Plasmids pt 2. lecture 6 Flashcards
What are col plasmids?
- Conjugative plasmids
- encode bacteriocins. Which can produce proteins secreted by one bacteria to cause damage to another speices. related speices or different strains are killed
Are Ti plasmids (T-DNA) conjugative?
No, cuz its not between bacterial cells
Peptidoglycan is hydrophilic/phobic
hydrophilic 9sugar)
Gram negative cell walls are
lipidacious
How does bacteriocin affect gram positive differently than gram negative
gram (+): bacteriocins can come straight across the peptidoglycan layer -> can inhbit peptidoglucan sysntehsis and make pores -> lose material
gram (-): bacteriocins need to enter outer memebrane via pore -> once in can interact with protein structures of DNA, RNA polymerase - but none of this is creating a pore.
What is a metabolic plasmid that is conjugative?
D-plasmids: found in Psuedomonas. Which confer ability to enzymatically convert substances into nutrient or energy source..
Can degrade hydrocarbons such as petroloum, pestisides, herbicides, jet fuel.
one strain carrying deg plasmid can degrade crude oil at cold temperatures -> can use to clean up oil spills
What are lectins
A class of proteins found in plants
What are nif-nod plasmid (pSym)?
Found in the bacterium Rhbizobium
Encodes for Nif genes which do nitrogen fixation. (nitrogenase and nod genes (nodule formation)
Enter bacteria enters the cell (different species of Rhizbohium attach to diffeent receptors fo the root cell). Specifiity is due to lectins present in the capsule.
Enzymes needed for fixing nitrogen are usually inactivated by oxygen. HWat does the plant do
the plant leghamoglobiin binds oxygen in the rot nodules creating low oxygen and allowing nitrogen fixatin to happen.
what are virulence or pathogenicity plasmids?
Can produce toxins or other virulence factors and be conjugative.
Found in bacteria.
ROle is to increase the pathogenicity/viruelnce of the bacteria
What is pathogenicity:
The ability to inflict damage (disease)
What is a pathogen
An organism capable of causing disease
What is virulence
The degree of pathogeniviyt
What is a virulence factor
Factor that contributes to the virulence of a pathogewn (but does not cause direct damange to a host)
Purpose of capsules in bacteria
protect against toxic compounds (such as antibiotics)
- desiccation
- better adherance to surfaces
What do factors that contribute to pathogenicity generally do?
-Cause direct dmdnage to the host.
- SYntheiss of one or more toxins, degradative enzyems like lipases, DNAses that damage the host cell.
Factors that contirbute to virulence
affect pathogenicity indirectly
- Presence of antibiotic resistance genes
- Sticky fimbriae: allow to adhrere better
- capsule or certain cell wall components
What does morbillia pertussis do?
cause whooping cough (capsule)
What are some virulence factors that affect pathogenicity directly
- Endotoxins:
soluble heat-labile toxic proteins which are secreted from bacteterial cells as it grows -> target host.
can be many types usch as neurotoxins (nerve tissues), enterotoxis ( intesntinal mucosa), cytotoxin (many tissues), can cause cell lysis - Endotoxins: not ormally released from undamaged bacteiral cells.
not proteins but the liposaccahride composnent on gram negative cell walls (so if u consume a dead bacteria)
What is heat labile
heat-labile protein is one that can be changed or destroyed at high temperatures.
What are enterotoxins
Affect intestinal mucosa
Are ENT plasmids and K88 plasmids conjugative?
yes, can share with a e. coli that does not have it
What are ENT plasmids
plasmids in E. coli strains.
encode exotoxins, can cause lesions, permeability/cause haemorrhage in the epithelium lining of the intestine. E. coli containing these are enterotoxigenic
What is the K88 plamids
in pathogenic E. coli strains
- require adhseive fimbriae (pili)
- fimbriase allows the aroganism to adhre to and invade hte lininff of the gut.
can induce a significant endotoxic respsone (non-secreted toxin part of the gram negative cell wall)
e. coli containing this are enterovasive.
What are enterotoxigenic E. coli
cotaining ENT plasmid
What are enterovasive E. coli
E. coli containign K88 plasmid.
toxins can create pathogenicity because of presence in
- a plasmid
- or a virus
- transposons
Examples of toxins
- plasmid encoded anthrax toxin: anthrax - cytoxin
- plasmid encoded tetnatnus toxin causes tetanus - neurotoxin
- virus encoded botulinum (BOTOX) causes botulims - neurotoxin
- virus encoded diptheria toxin - causes diptheria - cytotoxin
plasmid encoded toxin - plqaue (yesternia presstis)
virus endoded chlorea toxin - enterotoxin
whats the common structure of toxiins whether endoded by gram postive, gram negative, arobe, anaerobc. Exotoxins
AB exotoxins.
polypeptide B of toxins binds to host
Polymepetide A has enzyme activity (causes the toxicxity)
Whats a subclass of AB exotoxins
enterotoxins
Are all secreted toxins AB toxins
no
How does botulinum toxins act?
B binds to gangliosides on presyntamitc neurosn at neuron muscular junctions. A is endopeptiases which cleaves protein for neurotransmitter (acetylchlolide) -> paprapjlusis
What does the chlorea enterotoxin do
Lead to loss of fluid from the intestine
How does the cholera enterotoxin lead to loss of fluid from the intestine
when it binds causes colonization -> clocks movement form Na, Cl moved to the lumen.
causing massive water movement to the lumen, cholera symtoms
What can botox do
its one of the most serious toxins known, can cause death, known to treat migraine headaches, muscle spasms and wrinkles
Difference in freezing of botox and tetanus
Botox:
Acetylcholine indeuces contraction of muscle
but botulium toxin blcoks release of A, casuning contracting
-> freeze in a relaxed sate
Tetatnus:
Normal: glycine release stops acetyline reelase and alows muscles to relax
Tetanus toxin binds and prevents release of glycine -> muscles cant releax
-> freeze in a contracted state
What are membrane-disrupting exotoxins
- Do not have separate A and B subunits
- Have 2 types
+ pore forming exotoxins
+ phospholopases
Whats S. pyogenes?
Streptococcus pyogenes flesh-eating bacteria
What are some membrane disrupting exotoxins produced by streptococus and staphyloccous?
- They produce white blood cell killing toxins called leukocidins which kill phagocytic cells.
Hemolysins that form pores in the lasma memerbane of red blood cells, leukocytes and other cells
what are some hemolysiins produed by S. pyogenes
- Strepotlysin-O inactivated by oxygen
- Streptolysin S : insolible and bound to the bacterial cell (is oxygen stable)
What are phospholipases?
Memebrnae-disrupting exotoxins
- phospholipases which attack the phospholipid of the host cytoplasmic memebrnae
What are streptokinases
Produced by S. puogenes and stpahylo occi: breaks up blood clots by binding to plasminogen at site of infection. Help the infection stay local
how does membrane disrupting exotoxins cause killing of the cell?
cause pores and allow thigns to go uncontraallbky in and out -> kill the cells
How to make conj plasmids on conjugative
tra genes, oriT
What are engineerined plasmids
vectors used in cloning, gene experssion to express foreign DnA sequences
- non conjugative, and therefore must be inserted into host cells by transformation that adds Ca++ to alter the charges on the cell wall
A host cell that has taken up a vector is called
transformant
Cells tht tkae up plasmid DNA are called
Competent
To be useful cloning vectrs must:
- contain useful sites for insertion of DNA into the vector (MSC)
- replicate independetly of the host chromosome
- contain selectable markers
- be easy to isolate and manipulate
-> many of these properties are found in bacteriophage cloning vectors
What are some vectors
Plasmids, bacteriophasge
What is a multiple cloning site
a short segment of DNA which contains many (up to ~20) restriction sites
How are autonomous plasmids isolated from bacteira
- using an alkaline lysis procedure: lysozyme which degrades peptidolgycan
in the presecne of EDTA which chelates CA and Mg, wekaesn the gram negative cell wall and assist in isolation
What are resitrction endonuclases
DNA endonucleases produced by bacteria that cut both poynucleotide strands of DNA at specific internal sites. Bacteria have these enzymes so they can destroy forein
how can isolated plasmid DNA be analyzed
- ioslated plasmid DNA can be analyzed by electrophoresis on agarose gels
- the native plasmid DNA can be isolated or restriction endocnucleases can be used to generate fragments of the plasmid DNA
Where can transposons go from and to
can be on the same DNA, on another DNA, can go from plasmid to chormoosme
What are mobile DNA
- transposable elements
- only fon=und within anothe rnucleic acid molecule such as chromosomal DNA, viral DNA, RNA
- can move form one site (donor site) to another site (target site)
- target site can be the same or another DNA moclule
- transposon-like elemnts may be prsent in viron RNA but can only move through a DNA intermediate - jumping genes
What are 2 categoreis of transposons
transposn and
cut and paste
- ioslated plasmid DNA can be analyzed by electrophoresis on agarose gels
- the native plasmid DNA can be isolated or restriction endocnucleases can be used to generate fragments of the plasmid DNA
copy and pasete
What is a transformant? What are competent cells?
cells that have taken up a vector.
Cells that can take up plasmid DNA -> competent
Whats the difference between transposons and retrotransposons
Transposons: Move as DNA by the enzyme transposase encoded by the transposon
- common in prokaryotes
retrotransposon:
- transpose via an RNA intermediate i.e. retrotransposon is first transcribed into RNA internmeidate, than convreted back into DNA by a reverse transcriptase which is encoded by the retrotransposon.
0 common in eukatuotes
The frequency of transposition is a funciton of
The specific transposable elemnt
the cell enviornmemnt (cell stress increases frequency)
Are direct repeats part of the transposon?
No, they are on the target DNA. transposon leaves it when it gets out
How do transposons work?
First. The transposase gene is translated, and transposase recognizes the inverted repeats and exicsises it on both ends. which then comes and inserts into the target DNA. the target DNA is cleaved slanted -> generating direct repeats.
As a result, the donor site contains evidence of transpostion (direct repeats), and new direct repeats are made at the new site
Structure of transposon
tnp gene, flanked by inverted repeats and direct direepats (from target DNA). (transposase gene)
function of transposases
excisses IS at their inverted repeats, and also recognize & cut the insertion sequence staggeered of target DNA
What does replicative tranpsosition do?
Make 2 copies of the plasmid.
make original plasmid with transposon and target plasmid with new copy of transposon
What are insertion sequences (IS) and compositte transpsones
insertion seuqneces are the simpletest transosnones: contain only the genes encoding enzymes need for transposisiton (transposase)
Composite transpsostiosn: sometimes have an additional gene such as antibiotic resistance or toxin genes: mechanism for acquiring antibiotic resistance
What does the transposase gene do
recognize the ends of the IS or composite transposon and allow it to be accurately removed and recognizes the target sit to which the transposon will be added
the tnp gene actually encodes at least 2 proteins that catalyze transpisiton: tgt: transposases
What extra tnp gene do composite transposons have
encode a accessory factor called resolvase thats needed for replicative transposition
What happens if there are multations in the sequences
> result in an immobile transposon
What are the boundary at both ends of the transposon
ivnverted terminal repeats -> characteristic
each IS elemtn also has direct termianl repeats (not part of the transposon but foun din the target DNA and formed when transposons insert into the target site)
Most bacterial transposons are DNA /RNA
DNA, no RNA intermediate
Are all transposons conservative
not all lead to lost by donor and gained by the recipeient.
Many are replicative where the transposon is replciated in absence of excision and the donor retains a cop
In the F factors, there is IS2 and IS2 what does this lead to
smae structure of IS elements, but will have different nucleotide seuqence in inverted repeats -> therefore have several different IS sequences on both the reistn plasmid and bacterial chromosome
Strucut reof hte types of trnaspsonse
IS: transpoases gene (a chunk), IR, DR
Composite tranpsosite: transposase gene, antibiotic resistant gene, IR, DR
replciative transpsoon:
contains: transpossase, resolvase, IR, IR
