Lecture Outline

I Introduction
Early Developments
Genetic Material and Code

II Variation and Population Dynamics

Adaptation vs. Mutation

Types of Mutants and Mutagens
Mutant Methodology
DNA Alteration and Repair

EXAM 1
 Lecture Outline
III. Plasmids and Transposons

IV. Genetic Exchange and Recombination

Ø Mechanisms of Gene Transfer

q Transformation
q Conjugation
q Transduction
q Protoplast Fusion
q Parasexual Cycle and Haploidization

Second Exam
 Expected Learning Outcomes
At the end of the lecture the student will be able to:
1. determine the various roles of plasmids in the host
cell and in the development of molecular biology
2. differentiate between vertical and horizontal gene
transfer
3. distinguish the mechanisms of genetic exchange in
microorgansims
4. relate gene transfer methods to evolution of
microorganisms
4
PLASMIDS
PLASMIDS
 PLASMIDS

§ closed circular or linear DNA molecules that replicate

independently of the bacterial chromosome

§ code for functions involved in their own life cycles and for
functions which affect the physiology of the host cell

§ pathogenicity in plants and animals

§ resistance to a wide spectrum of antimicrobial compounds

(antibiotics, heavy metals; R plasmids); resistance to
mutagenic agents (ethidium bromide), and disinfectant
(formaldehyde)

§ 2 kb to 100 kb in size
Actis et al., 2000. Front Biosci. 4: D43-62.
 Don't nod
Dogma: I am God
Never odd or even
Too bad – I hid a boot
Rats live on no evil star
No trace; not one carton
Was it Eliot's toilet I saw?
Murder for a jar of red rum
May a moody baby doom a yam?
Go hang a salami; I'm a lasagna hog!
A Toyota! Race fast... safe car: a Toyota
Straw? No, too stupid a fad; I put soot on warts
Are we not drawn onward, we few, drawn onward to new era?
Doc Note: I dissent. A fast never prevents a fatness. I diet on cod
No, it never propagates if I set a gap or prevention
Anne, I vote more cars race Rome to Vienna
Sums are not set as a test on Erasmus
Kay, a red nude, peeped under a yak
Some men interpret nine memos
Campus Motto: Bottoms up, Mac
Go deliver a dare, vile dog!
Madam, in Eden I'm Adam
Oozy rat in a sanitary zoo
Ah, Satan sees Natasha
Lisa Bonet ate no basil
Do geese see God?
God saw I was dog
Dennis sinned
 PLASMIDS

http://www.mun.ca/biochem/courses/4103/figures/Snyder-Champness/T4-1.jpg
 Enzymes of
phytohormone synthesis

Virulence
Region
DNA processing
and transfer
RK 2 was first isolated in connection with an outbreak of antibiotic-
resistant Pseudomonas aeruginosa and Klebsiella aerogenes
in Birmingham in 1969, as one of a family of plasmids implicated in
transfer of Ampicillin resistance between bacterial strains.
• isolated from a piglet with diarrhea; a hybrid plasmid – carries
enterotoxin genes (Ent plasmid) and resistance genes (R plasmid)
 PLASMIDS

Copy number
§ varies from 1 or 2 per chromosome (e.g. F, P-types) to
over a hundred (e.g. pUC series)

Maintenance or host range

§ the ability of a plasmid to survive in the host bacterium
§ narrow (e.g. ColE1, pBR322, pUC18)
§ wide (e.g. P-type plasmids)
 Some plasmids and their properties
Mass Copies/ Self-
Plasmid X 106 Chrom transmissible Phenotypic features

Col Plasmids
Col1E1 4.2 10-15 No Colicin E1 (membrane changes)
ColE2 (Shigella) 5.0 10-15 No Colicin E2 (DNAse)
Sex Plasmids
F 62 1-2 Yes F pilus
F’lac 95 1-2 Yes F pilus; lac operon
R plasmids
R 100 70 1-2 Yes Camr, Strr, Sulr, Tetr
R6K 25 12 Yes Ampr, Strr
pSC101 5.8 1-2 No Tetr
Recombinant plasmids
pBR322 2.9 ~20 No High copy-number
pBR345 0.7 ~20 No ColE1-type
 PLASMIDS

Transferability
§ Conjugative Plasmid
§ transfer themselves from host to host (e.g. F & P-types,
most R-plasmids)
§oriT, tra genes

Mobilizability
§cannot transfer themselves (e.g. ColEl), but can be
mobilized by transmissible plasmids (e.g. F or ColV)
§oriT
 PLASMIDS
Incompatibility (Inc) Groups

§Failure of two plasmids to be stably inherited and

maintained, in absence of a selective pressure, in the same
cell line

§connected with regulation of copy number and

segregation

§plasmids of one incompatibility group have the same

replication control functions (trans-acting)

• each can control replication of the other

• one of the two plasmids is lost during random
partitioning
 INCOMPATIBILITY GROUPS

Incompatibility group Plasmids

FI F, R386
FII R1
FIII Col B-K99, Col B-K166
FIV R124
I R62, R64, R483 (at least 5
subgroups)
J R391
N R46
O R724
P RP4, RK2
Q RSF1010
T R401
W R388, S-a
 PLASMID
REPLICATION

Rolling circle

To see animation

http://highered.mcgraw-
hill.com/sites/0072556781/student_view0/chapter13/
animation_quiz_6.html
Rolling circle

Transfer of plasmid
from donor to recipient
Theta replication
 CONTROL OF PLASMID REPLICATION
AND COPY-NUMBER

§ plasmid copy-number is determined by regulating the

initiation of plasmid replication
§ control of initiation of plasmid replication
§ regulating the amount of available primer for the
initiation of DNA replication
§ regulating the amount of essential replication proteins
§ regulating the function of essential replication proteins
 CONTROL OF PLASMID REPLICATION
AND COPY-NUMBER

§ Two mechanisms to control the initiation of plasmid

replication
1. Regulation by antisense RNA
2. Regulation by binding of replication proteins to repeated
18-22 bp sites called iterons.
 Regulation of plasmid colE1
copy number by antisense RNA

http://www.mun.ca/biochem/courses/4103/figures/Snyder-Champness/T4-1.jpg
 Regulation of plasmid R1 copy number by antisense RNA

repA – initiator protein High concentration of plasmid,

accumulation of copA andCopB
copB
copB – repressor protein
cop A- antisense RNA

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/plasmid-repln.html
 Regulation of plasmid copy number by iterons
(iterated RepA binding sites )

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/plasmid-repln.html
 Plasmid replication control

Lestas et al. Nature 467, 174-178 (2010) doi:10.1038/nature09333

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/plasmid-repln.html
Jensen and Gerdes. 1999. The EMBO Journal.
18:4076-4084
 F FACTOR/PLASMID
(FERTILITY FACTOR/PLASMID)
§ OriT (origin of transfer)
§ OriV (origin of replication)
§ tra-region (transfer genes)
§ IS (insertion elements)

F factor in Escherichia coli

 States of the F plasmid

F+: a plasmid independent of the bacterial genome;

contains only F factor DNA

Hfr (High frequency of recombination): F factor

integrated into the bacterial genome

F’: F factor exists as an extrachromosomal element

but contains some sections of the chromosomal
DNA

F- - strain with no F factor

 Formation of Hfr

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/Hfr.html
Multiple IS insertions are present in many bacterial
chromosomes. For example, wild-type E. coli K-12 has 8
IS1 insertions, 6 IS2 insertions, and 5 IS3 insertions.
 Formation of F’

Illegitimate recombination

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/Hfr.html
 States of the F plasmid

F+: a plasmid independent of the bacterial genome;

contains only F factor DNA

Hfr (High frequency of recombination): F factor

integrated into the bacterial genome

F’: F factor exists as an extrachromosomal element

but contains some sections of the chromosomal
DNA

F- - strain with no F factor

Illegitimate recombination
 SUICIDE PLASMID

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/Hfr.html
 PLASMIDS AS CLONING VECTORS

Construction

http://www.mun.ca/biochem/courses/4103/topics/plasmids.html
 Cloning Vectors
Cloning vector: a DNA molecule that carries foreign DNA into
a host cell, replicates inside a bacterial (or yeast) cell and
produces many copies of itself and the foreign DNA.
Three features:
1. sequences that permit the propagation of itself in bacteria
(or in yeast for YACs)
2. cloning site to insert foreign DNA; the most versatile
vectors contain a site that can be cut by many restriction
enzymes
3. method of selecting for bacteria (or yeast for YACs)
containing a vector with foreign DNA; usually
accomplished by selectable markers for drug resistance
 Types of Cloning Vectors

Plasmid: cloning limit: 100 to 10,000 base pairs or 0.1-10

kilobases (kb).
Phage: Cosmid: an extrachromosomal circular DNA molecule that combines
features of plasmids and phage; cloning limit - 35-50 kb;
Phagemids: plasmids that can be induced to produce phage particles
containing single-stranded DNA.
Bacterial Artificial Chromosomes (BAC): based on bacterial mini-F plasmids.
cloning limit: 75-300 kb.
Yeast Artificial Chromosome (YAC): an artificial chromosome that contains
telomeres, origin of replication, a yeast centromere, and a selectable marker
for identification in yeast cells; cloning limit: 100-1000 kb.
 Cloning Vectors: Plasmids

qNatural plasmids simplified & modified for DNA cloning

qRequirements:
ü Autonomous replication (ori site)
ü Selectable marker (drugR)
ü Multiple unique restriction enzyme cut sites in non-
essential regions
ü Small size (~4 kb): easier to isolate & use & can add
larger DNA fragments
ü High copy number: ensure high yield of clones
 PLASMIDS AS CLONING VECTORS

Construction

http://www.mun.ca/biochem/courses/4103/topics/plasmids.html
 Cloning Vector pBR322

q ColE1-based E. coli plasmid

q Genes for ori & DNA
replication, but not
conjugation
q Markers: ApR (ampicillin) &
TcR (tetracycline) genes
q Multiple unique restriction
sites for cloning—note that
some will abolish antibiotic
resistance
from 3 main sources – the tetracycline resistance gene of pSC101, the ampicillin resistance gene of
RSF 2124, and the replication elements of pMB1, a a close relative of the ColE1 plasmid
Brock Biology of Microorganisms 10/e
Madigan/Martinko/Parker
2003 Benjamin Cummings
PLASMIDS AS CLONING VECTORS
MCS = multiple cloning site (poly-linker)
Blue-white screening

lacZ  beta-galactosidase

X-gal  blue product

Gene inserted into lacZ

 Disrupts gene

 defective protein
(disrupted b-gal activity)

 White colonies
PLASMIDS AS CLONING VECTORS

http://www.mun.ca/biochem/courses/4103/topics/plasmids.html
 Blue-white screening

http://www.sigmaaldrich.com/prodimages/b/b3928.jpg
 SHUTTLE VECTORS
• two origins of replication

http://2008.igem.org/Team:University_of_Washington/Project
http://www-archbac.u-psud.fr/labhome/YZivanovic/EstorilPoster96.html
 VECTORS FOR ARCHAEAL GENETICS

Typical integrative and shuttle-plasmid vectors for archaeal genetics (in this case, Haloferax
volcanii), with relevant features.
Allers and Mevarech. 2005. Nat. Rev. 6:59-73.
EXPRESSION VECTORS
Anatomy of an expression vector