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Lecture 24

MICRB316 Lecture Notes - Lecture 24: Selfish Dna, Dna Replication, Antimicrobial Resistance


Department
Microbiology (Biological Sciences)
Course Code
MICRB316
Professor
Jonathan Dennis
Lecture
24

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Plasmids ii
More Replication
Many plasmids use non-stable antisense RNAs as principal regulators. ½ life = ~1 min
These RNAs bind to their target RNAs and through a variety of mechanisms, prevent
initiation of plasmid replication.
These RNAs bind to their target RNAs and through a variety of mechanisms prevent
initiation of plasmid replication.
Because of the complementarity of anti-sense and target RNA it is suggested that RNA
duplexes are the inhibitory structures.
Where rep binds DNA: iterons, not conserved. Yet, all evolved to control DNA replication
of plasmids.
R1: plasmid
Origin of replication of R1: a lot of factors can go into replication control of a plasmid.
oriR, gene for repA, promoter for repA. ORF is next.. once repA is produced and
translated binds oriR and opens up DNA. Two RNAs are produced. Promoter for repA
(PrepA), upstream on longest mRNA gene for copB, translated to protein: multimirizes
and binds to operator which shuts down repA transcription. This is the first mech of
control.
RNA mechanism: copA- anti sense RNA and you can see that it runs on the opposite
strand of repA promoter, mRNA is produced which is anti sense stops translation of
Repa protein. RNA repressors of the opposite strands of DNA.
Fig 2. Plasmids are selfish genetic elements
The importance of regulation is especially evident in low copy # plasmids of Gram
negative bacteria:
Sloppy control results in a high frequency of plasmid loss.
Too low copy number- some daughter cells won’t get plasmid: cell death
Too high: runaway replication cell death by competition with other cells.
For cell- critical to maintain right number of plasmids
Should regulators be unstable?
In most examples, negative feedback loop where inhibitor acts upon a function for
replication.
For copA- intracell concentrations correlate with gene dosage.
2x plasmid #--> 2 x inhibictor con. ½ life= 1 min ish
need unstable inhibitior: everything gets screwed up if you don’t… next picture: shows
what happens without the right machinery.
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true for all plasmids: ColE1 and R1 (copA)
plasmid frequency
picture:
1. 2 cells that are dividing when they do, one doesn’t get enough plasmids, the
other one too many
2. red dots: inhibitors
3. unstable inhibitors- inhibitor concentration increases when there is lots of
plasmids
4. next, inhibitor decreases if you don’t have enough plasmids… inhibitor degrades,
turn up amount of replication normal # of plasmids
stable inihibitor
1. daughter cells without enough plasmids or with many
2. not enough- no turn up of rep, too many inihibitors, not many plasmids, daughter
won’t get plasmid – really hard for small # of plasmids to get to normal copy #.
When cell divides it is possible for some daughters to not get plasmid. Leads to
loss of plasmid/ cell death if it’s an “addictive” plasmid.
3. too many plasmids- lots of inhibitor- inhibitors keep on and you get right amount
of plasmids on one side.. normal.
Red dots- inhibitors blue: plasmids
Picture: Shows biochem
Initial “kissing” interactions between antisense and target RNAs often occur between
loop structures and precede the formation of stable complexes
CopT and CopA RNAs- ss: fold on each other- complementary bases fold up and
produce stem loops – interact at the loop part and unzipper stems and fold together-
this is the kissing interaction of liios
Complementarity between antisense and target RNA should thermodynamically
drive the pairing to completion.
What do you find in plasmids?
Building blocks: plasmids like chromosomes are made of building blocks.
The plasmids that have been sequenced suggest that there are common families of
related plasmids with many different functions.
e.g. Pdtg1 (naphthalene degradation) has similar plasmid backbone to pWW0
(toluene degradation) both IncP-9.
“Backbone” of these plasmids are highly homologous* both are incompatability
group 9- backbone is the same but have other variability.
Eg. E.coli F fertility plasmid is similar to R resistance plasmids.
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Picture: right: Pnd6 plasmid- nah operon and sal operon- for pollutant degradation:
very homologous to the plasmid pDTG1…! The rest of the plasmid don’t match the
backbone- value added genes.
Plasmids are evolving… start with backbone and evolve from there.
Bottom: Pww0: some tra genes + backbone genes… loks like backbone at some
poin (front and back). But nah operon is not there… sal operon is there! The rest of
the degradation genes are not there.
Looks like combination of value added genes and backbone genes of two different
plasmids.
Fig 4.1 Bacterial Plasmid
How people used to know how plasmids were related- took ds circle of DNA,
denature it, then reanneal with another plasmid that was ss. Where the two ss would
reanneal there would be complementary bases- so plasmids were related. No
homology: plasmids didn’t go together.
Here: F plasmid and R plasmid (From E.coli and resistance plasmid).
No annealing between the two ss DNA: bases are non homologous in the left region.
Where the genes are homologous- the r plasmid has backbone of F. some loops are
formed: differences in insertions but long stretches are identical in the F/R plasmids.
Tra genes are homologous: for pilus assembly/DNA processing.
Except for the antibiotic resistance genes the backbones look the same.
F with R1: other antibiotic resistance genes, backbone aligns: identical tra genes but
antibiotic resistance genes are different.
Combine R6-5 and R1: R determinants are similar, some new but similar… the rest
of the DNA is highly homologous- only some genes have been inserted in one and
not in the other one. These plasmids are VERY homologous and they have
backbone related to F.
This is the old fashion way of doing DNA sequence comparisons- based on
annealing properties of the whole genomes.
Do we know what to expect on a given plasmid?
1. If PCR of homologs fails, and hybridization fails: plasmids are not
similar. BUT DNA seq. shows similar mechanism: convergent evolution.
Function is evolving together.
2. Like chromosomes and viruses, plasmids have complex
recombinational history: A: homologous DNA exchanges B: transposons
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