli
71
What are conjugative plasmids?
Plasmids that can transfer DNA from one host cell to another via a process encoded by the plasmid.
72
What genes are essential for conjugation?
Transfer (tra) genes.
73
Conjugative plasmids require a donor and a recipient. If 2 donors/recipients are together
Nothign will happen. Since the receiver needs specific genes, and the donor needs
74
What are Col factors?
Plasmids that produce bacteriocins, toxins that kill other bacteria for competitive advantage.
75
How do bacteriocins work?
They can form channels and increase membrane permeability, degrade DNA, RNA, or cell walls.
76
How does plasmid transfer typically occur?
One copy remains in the donor, and another is transferred to the recipient.
77
What is oriT?
The origin of transfer used during plasmid DNA transfer.
78
What happens when a plasmid integrates into a host chromosome?
It can transfer part of or all its chromosone into another cell
79
The ability of conjugative plasmids to promote cell to cell transfer depends on
The prescnece of transfer (tra) genes and the presecne of an alternative type of oriign of replication
80
What is an F+ E. coli cell?
A cell with an autonomous F plasmid (donor)
81
What is an F factor
Feterility factor/Plasmid. Conjugatie plasmid. Is an episome (can either exist as an automnomous or integrated)
82
What is an Hfr cell?
A cell with an integrated F plasmid capable of high-frequency recombination.
83
What is an F- cell?
A cell that lacks the F plasmid and can receive genetic material from F+ or Hfr cells.
84
85
Is plasmid transfer polar or non-polar?
It is polar; donor and recipient must be compatible.
86
How does the F plasmid integrate into the host chromosome?
Through homologous recombination at specific insertion sequences. There is insertion site sequences found on both the plasmid and chromosone. It cant just insert anywhere. The sequences are transposons
87
What are insertion sequences?
Transposable elements like IS2, IS3, and Tn1000 that facilitate insertion and excision.
88
What is OriV? OriT
The origin for circular DNA replication OriT: site for rolling circle replication
89
What does the map of the F plasmid look like?
Has a region of the tra region, Origin of replication for circular DNA, oriT repication for rolling replciation, and transposon elements (sequences of exision and insertion from plasmid to chromondos)
90
What is the function of the tra gene cluster?
Encodes proteins for pilus synthesis and conjugation.
91
What is the size of the F plasmid?
Approximately 100 kb.
92
Where are genes for conjugation encoded?
On the F plasmid, especially in the tra gene region.
93
What happens during rolling circle replication?
A single DNA strand is transferred to the recipient. The recipient makes it double stranded. In the donor, it is double stranded
94
What is circular (theta) replication?
OriV (oriS) is used when the plasmid is replicating autonomously (no DNA transfer)/ A bidirectional replication where two forks move around DNA theta shaped intermediates. Forks meet at the end to form 2 separate circular ds DNA moleculees.
95
Bacterial chromosome (or plasmid) is a ___ replicon
Single
96
When is OriV used?
During autonomous replication of plasmids.
97
Steps in rolling replication
One strand is nicked at the oriT. The free 3’ hydroxyl end is extended by replication enzymes. As the 3’ is lengthened, the 5’ end is displaced by a lenghtehning tail. The displaced 5’ end is transfereed to the recipient cell. Complementary strand synthesis takes place in the recipient cell.
98
What is rolling replication also used in
Viruses in the lytic ceycle
99
What is the first step of rolling circle replication?
A nick is made at the oriT site in one DNA strand.
100
What happens to the displaced 5’ end?
It forms a tail and is transferred to the recipient cell.
101
What occurs in the recipient during rolling circle replication?
The single-stranded DNA is converted to double-stranded DNA.
102
How do donor and recipient cells come into contact?
Through sex pili encoded by tra genes.
103
What happens if the connection breaks prematurely?
The full plasmid or chromosome may not be transferred, affecting transfer success.
104
What is type IV secretion?
A mechanism that moves DNA and effector molecules between cells, aiding in biofilm formation or virulence.
105
How can conjugative plasmids integrate into chromosomes?
Via insertion sequences and homologous recombination.
106
How does the Type 4 secretion system work? What does it do?
It moves DNA and effectors such as toxins.
107
Can bacteriocins be delivered by type 4 secretion contact-dependent interkingdom
No, it requires contact
108
What are the different types of type IV srecertions
1. Contant deppdnet interkingdom: requires the bacteria to enter a eukaryotic cell cell. Then it can use T4 to secrete DNA. 2. Contact depedent interbacterial: can transfer DNA via conjugation, or deliver toxins 3. Contact independent DNA uptake or release and biofilm formation
109
What does Hfr stand for?
High frequency recombination, referring to a cell with an integrated F plasmid capable of transferring chromosomal genes.
110
Can the F plasmid transfer the entire chromosome?
Not usually; the connection often breaks before complete transfer.
111
It is important that the pili remains in contact because
Without it, the F factor would not be transferred
112
How is F+ (donor) transfered to F- (recipient)
Receipient does not have surface molecules. F plus cell recognizes surface molecules. Pili forms and retracts pulling the cells together, cell pairs are stabilized. F plasmid nicks 1 strand. Transfers one strand from F+ cell to F- cell. F plasmid simultanoeusly replicated in F” cell (complex protein sysmte). Synthesis of completmentary strand beigns in recipient cell. After the DNA transfer and synthesis. Cells separaet. Becoming both F+ cells.
113
What happens if the F factor is not transferred from Htf strains?
The recipient remains F-.
114
How do htf strains transfer chromosomal genes?
Nick happens at the oriT. Part of the conj plasmid is on one side and the other is on the other side. -> so oyu need replication to happen the full cycle. In order for donor to receive the DNA. Sex pili needs to be infact -> but chances it remains intact is low -> still forms forms F-
115
Can conjugative plasmids integrate into host chromosome?
Yes, via insertion sequences via homologous recombination (in bith the plasmid and the chomromosome). Remember, is the transposon elemnets (insertional elements), not the whole plasmid thats inserted.
116
What happens to the genes when the Htf is first added to recipient
Initially, the recipient cell is diploid for the genes obtained, but can udnergo degradation oncluding part of the bacterial chromosome. but the receipient can undergoreocmibantion with new genes resulting in changes to the recipient genome.
117
If F plasmid is obtained in transfer while integrated, htf can/cannot replicate autonomoglu? But what can happen
Cannot replicate autonomously. But F plasmids can be exicsed froom the chromosome and transfer part of the genome to another cell. -> beocomes F’
118
Even If theres issues with conjugation, can new genes still be quired
Yes, and they can be transferred to another gene
119
Can F plasmid when integrated replicated autonomously?
No, as Hft they cannot. But the plasmid can be exised.
120
Can integrated plasmids be donated?
Yes, but the whole chromosone would need to be replicated. Also, if it wants to replicate autonomously -> get out of the chromosome.
121
Can F plasmids excise incorrectly?
Yes, leading to F’ plasmids that carry extra chromosomal genes.
122
What does the F factor encode for?
encodes genes for sexual pili, thin rod-like structures with which F-carrying (male or donor) bacteria attach to F− (female or recipient) cells for conjugative transfer
123
What is an F’ plasmid?
A plasmid formed by incorrect excision of an integrated F plasmid, containing host chromosomal genes.
124
Can plasmids transfer between kingdoms?
Yes, e.g., Ti plasmid from Agrobacterium transfers to plants.
125
What does the T-DNA region of the Ti plasmid do?
It integrates into plant cells causing crown gall tumors.
126
How does T-DNA affect plants?
It induces cell division and tumor formation, which can impair plant functions like water transport.
127
How are Ti plasmids used in biotechnology?
Genes of interest are inserted into T-DNA to create genetically modified (transgenic) plants.
128
How is the tumor-inducing function of Ti plasmid suppressed?
By inactivating the T-DNA tumor-causing genes when inserting the desired gene into the T region
129
So what is it that causes the galls?
Its the plasmid not the chromsome DNA.
