微生物遺傳與生物技術
(Microbial Genetics and
Biotechnology)
金門大學
食品科學系
何國傑 教授
Autonomously replicating
genetic entities
(1) the plasmid and bacterial
conjugation
I. What is a plasmid?
1. In addition to chromosome, bacteria cells often contain other
DNA molecules called plasmids. It is an extrachromosomal
DNA which can replicate independent of chromosome.
2. Naming plasmid
(1) previously plasmids are named by the gene function they
carry, for example, R-factor plasmids contain genes for
resistance to several antibiotics.
(2) The naming of plasmids is now standardized: A small ‘p’ for
plasmid, precedes letters that describe the plasmid or
sometimes give the initials of the person or persons who
isolated or constructed it. These letters are often followed
by numbers to identify the particular construct.
For example, pBR322 was constructed by Bolivar and
Rodriguez, and 322 of the plasmid they constructed.
3. Functions encoded by plasmids
(1) Unlike chromosomes, plasmids generally do not encode
functions essential to bacterial growth. Instead, plasmid
genes usually give bacteria a selective advantage under
I. What is a plasmid?
(2) Why many nonessential functions are encoded on plasmid
and not on chromosome?
4. Plasmid structure
(1) Most plasmids are circular with no free ends, although a few
known plasmids are linear.
(2) Plasmid DNA can be supercoiled because it is a covalently
closed circular, and are usually negatively supercoiled
(3) The negative supercoiling introduces stress and this stress
is partially relieved by the plasmid wrapping up on itself. In
the cell, the DNA wraps around proteins, which relieves
some of the stress. The remaining stress facilitates some
reactions involving the plasmid, such as separation of the
two DNA strands for replication or transcription.
5. Properties of plasmids
(1) replication – A plasimid is a replicon that can replicate
autonomously in the cell. Plasmid encodes only a few of
the proteins required for their own replication.
Supercoiling of a covalently closed
circular plasmid
Less EtBr can bind to a covalently closed
circular DNA than to a linear or nicked
circular DNA
Separation of covalently closed circular
plasmid DNA from linear or nicked
circular DNAs on EtBr-CsCl gradient
I. What is a plasmid?
2. Each type of plasmid replicates by one of two general
mechanisms:
i. Theta (θ) replication – In this process, two strands of DNA
are opened at ori and an RNA primer begins replication,
which can proceed in one or both direction(s).
ii. Rolling-circle replication:
(i) A Rep protein recognizes and binds to a palindromic
sequence which contains the double-strand origin (DSO)
on the DNA.
(ii) Rep protein mightallow the formation of a cruciform
structure by base pairing between the inverted repeated
sequences.
(iii) Rep protein makes a nick and remains covalently
attached to the phosphate at 5’ end of DNA through a
tyrosine in one copy of the dimmer Rep.
(iv) The DNA polymerase III uses the free 3’ OH end at the
break as a primer to replicate around the circle,
displacing one of the strand.
Uni- and bi- directional replication
A. Unidirectional replication: Replication terminates when the replication fork gets
back to the origin.
B. Bidirectional replication: Replication terminates when the replication forks meet
somewhere on the DNA molecule opposite the origin.
Rolling-circle replication
Rolling-circle replication
1. A nick is made at the double-stranded origin (DSO) by plasmidencoded Rep protein, which remains bound to the 5’ phosphate
end.
2. The free 3’ end serves as a primer for Pol III that replicate around
the circle, displacing one of the old strands as a single-stranded
DNA.
3. Rep make another nick, releasing the single-stranded circle, and
also joins the ends of new DNA to form a circle by
phosphotransferase reaction.
4. The DNA ligase joins the ends of the new DNA to form a doublestranded circle.
5. The host RNA polymerase makes a primer on the single-stranded
DNA origin (SSO), and Pol III replicates the single-stranded (SS)
DNA to make another double-stranded circle.
6. DNA pol I removes nthe primer, replacing it with DNA, and ligase
joins the ends to make another double-stranded DNA
7. CCC DNA: covalently closed circular; SSB: single-stranded-DNAbinding protein
(3) Replication of linear plasmids
i. Linear DNA replication faces a problem with replicating the
lagging strand. There is no upstream primer on this strand
from which to grow. Different linear DNAs solve the primer
problem in different ways.
ii. Some linear plasmids have hairpin ends, which means that
the 3’ end is attached to the 5’ end on the other antiparallel
strand. The plasmid replicates from an internal origin of
replication to form dimeric circles, composed of two
plasmids joined head to tail to form a circle. These dimeric
circles are then resolved into individual linear plasmid DNA
by prototelomerases.
iii. Some linear plasmids have extensive inverted repeated
sequences at their ends and a terminal protein attached to
the 5’ ends. They might use some sort of slippage
mechanism, using the terminal protein as either a
recombinase or a primer or both.
(4) Functions of the ori region
In most plasmids, the genes for proteins required for replication
are located very close to the ori sequences at which they act. The
genes in the ori region often determine many other properties of
the plasmid. Therefore any DNA molecule with the ori region of a
particular plasmid will have most of the characteristics of that
plasmid, such as
i. Host range – Some plasmids, such as those with ori regions
of ColE1 plasmid type have a narrow host range. Other
plasmids have a broad host range. These plasmids of broad
host range must encode all of their own proteins required for
initiation of replication and so do not have to depend on the
host cell for any of these functions.
ii. Copy number – Plasmids that have high copy numbers,
called relaxed plasmids, such as ColE1 plasmids need only
have a mechanism that inhibits the initiation of plasmid
replication when the number of the plasmids in the cell
reaches a certain level. Low-copy-number plasmids, called
stringent plasmids, such as F plasmids must have a tighter
Incompatibility
iii. Incompatibility – refers to the ability of two plasmids to
coexist stably in the same cell. If two plasmids can not
coexist stably, they are said to be members of the same
incompatibility (Inc) group. If they can coexist stably, they
belong to different Inc groups.
iv. There are a number of ways in which plasmids can be
incompatible:
(i) Due to shared replication control – Each plasmid regulate
the other’s replication.
(ii) Due to partitioning – Two plasmids share the same Par
(partition) system.
Coexistence of two plasmids from
different Inc groups
A. Coexistence of two
plasmids of
different Inc groups.
After division, both
plasmids will
replicate to reach
their copy number.
B. Curing of cells of one of two plasmids when they are members of the
same Inc group. The sum of the two plasmids will equal the copy
number, but one may be underrepresented and lost in the subsequent
divisions. Eventually, most of the cells will contain only one or the
other plasmid.
(5) Control mechanisms of plasmid
replication
(5) (using ColE1-derive plasmid as an example)
i. Replication is regulated mostly through a small plasmidencoded RNA I which interferes with the processing of RNA II
ii. RNA II is cleaved by the RNA endonuclease RNase H,
releasing a 3’ hydroxyl group that serves as the primer for
replication first catalyzed by DNA polymerase I.
iii. RNA I can form a double-stranded RNA with RNA II because
they are transcribed from opposite strands in the same region
of DNA. Initially, the pairing between RNA I and II occurs
through short exposed regions on the two RNAs that are not
occluded by being part of secondary structure.
This initial pairing is very weak and has been called “kissing
complex”. Rop protein (sometimes
called Rom) is known to help the formation of
kissing complex.
Mutations that inactivate Rop cause only a moderate increase
in plasmid copy number.
Control mechanisms of plasmid
replication
iv. Formation of the double-stranded RNA prevents the
RNA II from forming the secondary structure
required for it to hybridize to the DNA before being
processed by RNase H to form the mature primer.
v. RNA I is synthesized from the plasmid, more RNA I
is made when the concentration of the plasmid is
high (up to 16 copies) and will interfere with
processing of most of the RNA II. This mechanism
provides an explanation for how the copy number
of ColE1 plasmids is maintained.
vi. A single-base-pair mutation in the RNA I coding
region of plasmid will effectively change the Inc
Control mechanisms of plasmid
replication
vii. The ColE1-derived plasmids are unusual in that
they do not required the plasmid encoded protein,
Rep to initiate DNA replication at their oriV region,
only an RNA primer synthesized from plasmid.
The Rep protein is required to separate the DNA
strands of DNA at the oriV region, often with the
help of host proteins including DnaA. The copy
number of some plasmids can be controlled, at
least partial, by controlling the synthesis of the
Rep protein, such as R1 plasmid.
Pairing between an RNA and its
antisense RNA
Regulation of replication of IncFII
plasmid R1
A. The locations of
promoters and
genes, and gene
products involved
in regulation.
B. Immediately after the plasmid enters the cell, most of the repA mRNA is made
from promoter PrepA until the plasmid reaches its copy number.
C. Once the plasmid reaches its copy number, CopB protein represses transcription from
PrepA. Now, repA is transcribed only from PcopB.
C’ The antisense RNA CopA hybridizes to the leader peptide coding sequence in the repA
mRNA, and the double-stranded RNA is cleaved by RNase III. This prevents translation of
RepA, which is translationally coupled to translation of the leader peptide.
Iteron plasmids
• The plasmids whose oriV region contains several
repeats of a certain set of DNA bases called iteron
sequence, such as pSC101, F.
The ori region of pSC101, R1, R2, and R3 are the
three iteron sequence (CAAGGTCTAGCAGCA
GAATTTACAGA for R3) to which RepA binds to
handcuff two plasmids. RepA autoregulates its
own synthesis by binding to the inverted
repeats IR1 and IR2.
The “handcuffing” or “coupling” model
for regulation of interon plasmids
Left: At low concentrations of plasmids, the RepA binds to only
one plasmid at a time, initiating replication.
Right: At high plasmid and RepA concentrations, the RepA may
dimerize and bind to two plasmids simultaneously,
handcuffing them and inhibiting replication.
Molecular genetic analysis for the
regulation of interon plasmids
A. The RepA protein
is expressed from a
clone of the RepA
gene in an unrelated
cloning plasmid
vector.
B. Additional iteron sequences in an unrelated plasmid can
cower the copy number of an iteron plasmid.
6. Mechanism to prevent curing of
plasmids
Cells that have lost a plasmid during cell division are said to be
cured of the plasmid. Several mechanisms prevent curing,
including plasmid addition systems, site-specific recombination
and partitioning systems.
i. Resolution of multimeric plasmids –
(i) A possibility that a cell will lose a plasmid during cell
division is increased if the plasmids form dimmers or
multimers during replication due to segregating into only
one daughter cell. because of with more than one par site.
(ii) Dimers or multimers can occur as a recombination
between monomers or the termination of rolling-circle
replication after each round of replication is not efficient.
(iii) Multimers may replicate more efficiently than monomers,
perhaps because they have more than one origin of
replication.
(iv) To avoid the problem, many plasmids have site-specific
recombination systems that resolve multimers as soon as
6. Mechanism to prevent curing of
plasmids
ii. The most effective mechanism that plasmids have to avoid
being lost from dividing cells is their set of partitioning
systems. The following is the example of R 1 plasmid:
(i) The partitioning system of R 1 plasmid consists of two
protein-coding genes, parM and parR , as well as a
centromere-like cis-actin site, parC.
(ii) Protein ParM can bind to ParR only a few dimmers of ParR
protein bound to parC site. The ParR-parC complex serves
as a sort of nucleation site for the assembly of ParM.
(iii) While plasmid is replicating, this complex of the two ParM
and ParR proteins is localized to the midpoint of the cell
and thereby localizes plasmid to this point.
(iv) When replication is completed, the ParM protein, in ParMATP form begins to polymerize into helical filaments that
extend from the center of the cell toward the cell pole by the
addition of ParM subunits to the end.
(v) After the plasmid copies have been pushed to the ends, the
ParM-ADP dissociate and the filaments disappear.
Partioning
of the R1
plasmid