General Structure of Plasmid Origins of Replication

• Replication starts from the origin of replication (ori).
• It refers to the portion of the sequence that is targeted by replication initiation factors.
• Origin of replication or orito refer to the cis-ori, and replicon to refer to basic or minimal replicons.
• Rep protein helps in initiation during the replication process.
• But some theta plasmids depend on the host initiation factors for replication.
• Rep recognition sites typically consist of direct repeats or iterons.
• Its specific sequence and spacing are important for initiator recognition.
• Two Rep proteins are present:
• π of R6K
• RepA of ColE2

Replication Initiation: Duplex Melting and Replisome Assembly

• Duplex melting is dependent on transcription.
• It can be mediated by plasmid-encoded trans-acting proteins (Reps).
• When the Rep protein binds the ori region then a nucleoprotein complex is formed.
• At the A+T-rich segment, the DNA duplex is opened.
• The opening of the two strands of the DNA is important.
• In Theta-type plasmid, the assembly of the replisome can be:
  • DnaA-dependent
  • PriA-dependent
• DnaA-dependent assembly closely resembles replication initiation at oriC. It is the site where the chromosomal replication initiates
• PriA-dependent assembly parallels replication restart following replication fork arrest. It depends on D-loop formation with the extra DNA strand supplied by homologous recombination.
• In the theta-type plasmids, the Rep protein unwinds the two strands.
• The replication fork is formed where the DnaB is loaded in it. DnaA helps with this loading.
• Some plasmids depend on transcription for duplex melting, i.e unwinding of the two strands.
• The transcript itself can be processed in it and become the primer for an extension.
• When the primer is extended continuously, it leads to the synthesis of a leading strand.
• It facilitates  the formation of a Displacement loop or D-loop
• The nascent ssDNA strand separates the two strands of the DNA duplex and hybridizes with one of them.
• PriA (initiator of primosome assembly) can be recruited to the forked structure of the D-loop.
• Alternatively, PriA can be recruited to a hairpin structure. It forms when the double-stranded DNA opens.
• PriA helps in the unwinding of the lagging-strand arm.
• It also helps in the assembly of two additional proteins (PriB, and DnaT) to load DnaB onto the lagging strand template.
• The loading of DnaB is independent of DnaA in this case.
• After loading DnaB, both DnaA-dependent and –independent modes of replication converge.
• Other protein and enzymes involved in it are:
  • SSB (single-stranded binding protein)
  • DnaB (helicase)
  • DnaC (loading factor)
  • DnaG (primase)
  • DNA polymerase III (Pol III) holoenzyme.
• SSB protein binds the single-stranded DNA and helps in its stabilization.
• Then in the replication fork, DnaB is loaded in the form of a complex with DnaC.
• Then, DnaG (the primase) synthesizes RNA primers for the synthesis of lagging-strand synthesis.
• Then Pol III holoenzyme is loaded.
• The holoenzyme contains:
  • a core (with α, a catalytic subunit, and ε, a 3’→5’ exonuclease subunit),
  • a β₂ processivity factor
  • a DnaX complex ATPase.
• DnaB helicase activity is stimulated through its interaction with Pol III and modulated through its interaction with DnaG.
• It facilitates the coordination of leading-strand synthesis with that of lagging-strand synthesis during slow primer synthesis on the lagging strand.
• In the Gram-negative bacteria, single replicative polymerase (Pol III) is present.
• In the Gram-positive bacteria, two replicative polymerases are present:
  • PolC
    • PolC polymerase helps in the synthesis of the leading strand.
  • DnaE extends DnaG-synthesized primers before handoff to PolC at the lagging strand.
• In theta plasmids, lagging-strand synthesis is discontinuous and coordinated with leading-strand synthesis.
• The replicase extends a free 3’-OH of an RNA primer, which can be generated by DnaG primase (in Gram – bacteria)
• It is done by the concerted action of DnaE and DnaG primase (in Gram + bacteria)
• It can also be done by alternative plasmid-encoded primases.
• Discontinuous lagging-strand synthesis involves repeated priming and elongation of Okazaki fragments.
• DNA polymerase I (Pol I) contributes to plasmid replication in several ways.
• In ColE1 and ColE1-like plasmids, Pol I can extend a primer to initiate leading-strand synthesis.
• Then it opens the DNA duplex.
• This process can expose a hairpin structure in the lagging-strand, known as single-strand initiation (ssi) site or primosome assembly (pas) site, and/or generate a D-loop.
• Both hairpins and forked structures recruit PriA. It is the first step in the replisome initiation complex.
• Then, Pol, I help in the synthesis of the discontinuous lagging strand.
• It removes RNA primers through its 5’→3’ exonuclease activity and fills in the remaining gap through its polymerase activity.
• Pol I can functionally replace Pol III in  coli.
• There are three modes of replication for circular plasmid replication. They are:
  • Theta
  • strand-displacement
  • rolling circle.

Theta Plasmid Replication:

• Theta mode of replication is similar to chromosomal replication.
• There is the synthesis of leading- and lagging-strand.
• Lagging-strand is discontinuous.
• No DNA breaks are required for this mode of replication.
• There is the formation of bubbles in the early stages of replication.
• It resembles the Greek letter θ.
• Theta replication is of 4 types:
  • θ class A
  • θ class B
  • θ  class C
  • θ  class D

Class A Theta Replication

• Class A theta plasmids include:
  • R1
  • RK2
  • R6K
  • pSC101
  • pPS10
  • F
  • P
• For the replication initiation, all these plasmids depend on Rep protein:
  • RepA for R1, pSC101, pPS10, and P1
  • Trf1 for RK1
  • π for R6K
• Rep proteins bind interons (direct repeats) in the plasmid origin of replication.
• In plasmid P1, RepA monomers contact each iteron through two consecutive turns of the helix.
• It leads to in-phase bending of the DNA, which wraps around RepA.
• In R6K plasmids, the π binding of its cognate iterons bends the DNA and generates a wrapped nucleoprotein structure.
• The two exceptions to the presence of multiple iterons in class A theta plasmid origins of replication are:

(a) Plasmid R1, which features two partial palindromic sequences instead of iterons. R1 palindromic sequences are recognized by RepA.

(b) The R6K plasmid, which has three origins of replication:

• γ (with 7 iterons)
• second origin (α) features a single iteron
• third origin (β) only has half an iteron.
• γ oriis an establishment origin. It allows replication initiation immediately the following mobilization when levels of π protein are low.
• α and β oris would be maintenance origins in cells inheriting the plasmid by vertical transmission.
• γ ori acts as an enhancer which favors the long-range activation of α and β oris by transfer of π.
• α and β oris are still dependent on the multiple iterons present in ori γ.
• Rep binds the ori region and duplex DNA melting occurs.
• Rep-DnaA interaction is frequently involved.
• In plasmid pSC101, RepA helps to stabilize DnaA binding to distant dnaA It leads to strand melting.
• Plasmid P1’s ori has two sets of tandem dnaA boxes at each end.
• DnaA binding loops up the DNA which leads to preferential loading of DnaB to one of the strands.
• RK2’s TrfA mediates the open complex formation and DnaB helicase loading in the absence of dnaA
• The presence of DnaA protein is still required.

Class B Theta Replication:

• Class B theta plasmids include ColE1 and ColE1-like plasmids.
• Class B plasmids rely on host factors for both double-strand melting and primer synthesis.
• The DNA duplex is opened by transcription of a long (~600 bp) pre-primer called RNA II.
• It is transcribed from a constitutive promoter P2.
• The 3’ end of the pre-primer RNA forms a stable hybrid with 5’ end of the lagging-strand DNA template of ori.
• This stable RNA-DNA hybridization (R-loop formation).
• The pairing of the G-C between the transcript and lagging strand DNA template facilitates it.
• It forms a hairpin structure between the G- and C-rich stretches.
• Then the RNA pre-primer is processed by RNAse H producing a free 3’ -OH end
• It recognizes the AAAAA motif in RNAII.
• Extension of this RNA primer by Pol I initiates leading-strand synthesis.
• The point where the RNA primer is extended (known as RNA/DNA switch) is considered the replication start point.
• The nascent leading-strand separates the two strands of the DNA duplex and can hybridize with the leading-strand template, forming a D-loop.
• PriA is recruited to the forked structure of the D-loop.
• Alternatively, PriA can be recruited to hairpin structures forming on the lagging-strand template when the duplex opens.
• priA strains do not support ColE1 plasmid replication.
• The hypomorphic mutations in priA priBresult in a reduced ColE1 plasmid-copy-number.
• When the Pol III holoenzyme is loaded, this polymerase continues leading-strand synthesis.
• Then it initiates lagging-strand synthesis.
• Pol III replication of the lagging strand toward RNA II sequence is arrested 17 bp upstream of the DNA/RNA switch, at a site known at terH.
• It is unidirectional replication.
• Lagging-strand replication by Pol III appears to end a few hundred nucleotides upstream of the terH site, leaving a gap that is filled by Pol I.

R-loop formation:

• R-loop formation is essential in process of replication initiation.
• Deficits in RNAse H and/or Pol I do not prevent initiation.
• In the absence of RNAse H, unprocessed transcripts can still be extended with some frequency.
• In the absence of Pol I, the Pol III replisome can still be loaded on an R-loop formed by the transcript and lagging-strand template.
• R-loop formation occurs as a result of local supercoiling in the trail of the advancing RNA polymerase during transcription and is highly deleterious.
• It is because R-loops block transcription and the elongation step during translation.
• So, the unscheduled R-loop formation is suppressed by the cell.

Hybrid Classes of Theta Replication (Class C and D):

• The specialized priming mechanisms are present in these two classes which are combined with elements of class A and class B replication.
• Rep protein is present in class C and D plasmids.
• They initiate the leading-strand synthesis by Pol I extension of a free 3’-OH.
• They have termination signals in the 3’ direction of lagging-strand synthesis.
• Replication of these plasmids is unidirectional.
• Class C and D have evolved the specialized priming mechanisms.
• Class C includes ColE2 and ColE3 plasmids.
• The oris for these two plasmids are the smallest and differ only at two positions.
• One of them determines plasmid specificity.
• ColE2 and ColE3 oris have two iterons and show two discrete functional subregions.
• One is specializing in the stable binding of the Rep protein (region I)
• another one specializing in the initiation of DNA replication (region III).
• The Rep protein in class C plasmids has primase activity.
• It synthesizes a unique primer RNA (ppApGpA) which is extended by Pol I at a fixed site in the origin region.
• Class C replication is unidirectional.
• The Rep protein may stay bound to the ori after initiation of replication, blocking the progression of the replisome synthesizing the lagging strand.

Class D:

• It includes large, low-copy streptococcal plasmids.
• Replication occurs in a broad range of Gram-positive bacteria.
• Examples:
• Enterococcus faecalispAMβ1
• pIP501 from Streptococcus agalactiae
• pSM19035 from Streptococcus pyogenes
• It requires transcription across ori sequence, Pol I extension, and PriA-dependent replisome assembly.
• The transcript is generated from a promoter controlling expression of rep.
• Replication depends on transcription through the origin.
• Rep binds specifically and rapidly to a unique site.
• Denaturation of AT-rich sequence occurs and forms the open complex.
• This binding denatures an AT-rich sequence immediately downstream of the binding site to form an open complex.
• RepE also has an active role in primer processing.
• As melting increases RepE binding and RepE can cleave transcripts from the repE operon close to the RNA/DNA switch.
• Class D replisome assembly is PriA-dependent.
• A replisome assembly signal can be found 150 nt downstream from ori on the lagging-strand template.
• Replication arrest is induced by Topb, a plasmid-encoded topoisomerase.
• A second replication arrest is caused by collision with a site-specific resolvase, Resb.
• It is a plasmid-borne gene responsible for plasmid segregation stability.

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• The inability of two or more plasmids to coexist stably in the same cell is known as Plasmid incompatibility.
• This function of plasmids is controlled by the ori
• Many bacteria contain more than one type of plasmid. However, These types of plasmid, they coexist stably in the bacterial cell. Even after many cell generations, they may remain in it.
• Sometimes two plasmids of different types cannot coexist stably in the same cell.
• As the cells multiply, one or the other plasmid is lost in this case.
• When two plasmids cannot coexist stably, then they are said to be members of the same incompatibility (Inc) group.
• They will belong to different Inc groups if two plasmids can coexist stably.
• Plasmids can be incompatible in many ways:
  • If they can each regulate the other’s replication plasmid
  • if they share the same partitioning (par) functions.
• In such a case, for the utilization of initiator protein, competition may occur.
• Plasmids are usually classified by the Inc group to which they belong. Example:
  • RP4 (also called RK2) is an IncP (incompatibility group P) plasmid.
  • RSF1010 is an IncQ plasmid.
• Plasmid incompatibility was first described for the F plasmid in E. coli strains in the early 1960s.
• When any new plasmid is identified, it will be classified based on the incompatibility relationship with the known plasmids.
• This may even lead to the creation of an entirely new class of incompatibility groups.
• Plasmid incompatibility is an important consideration in designing any plasmid transfer experiment.
• It is also crucial in selecting a cloning vector for genetic engineering.

Incompatibility Due to Shared Replication Control:

• Two plasmids can be incompatible if they share the same mechanism of replication control.
• Since the two different plasmids cannot be recognized as different by the replication control system, so either plasmid may be randomly selected for replication.
• The total copy number of the two plasmids will be the same, at the time of replication.
• But one may be represented much less than the other.
• Example:
  • A cell contains two types of plasmids, suppose, type A and type B. At the time of replication, randomly one type will be picked up. If type A is replicated, then, there will be two copies of type A and only one of type B plasmid. In the next round, type A will have a higher probability of being replicated and plasmid type B may gradually be lost.

Incompatibility Due to Partitioning

• If two plasmids share the same Par (partitioning) system then they can be incompatible.
• During the cell division, Par systems help in the segregation of plasmids or chromosomes into daughter cells.
• In the normal condition, this system helps to ensure that both daughter cells get at least one copy of the plasmid.
• It ensures neither daughter cell is cured of the plasmid.
• But if the same Par system is shared by the coexisting plasmids, then, during the division one or the other is always distributed into the daughter cells.
• Sometimes one daughter cell will receive one plasmid type and the other cell gets the other plasmid type.
• It can produce the cured cells of one or the other plasmid.

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• The copy number plasmid means the number of plasmids that are normally present in a single bacterial cell.
• The size and copy number of a plasmid is important.
• Cloning is concerned with it.
• Plasmid size that less than 10 kb is desirable for a cloning vector.
• Some plasmids, especially the larger ones, are stringent.
• Larger plasmids and have a low copy number which might be just one or two per cell.
• Others are called relaxed plasmids which are present in multiple copies of 50 or more per cell.
• Large quantities of the recombinant DNA molecule can be obtained when multiple copies of the cloning vector are present in the cell.
• Naturally occurring plasmids are usually stably maintained in their bacterial hosts.
• This stability often must be accomplished despite a very low number of plasmid copies per cell.

Plasmid replication and control:

• Many plasmids are replicated as double-stranded circular molecules.
• The replication of the double-stranded molecule is the same as that of the chromosome.
• Replication starts from the origin of replication which is known as
• It may be unidirectional ( single directional ) or bidirectional ( both directions).
• When the whole circle is completed, then it segregates.
• However, there are some aspects of replication that differ from that of the chromosome, especially for the multicopy plasmids.

Mechanism of regulation of plasmid copy number

• Regulation of plasmid number is important to control the copy number of the plasmid.
• The mechanisms used by some plasmids to regulate their copy number are :

1) ColE1-derived Plasmids:

• It is the regulation of the processing of primer by complementary RNA
• The mechanism of copy number regulation of the plasmid ColE1 was one of the first to be studied.
• The mechanism of regulation of ColE1-derived plasmids is shown in:
  • Replication starts always from the origin of replication (oriV)
  • RNA II forms the primer for the plasmid DNA replication.
  • At the origin of replication, an RNA-DNA hybrid is formed.
  • RNA is RNA II and that DNA is the DNA of plasmid.
  • Then RNase cleaves the RNA II.
  • RNase H has endonuclease activity which means it can capacity to cleave.
  • At the cleaved or cut portion, 3 ʹOH is exposed where the nucleotides will be added by DNA polymerase I.
  • Thus the plasmid is replicated.
  • But when there is a sufficient number of plasmid its replication needs to be stopped.
  • This process is regulated by the RNA I and the Rop protein.
  • The Rop protein (sometimes called Rom)  helps RNA I to pair with RNA II.
  • RNA I is transcribed from the opposite strand to RNA II and is complementary to the first 108 bases of RNA II.
  • When there is pairing between RNA I and RNA II, RNAse can not cut the strand.
  • When the strand isn’t cleaved further replication is halted.
  • Therefore, it inhibits DNA replication.
  • The inhibition of replication is almost complete when the concentration of the plasmid reaches about 16 copies per cell which is the copy number of the original ColE1 plasmid.
  • Mutations that inactivate Rop cause only a moderate increase in the plasmid copy number.

  • 

2. R1 plasmid:

• R1 plasmid is a member of the IncFII family of plasmids.
• Plasmids belonging to the INFII group (R1, R6-5, R100) are self-transmissible, conjugative plasmids found in Enterobacteriaceae.
• R1 plasmid regulates its copy number by regulating the amount of the Rep protein.
• Plasmid requires the Rep protein to initiate the replication.
• It is different from ColE1-derived plasmids which do not require Rep protein. Only the RNA primer was enough for it to start the replication process.
• The Rep protein is required to separate the strands of DNA at the oriV region, often with the help of host proteins, including DnaA.
• Opening the strands is a necessary first step of replication.
• The Rep proteins bind specifically in the DSO of DNA
• By controlling the synthesis of the Rep protein, the plasmid copy number can be controlled.
• The R1 plasmid uses its complementary RNA to inhibit the translation of the mRNA that encodes the Rep protein and inhibits the replication of the plasmid DNA.

• The plasmid-encoded protein RepA is the only plasmid-encoded protein that is required for the initiation of replication.
• Two promoters transcribe the repA gene:
  • pcopB
  • pCopA

CopB:

• CopB, codes for a protein that represses transcription of the repA gene.
• When the plasmid first enters a bacterial cell, the absence of CopB allows expression of RepA and so there is a burst of replication until the level of CopB builds up to repress this promoter.
• From then on, expression of RepA occurs at a low level from the copB promoter

CopA:

• The second regulatory gene, copA, then regulates the expression of RepA.
• This gene codes for an 80–90-nucleotide untranslated RNA molecule.
• The copA gene is within the region of DNA that is transcribed for the production of RepA but is transcribed in the opposite direction (it is an antisense RNA).
• The copA RNA is therefore complementary to a short region of the repA transcript and will bind to it, interfering with the translation of the RepA protein.
• When the plasmid replicates, the number of copies of the copA gene is doubled and the amount of the copA RNA will therefore increase.
• This causes a marked reduction in further replication initiation, until cell division

• 

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• Those DNA molecules which can replicate autonomously are known as replicons. Example: Plasmids, phage DNA, chromosomes.
• Plasmid can replicate independently.
• Replicons have at least one origin of replication, or ori site, where replication begins.
• The cell also contains the proteins that help to initiate the replication.
• For their replication, plasmids encode only a few of the proteins.
• Many encode only one of the proteins needed for initiation at the ori
• Other required proteins are DNA polymerases, ligases, primases, helicases, etc.
• The plasmid replication origin is often named oriV for ori vegetative, to distinguish it from oriT. oriT is the site at which DNA transfer initiates in a plasmid.

Mechanism of Plasmid replication:

• There are two methods for the replication of plasmids. Among the two mechanisms, replication can occur by any one of the mechanisms:

1. theta mode for both unidirectional and bi-directional pathways
2. rolling circle mode.

1. Mechanism of Theta Plasmid Replication:

• DNA unwinds at the ori site from where the replication begins.
• It then creates the structure where the whole replicational machinery assembles.
• Since the structure resembles the Greek letter theta (θ), its name has been derived from it.
• The process gets initiated by the RNA primer.
• Then deoxyribonucleotides are added which extends the process.
• The replication process may proceed in one (unidirectional) or both directions (bi-directional).
• In the first case (unidirectional), a single replication fork moves around the circle until it returns to its point of origin. and then the two daughter DNAs separate.
• In the other case (bidirectional replicational) two replication forks begin at ori then it travels to the opposite until they meet at some point on the other side of the molecule.
• This is the most common mode of DNA replication.
• The theta mechanism is the most common form especially in Gram-negative bacteria like the proteobacteria.
• Commonly used plasmids, including ColE1, RK2, and F, as well as the bacteriophage P1, use this type of replication
• 

2. Mechanism of Rolling Circle Plasmid Replication

• It is called rolling-circle (RC) replication because it was first discovered in a type of phage where the template circle seems to “roll”.
• It is a unidirectional process (one direction only).
• Plasmids that replicate by this mechanism are sometimes called RC plasmids.
• This type of plasmid is found in the largest groups of bacteria, as well as in archaea.
• To perform this rolling-circle mode of replication, genetic material needs to be circular.
• In this method, one strand comes out while the other strand is being synthesized.
• Replication starts at the ori site that is the origin of replication where the Rep protein attaches one of the strands.
• Rep protein is the dimer that is formed of the two monomers.
• It has the tyrosine as the active group.
• First, the Rep protein recognizes and binds to the strand that contains the double-strand origin (DSO) on the DNA.
• Then the Rep protein can make a nick in the sequence.
• When the Rep protein has made a break in the DSO sequence, two ends will be formed in the DNA.
• At the 3’ end, there is the presence of OH group while at the 5’ end there is the presence of phosphate group.
• Rep protein will remain attached to the phosphate at the 5’ end of the DNA.
• Then the DNA polymerase III which is the replicative polymerase uses the free 3′ hydroxyl end at the break as a primer to replicate around the circle.
• For the separation of the strand, it may use a host helicase.
• The Rep protein itself may have the helicase activity, depending on the plasmid.
• Once the circle is complete, the 5′ phosphate is transferred from the tyrosine on the Rep protein to the 3′ hydroxyl on the other end of the strand. Then a single-stranded circular DNA is produced.
• This process is called a phosphotransferase reaction and requires little energy. The same reaction is used to re-form a circular plasmid after conjugational transfer.
• The displaced circular single-stranded DNA now replicates by a completely different mechanism using only host-encoded proteins.
• The RNA polymerase of the host cell recognizes the SSO ( single-strand origin) on the DNA.
• Then the RNA polymerase makes a primer.
• Then, replication occurs around the circle by DNA polymerase III.
• The RNA polymerase does not make this primer until the single-stranded DNA is completely displaced during the first stage of replication.
• When the entire complementary strand has been synthesized, the DNA polymerase I remove the RNA primer with its 5′ exonuclease activity while simultaneously replacing it with DNA.
•  DNA ligase joins the ends to make another double-stranded plasmid.
• Finally, The two new double-stranded plasmids are synthesized from the original double-stranded plasmid.
• 

3. Replication of linear plasmids:

• Some plasmids and bacterial chromosomes are linear rather than circular.
• The linear DNAs face two problems in all organisms.
• One problem is, at the end of linear fragments, the cell must distinguish the “normal” DNA ends from the ends formed when DNA double-strand breaks occur.
• This condition could be lethal to the cell and must quickly be repaired.
• A second problem with linear plasmids and chromosomes has to do with replicating the lagging-strand template.
• It is the strand that ends with a 5′ phosphate, all the way to the end of the DNA.
• Since DNA polymerases cannot initiate the synthesis of a new strand of the DNA, this has been the “primer problem”.
• They can only add nucleotides to a preexisting primer.
• In a linear DNA, there is no upstream primer on this strand from which to grow.
• The primer problem is solved by the different linear DNA in different ways.
• Some linear plasmids have hairpin ends. They have the 5′ and 3′ ends joined to each other.
• These plasmids replicate from an internal origin of replication to form dimeric circles.
• These dimeric circles are composed of two plasmids joined head to tail to form a circle.
• These dimeric circles are then resolved into individual linear plasmid DNAs with closed hairpins at the ends.
• The hairpin ends are previously not recognized as DNA double-strand breaks.
• It is because they are not targets for exonucleases in the cell.
• A completely different mechanism is also used to maintain linear plasmids in some systems.
• With this mechanism, a special enzyme called a terminal protein attaches to the 5′ ends of the plasmid DNA.

It is interesting that bacteria with linear plasmids also often have linear chromosomes, and the two DNAs replicate by similar mechanisms

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• Plasmids are the extrachromosomal genetic elements found in bacteria.
• They are circular pieces of DNA that are extra genes.
• About 1-20 copies of plasmids are present in one bacterial cell.
• Episomes are the type of plasmid that can be inserted into the bacterial chromosome and can replicate with it.
• For normal life and functioning, a plasmid is not required in the bacteria. But their presence confers new properties in the bacteria. Example: Drug resistance, toxigenicity

Properties/Characteristics of bacterial plasmids:

1. Physical properties:
   • Plasmid is a double-stranded circular and supercoiled DNA.
   • Within a cell, it can exist autonomously. It can replicate independently of the bacterial chromosome.
   • It has a molecular weight of 10⁶-10⁸ which may encode from 40-50 genes.
   • It has about 1-3% of the weight of the bacterial chromosome consisting of 1500-400,000 base pairs.
   • Plasmid as large as 2 million base pairs can occur in some bacteria.

2. Replication:
   • It contains genes for self-replication.

3. Curing:
   • It can be lost spontaneously or by curing agents.

4. Incompatibility:
   • In the same cell, two members of the same group cannot co-exist.

5. Transferability:
   • Some plasmids are self-transferable.

6. Recombinations:
   • Episome can integrate with host chromosome.

7. Mobilisation:
   • By the process of integration, the self-transferable plasmid can mobilize the chromosomal gene or other plasmids.

Types of plasmid:

• Based on their function, plasmids are of five types:
• Resistance ( R ) plasmid
• Fertility (F) plasmid
• Bacteriocinogen or Col plasmid
• Degradative plasmid
• Virulence plasmid

 1. R-plasmid (R-factor):

• They are circular with double-stranded plasmid.
• R factor occurs in two sizes:
  • large plasmids ( mol. wt. 60 million)
  • small plasmids ( mol. Wt. 10 million)
• Large plasmids are conjugative ‘R’ factors. To code for the conjugation process, it contains extra DNA.
• Small plasmids contain only the ‘r’ genes. They are not conjugative.
• It consists of two components.
  • Resistance transfer factor (RTF): carries the genes that govern the process of intercellular transfer.
  • Resistant determinant ( R-determinant): carries resistant genes for each of the several drugs.
• The drug resistance is not transferrable in the case when RTF dissociates from the R-determinant.
• For the spread of the multiple drug resistance in the bacteria, R factor plays a vital role.
• Antibiotics can be destroyed and the membrane transport system can be modified.
• R-factor may carry the resistance genes either one, two, or more than these.
• They may also carry the gene resistance for the metal ions.
• They also carry resistance to certain bacteriophages by coding for the enzymes.

2. F-plasmids:

• It is a transfer factor or F-factor.
• It contains genetic information, which controls the mating process of the bacteria during the conjugation.
• It contains the basic genetic information necessary for:
  • Extra-chromosomal existence
  • Self-transfer
  • Synthesis of sex-pilus.
• F-plasmid carries some fourteen genes which include the structural gene for the pilin.
• Pilin is the pilus protein that functions in sex pilus formation.
• Strains of bacteria having the F plasmid are called F₊ and function as donors.
• Strains of bacteria lacking the F plasmid are called F- and function as recipients.
• It is also called the conjugative plasmid.
• The conjugative function is determined by the cluster of at least 25 transfer (tra) genes.
• These genes determine:
  • Expression of pili
  • Synthesis and transfer of DNA during mating
  • Interference with the ability of F₊ bacteria to serve as recipients.

3. Bacteriocinogen or Col plasmid:

• Coliforms produce extracellular colicins.
• In the several species of coliform, the colicinogenic (col) factors are present.
• These bacterial factors are the lethal toxins for the closely related species or even for the different strains of the same species.
• Some bacterial substances are produced not only by the coliforms but also by the other bacteria.
• This group of substances is called bacteriocins.
• Colicins are produced by coli
• Pyocin are produced by Pseudomonas aeruginosa.
• Marscesins are produced by Serratia marcescens.
• Diphthericin is produced by Corynebacterium diphtheria.
• Bacteriocin produced by the different bacterial strains helps in the interspecies typing of organisms.

4. Degradative plasmids:

• From the dead plants and animals, degradative plasmid helps in the degradation and digestion of the dead organic matter.
• It is then used in the biosynthesis process.
• It will make energy and will recycle further.

5. Virulence plasmids:

• With the help of this plasmid, bacteria will be transformed into a pathogen.
• It carries the genes which are responsible for causing disease.

Based on the role in conjugation, plasmids are of two types:

• Conjugative plasmid
• Non-conjugative plasmid

i. Conjugative plasmids:

• These large plasmids (F plasmids) carry genes that are responsible for transferring themselves to other cells.
• It includes the genes that direct the synthesis of sex pilli.

ii. Non-conjugative plasmids:

• These plasmids are present in Gram-positive bacteria, especially in the Gram-positive cocci.
• It is also present in the Gram-negative organism. Example: Haemophilus influenza, Neisseria gonorrhoeae.
• They are usually small, 1-10 dal.
• In each bacterium, multiple copies (more than 30 ) may be present.
• When the same bacterium carries both the conjugative and non-conjugative plasmids, they can be mobilized for transfer to another cell.
• When the conjugation is established then the donor can transfer non-conjugative plasmids.

Functions/applications of plasmid:

• The main function of the plasmid is the spread of antibiotic-resistant genes. These resistant genes are carried within the plasmid and are transferred from one cell to another.
• Plasmid is used in recombinant DNA technology.
• To deliver the desired drug into the body, a plasmid is used.
• For the insertion of the human insulin on the body
• Insertion of human growth hormone in mammalian cells of animals.
• Plasmids are used in Gene Therapy:
  • For the insertion of the therapeutic genes in the human body. It helps to fight against diseases.
  • Easy manipulation and can be replicated in bacterial cells easily.
  • Targeting the defected cells easily and triggering the therapeutic genes in them.
• Plasmids carry the genes involved in metabolic activities. They aid in the digestion of pollutants from the environment.
• Plasmids can produce antibacterial proteins.
• Plasmid can carry genes that increase the pathogenicity of the bacteria.
• When the nutrients are scarce, the plasmid can help bacteria by:
  • Fix the nitrogen
  • Degrade organic compounds

Host Range of plasmid

• The host range of a plasmid means the types of bacteria in which the plasmid can replicate.
• It is usually determined by the ori region from where the replication starts.
• Plasmid having the narrow host range includes:
  • ColE1 plasmid type; Example: pBR322, pET, and pUC.
  • Replication of these plasmids occurs only in coli.
  • It may occur in Salmonella and Klebsiella also which are closely related bacteria.
• Plasmids having the broad host range includes:
  • RK2
  • RSF1010 plasmids
  • RC plasmids; Example: pBBR1MCS .
  • Plasmids with the ori region of RK2 can replicate in most types of Gram-negative proteobacteria.
  • Plasmids with the RSF1010-derived plasmids can replicate in Gram-positive bacteria too. Example: Firmicutes.
  • Replication of the same plasmid can occur even in the distantly related bacteria.
  • Broad-host-range plasmids do not depend on the host cell because they encode their proteins. These proteins are essential for the initiation of replication.
  • Broad host-range plasmids should be for gene expression in many types of bacteria.

Determining the Host Range

• It is sometimes difficult to ensure the particular plasmid will replicate on the other host or not. So, The actual host ranges of most plasmids are unknown.
• Initially, plasmids need to be introduced to the other bacteria.
• So, for this process, a system has been developed which is known as transformation.
• By this method, the plasmid can be introduced into the bacteria to see if it could replicate or not.
• But it has limitations as it can’t be applied to all types of bacteria.
• Similarly, to introduce DNA into cells, electroporation can be used.
• Plasmids can be introduced into other types of bacteria by the conjugation process.
• It is also found that the expression of the gene present in one plasmid does not function well or doesn’t get expressed in the other bacterium.
• Sometimes the selected gene can be introduced into the different bacteria.
• A bacterium might possess resistance to any antibiotic due to the presence of a particular gene. Such resistance property can be transferred to other bacteria too when they will uptake those resistant genes.
• For example, the kanamycin resistance gene, which is first found in the Tn5 It can be expressed in most Gram-negative bacteria. Then, it will make resistant to kanamycin antibiotic.
• By this property, a marker gene can be cloned in the plasmid. i.e making numerous copies.
• A transposon carrying a selectable marker into the plasmid can also be introduced by this method.
• Care must also be taken to ensure that the plasmid has not recombined into the host chromosome.
• Determining the host range of a plasmid is laborious too. Many barriers hinder the transfer of plasmid into the host. The same method can’t be approached to all sorts of plasmids and bacteria.

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• Induced mutations are induced by known factor- such as-physical (ionizing irradiation, ultraviolet light), chemical and biological mutagens (bacteria and viruses).

Mechanism of induced mutation:

• Induced mutations occurs by at least three different mechanisms. They are-
  • By replacing nitrogenous base with base analogs
  • By base alteration-altering a base so that it specifically mispair with another base
  • By distortion of DNA molecule- damaging a base so that it no longer pair with another base

I. Incorporation of base analogs:

• Some chemical compounds are sufficiently similar to the normal nitrogenous bases of DNA known as base analogs and they can incorporated into DNA in place of normal bases.
• These analogs can base pair with other nitrogenous bases but they induce insertion of incorrect nucleotide during replication causing mutation.
• Uracil is halogenated in the carbon-5 position to give 5-bromouracil, 5-chlorouracil, and 5-iodouracil which can be incorporated into DNA in the place of thymine.
• 5-bromouracil (5-BU) bromine is formed by bromination at the carbon-5 position of uracil. The resulting structure of 5-bromouracil is similar to thymine  but thymine has CH3 group at C5 .
• 5-Bromouracil is most effective analog to thymine because size of bromine has same van der Waals radius as the methyl group in thymine.
• 5-bromouracil is highly mutagenic and it pairs with Adenine in normal condition. In 5-BU, the bromine atom is not in a position in which it can hydrogen-bond during base pairing, so the keto form of 5-BU pairs with adenine.
• However the frequency of tautomeric shift of 5-bromouracil is much higher than Thymine. It changes from keto form to either enol form or an ionized from amino form to keto form when protonated. Now, 5-bromouracil form hydrogen bond with Guanine instead of complementary base Adenine which result in base pair transition from T=A to C=G in subsequent replication cycle.
• Another commonly used base is 2-Aminopurine which is analog to adenine and pairs with thymine by two hydrogen bond. After a tautomeric shift due to protonation, it can form pair with cytosine.
• Therefore, 2-aminopurine can induce base pair transition from A=T to G=C in subsequent replication cycle.

II. Base alteration: alkylation, depurination, deamination and hydroxylation

• In this case, the mutagens do not incorporate with the DNA, however they can causes base alteration in a specific way.
• Some of the base alteration are- alkylation, depurination, deamination, hydroxylation etc.

Alkylation:

• Alkylating agents are most widely used mutagens which carry one, two or more alkyl groups in reactive form.
• Alkylating agents transfer their alkyl group (methyl or ethyl) group to nitrogenous bases of DNA and to phosphate group (alkylation).
• The most commonly used alkylating agents are- ethyl-methane sulfonate (EMS), Dimethyl sulphonate (DMS), Diethyl sulphonate (DES) etc , nitrosoguanidine (NG)
• Mechanism of alkylating agents:
  • Transfer alkyl group to phosphate group of DNA and produces unstable phosphate trimester which hydrolyses to give alkyl group. But some alkyl group may remain attached to phosphate which interfere with DNA replication cycle as well as cause breakage of sugar phosphate back bone.
  • Transfer alkyl group to 6-oxygen and 7-nitrogen of DNA bases. Among these alkylation of nitrogenous base, 7-ethylguanine is most common derivative. 7-ethylguanine is base analog to cytosine and can form base pair with thymine which ultimately cause base transition from G=C to AT.
  • Some difunctional or polyfunctional alkylating agents can crosslink with DNA strand and interfere DNA replication leading to chromosome breakage.

Depurination:

• Alkylation of purine base (Adenine and Guanine) give rise to unstable quaternary nitrogenous base which interfere with glycosidic bond between nitrogenous base and deoxyribose sugar.
• The loss of purine bases from DNA is termed as Depurination.
• Once the base is depurinated, it cannot specify complimentary base to the original purine during replication which result in incorporation of wrong base.
• Therefore, this causes base pair substitution.

Deamination:

• Some mutagens react with nitrogenous base containing amino group and remove it with another functional group.
• Nitrous acid (HNO2) a chemical which reacts with amino group containing nitrogenous bases (Adenine, cytosine and guanine) and replace it.
• Deamination of adenine by nitrous acid yields hypoxanthine (H) which can base pair with cytosine. This deamination at the position of adenine result in base transition from A:T to G:C in successive replication.
• Similarly, deamination of cytosine by HNO2 results in uracil. Now uracil can base pair with adenine. Therefore, change of base from cytosine to uracil results in base transition from G:C to A:U and then A:T in successive replication cycle.
• Other example of deamination: conversion of 5-methylcytosine to thymine.

Hydroxylation:

• The mutagen hydroxylamine (NH2OH) reacts with amino group of cytosine causing hydroxylation to yield hydroxylcytosine.
• Now the hydroxylcytosine can form base pair with adenine instead of guanine. This results in base pair transition.

III. Distortion of DNA molecule:

• Some of the mutagens directly reacts with DNA molecules causing distortion and modify the structure.

i. Intercalating agents:

• Certain florescent acridine dyes such as acridine orange, proflavin causes DNA mutation by insertion or deletion of nitrogenous bases.
• Acridine dyes are planar (flat) molecule that mimic nitrogenous bases and at low concentration it can inserts or intercalates between subsequent nitrogenous bases in DNA molecule.
• Insertion of the agent stretches the distance between adjacent base pair by 0.68nm which is twice the normal distance. This distortion result in single nucleotide deletion or insertion at this position during recombination.
• Intercalating agents result in insertion of bases:
  • Intercalation of acridine dye between two nitrogenous bases in template strand result in stretch of DNA molecule.
  • During DNA replication, a new base can be inserted in the newly synthesized strand opposite of acridine molecule.
  • Now during replication of newly synthesized strand, a complimentary base in added opposite to newly added base.
• Intercalating agents result in deletion of bases:
  • Insertion of acridine molecule in DNA strand may block the base in the template strand and does not allow to base pair.
  • During replication, one base is deficient in newly synthesized strand.

ii. DNA damage: by radiation

• Various kinds of radiations can induce mutation. Mutagenic radiations are two types- non-ionizing radiation (UV rays) and ionizing radiation (X-rays, gamma rays):

a. Mutation caused by UV rays: Pyrimidine dimer formation

• UV rays have a wavelength of 10-390nm.
• When UV rays falls on the genetic materials, it is absorbed and due to increase in energy level electrons are excited.
• The most effective wavelength of UV rays for inducing mutation is 260nm.
• UV rays are non-ionizing radiation and have a penetration power lower than X-rays.
• Exposure of DNA to UV rays causes nitrogenous bases to become highly reactively unstable free radicals.
• One of the consequences of UV rays exposure is photochemical fusion of two pyrimidine that are adjacent to each other on a same polynucleotide chain.
• Pyrimidine dimer formation is the primary effect of UV rays on DNA molecule.
• In case of two thymine, the fusion is called thymine dimer. UV rays causes adjacent thymine on the same DNA strand to bond together by covalent linkage between carbon 5 and 6 of adjacent thymine molecule forming cyclobutane ring.
• In case of thymine adjacent to a cytosine, the resulting fusion is a thymine-cytosine dimer in which the thymine is linked via its carbon atom 6 to the carbon atom 4 of cytosine.
• This dimer structure cannot fit into DNA helix and cause distortion of DNA molecule which result in failure of DNA replication and sometime leads to lethal effect.
• Pyrimidine dimer can also formed by cross linking of pyrimidine bases of adjacent DNA strand.
• There are three types of thymine dimers
• Thymine-thymine dimer= 50%
• Thymine-cytosine dimer= 40%
• Cytosine-cytosine dimer=10%

b. Mutation caused by Ionizing radiation (IR):

• The effects of different ionizing radiation are qualitatively the same.
• In direct effect, IR breaks the phosphate ester bond in DNA. The breakage may take place at one or more points. As a result of breakage, segment of DNA either get lost or rearranged during repair. Sometimes the effect is fatal.
• In indirect effect, the ionizing radiation ionize water producing free radicals which is extremely reactive.
• The free radicals react with DNA molecule to alter its structure.

Mechanism of induced mutation: base analogs, base alteration, intercalation, pyrimidine dimer formation

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• Transduction is a method of gene transfer in bacteria from donor to recipient using bacteriophage.
• In transduction at first bacteriophage infects donor bacteria and then carries some part of donor genome with it. When this bacteriophage infects new bacterial cell, it transfer that DNA in to recipient cell.

There are two types of transduction

1. Generalized transduction
2. Specialized transduction

1. Generalized transduction:

• If all the fragments of donor DNA from any region of chromosome have a chance to enter into transducing bacteriophage then it is known as generalized transduction.
• In this type of transduction, at first bacteriophage infects donor cell and begins lytic cycle.
• When virus enter into bacterial cell, virus hijack host cell and synthesize virus components such as genome, enzymes, capsid, head tail and tail fibers. Then viral enzyme hydrolyses host cell DNA into small fragments.
• During assembly of virus component to form progeny viruses, sometime any of the fragments of donor DNA get incorporated into the virus capsid (bacteriophage head). Such abnormal bacteriophage when infects a new cell, it can transfer this donor DNA into new bacteria. Since this donor DNA is not viral DNA, it does not replicates inside recipient bacteria but undergoes homologous recombination with recipient cell’s chromosomal DNA forming recombinant cell.

2. Specialized transduction:

• In specialized transduction, bacteriophage transfer only a few restricted gene (DNA fragments) from donor bacteria to recipient bacteria. Specialized transduction is carried only by temperate bacteriophage which undergoes lysogenic cycle in donor cell.
• At first temperate bacteriophage enter into donor bacteria and then its genome gets integrated with host cell’s DNA at certain location and remains dormant and pass generation to generation into daughter cell during cell division. The bacteriophage which follows lysogenic cycle is known as temperate phage.
• When such lysogenic cell is exposed to certain stimulus such as some chemicals or UV lights, it causes induction of virus genome from host cell genome and begins lytic cycle.
• On induction from donor DNA, this phage genome sometimes carries a part of bacterial DNA with it. The bacterial DNA lies on sides of integrated phage DNA are only carried during induction.
• When such bacteriophage carries a part of donor bacterial DNA infects a new bacteria, it can transfer that donor DNA fragments into new recipient cell. So, in this specialized transduction only those restricted gene are situated on the side of integrated viral genome have a chance to enter into recipient cell.

Transduction: generalized and specialized transduction

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Hfr cell

• When F-plasmid (sex factor) integrated with chromosomal DNA then such bacteria is known as high frequency recombination (Hfr) bacteria.
• In the cross (conjugation) between Hfr cell and F- cell, frequency of recombination is very high but frequency of transfer of whole F-factor is very low.
• Hfr cell acts as donor while F- cell acts as recipient.
• At first F-factor makes sex pilus that joins donor and recipient cell then F- factor opens as replication origin then one strand is cut down. Now the 5’ end of this strand enters into recipient cell through conjugation tube.
• Since, replication origin lies somewhere in the middle of F- factor, portion of F-factor that lies at 5’ end enters first into recipient cell but the portion situated at 3’ end enters only when whole chromosomal DNA enters into the recipient cell.
• To transfer whole chromosomal DNA, it takes 100 minutes in E. coli. In most of the cases, sex pilus (conjugation tube) breaks before transfer of whole chromosomal DNA takes place. So, frequency of transfer of whole F-factor is very low. After the cross between Hfr cell and F- cell, recipient cell remains recipient.
• In this conjugation, chromosomal DNA is always almost transfer from donor to recipient cell together with portion of F- factor. So, frequency of recombination is high.

F –prime (F’) cell:

• Bacteria in which contains F-factor and a part of chromosomal DNA integrated in it is known as F-prime bacteria.
  F’ cells are formed from Hfr cell during induction of F- factor from chromosomal DNA in which F-factor carries a portion of chromosomal DNA along with it.
• In the cross (conjugation) between F-prime (F’) cell and F- cell, frequency of recombination is high as well as frequency of transfer of whole F-factor is also high.

High frequency recombination (Hfr) cell conjugation and F-prime (F’) cell

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• Conjugation in bacteria is a process in which plasmids are transferred by themselves alone or along with other DNA element from one cell to another cell through conjugation tube.  Conjugation occur by physical contact between cells.
• The cell which transfer plasmid is called donor and the cell which receive the plasmid is called recipient.
• The cell which has received the plasmid from the donor cell is called trans-conjugant.
• The phenomenon of conjugation in bacteria was discovered by laderburg and Tatum in 1946.

Types of Plasmids

1. Self-transmissible (F-plasmid) plasmid:

• These plasmid encodes all the functions necessary for their transfer as well as the transfer of other DNA element and mobilizable plasmid into recipient cell.
• The cell which possess self-transmissible plasmid are called donor cell and other cell which usually don’t possess self-transmissible plasmid are called recipient cell.
• These plasmid contains both Tra gene and Ori T sites
• These plasmid are known as F-factor or F-plasmid or conjugative plasmid
• Present in Pseudomonas, E. coli, Bacillus, streptococcus, Staphylococcus, Streptomyces etc

2. Mobilizable plasmid:

• These plasmid encodes only function for its transfer into recipient cell.
• mobilizer plasmid is not transfer by itself. It requires the help of self-transmissible plasmid for its transfer.

Mechanism of conjugation:

Conjugation is brought about by 2 genes in self transmissible plasmid, namely transacting gene (Tra gene) and Origin of transfer (Ori T) site.

I. Tra gene:

Tra gene consists of 2 components- Dtr and mpf

i. Dtr (DNA transfer and replication) component:

• Dtr component prepare plasmid for transfer.
• It includes components such as= relaxases, relaxosome complex and primase.

Relaxase:

• Relaxase is a site specific endonuclease which acts on plasmid at its OriT site.
• Relaxase also recyclizes the plasmid after it has been transferred to the recipient cell.
• Relaxase is transcribed along with the plasmid into the recipient

Relaxosome complex:

• It consists of group of proteins clustered around the Ori T site
• Relaxosome carries three basic functions-
  • It helps relaxase bind to the oriT site and initiates plasmid transfer,
  • relaxosome communicates with the coupling protein of Mpf component which signals relaxase when to cut the plasmid at Ori t site,
  • Helicase is a component of relaxosome which helps to separate the plasmid DNA strands during displacement and transfer of plasmid.

Primase:

• Primase has no role in replication replication of donor plasmid in donor cell
• The free 3-OH end created at the nick site acts as a primer in donor cell.
• Primase is trnasfered to the recipient cell and synthesizes a primer to complete the replication of another strand of plasmid DNA in recipient cell.

ii. Mpf (Mating pair formation) component:

• Mpf component holds the donor and recipient cell together, forms a channel through which DNA is transferred and signal Dtr component to initiate transfer.
• It has 3 components- pilus, channel and coupling protein

Pilus:

• Pilus holds donor and recipient cell together
• It is 10nm in diameter tubular structure with a central channel projecting out of the cell surface.
• Pili may be structurally long, thin and flexiable and it is encoded by F-plasmid in those cell
• Incompatibility F-plasmid (Inc F) is a long and rigid pili encoded by pKM101 (Inc N)
• Short, thick and rigid plasmid is encoded by RP4 (Inc P)
• Long, thin and flexible pili mediates conjugation in cell in liquid medium
• Short, thick and rigid pili mediates conjugation in cell fixed to solid support (Agar medium)
• Inc I plasmid (col 1BP9) encodes both long, thin, flexible pili and short, thick and rigid pilli, therefore it can mediate conjugation in both liquid and solid media

Channel:

• Channel are also encoded by Tra gene
• Channel mediates the transfer of DNA from donor to recipient cell

Coupling proteins:

• Coupling protein is associated with channel
• It signals the relaxase which then initiates the process of DNA transfer
• Coupling protein determines which protein are to be transported to the recipient cell ( relaxase and primase)

II. Ori T site:

• It is the site where plasmid DNA transfer initiates in donor cell and the site for recyclization in the recipient cell.
• It is the site which is specifically recognized by relaxase.
• Any plasmid that possesses Ori T site can be transferred with the help of self-transmissible plasmid
• Ori T site is a cis-acting site
• A known Ori T site of F-plasmid has around 300 bp and contains inverted repeats and AT rich sites.

Chromosome transfer by plasmid:

Plasmid can also mediate the transfer of chromosome because Ori T site and Tra gene are present in plasmid. For transfer of chromosome, plasmid must be integrated with chromosome. The integrated form of plasmid is known as Hfr or High frequency recombination.

Plasmid can integrate into chromosome through 2 mechanism.

1. Recombination: plasmid can recombine with chromosome when plasmid and chromosome share common sequences (homologous sequences). Although the sequences of plasmid are unique to that of chromosome they share homology at certain insertion sequences.
2. Transposition: plasmid can insert itself into chromosome by transposons and results in formation of Hfr.

 Steps of bacterial conjugation:

Step I: Pilus formation

• Donor cell (F⁺ cell) produces the sex pilus, which is a structure that projects out of the cell and begins contact with an F^– (recipient) cell.

Step II: physical contact between donor cell and recipient cell

• The pilus enables direct contact between the donor and the recipient cells forming conjugation tube

Step III: transfer of F- plasmid

• F-factor opens at replication origin (Ori T site).
• one strand of F-factor is cut down at origin and then 5’end of this strand enters into recipient cell.

Step 4: complementary strand synthesis

• In the last step, the donor cell and the recipient cell, both containing single-stranded DNA of F-plasmid
•  A complementary strand is then synthesized in both donor and recipient cell,
• Now the recipient cell also contain a copy of F-plasmid and become a donor cell.

Bacterial Conjugation: steps and mechanism of transfer of plasmid from donor to recipient cell

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• Transformation is the process of introduction of derived DNA fragments from a donor bacteria into a recipient bacteria.
• It is one of the cornerstone of molecular genetics.
• It is the transfer of naked DNA from donor cell to recipient cell.

Types of transformation

1. Natural transformation
2. Artificial transformation

Natural transformation:

• Most types of cell cannot take up DNA efficiently unless they have been exposed to special chemical or electrical treatment to make them more permeable.
• However, some types of bacteria are naturally transformable ie they can take DNA from environment without requiring special treatment.
• Bacteria that take up DNA are called competent.
• At least 40 species of naturally competent and transformable bacteria have been found.

Gram +ve : Bacillus subtilis, Streptococcus pneumonae

Gram –ve: Haemphilus influenzae, Neisseria gonorrahe, Helicobacetr pylori, Acenetobacter baylyi, Cyanobacteria

Competence in Gram positive bacteria: Mechanism of uptake DNA during transformation

Figure: competence in Bacillus subtilis

• The protein involved in transformation of these Gram +ve bacteria is a product of com
• In Bacillus subtilis , the com gene are organized into several operons.
• The product of com A and com K are involved in regulation of competence and other com E, com F and com G encodes structural protein for uptake of DNA.
• The first gene of com E operon, com EA encodes the protein that directly binds extracellulat double stranded DNA.
• The com F gene encodes the protein that translocate DNA into cell. for example; Com FA is an ATPase that translocate DNA into cell.
• The com G gene of comG operon encodes protein that form pseudopilus that helps to move DNA through channel.
• The com E, com F and com G operon are under transcriptional control of com K operon.
• Com K is a transcriptional factor that is regulated by com A
• Some other genes involved in transformation are nuc A gene that encodes nuclease enzyme which cuts extracellular dsDNA to single stranded,
• Single strand binding protein and Rec A gene which helps in recombination of transforming DNA with chromosome of recipient bacteria.

Competence in Gram negative bacteria: mechanims of uptake of DNA during transformation

• A variety of Gran Negative bacteria are capable of competence.
• Some examples are Acenetobacter calcoaceticus, Helicobacter pylori, Neisseria spp, etc
• H. pylori and Neisseria spp require specific DNA sequences for binding of DNA so these species take up DNA from same species only.

Gram-ve bacteria utilizes two different pathway for uptake of DNA

1. PSTC transformation pathway
2. Type IV secretion related pathway

PSTC transformation pathway

Figure: Competence in Gram negative bacteria by PSTC pathway

• The term PSTC has been applied to some protein indicating their multiple role in Pilus formation, Secretion, Twitching motility and Competence.
• These proteins form structure needed to transport DNA across the cell wall and cell membrane.
• Many of these proteins that function in DNA uptake in H. influenza, Neisseria, Acinetobacter, Vibrio etc are related to com G

Type IV secretion related pathway

• This mechanism is found in Helicobacter pylori.
• The DNA is translocated through cell wall and membrane with the help of protein similar to Agrobacterium for conjugation of Ti plasmid.
• Type IV system function transfer of DNA in two ways-moving in and out of cell.

Artificial transformation

                             figure: artificial transformation

Most of the bacteria are not natural transformable. These bacteria can be made competence by certain chemical treatment or by strong electrical shock.       

Some of the methods are-

Calcium treatment:

• Treatment with calcium ion (ca++) make same bacteria eg. E.coli, Salmonella, Pseudomonas etc competence.
• The Calcium ion increases the permeability of cell membrane
• Cell treated with calcium can take up both ssDNA as well as dsDNA, no matter circular or linear.

Electroporation:

• A strong electric shock is applied in the bacterial culture mixed with naked DNA
• The recipient bacteria should be wash with non-ionic (distilled water) solution to prevent osmotic shock.
• The strong electric field creates artificial pore of water lined by phospholipid head group. The DNA can pass through these artificial hydrophilic pore.

References

1. http://ars.els-cdn.com/content/image/1-s2.0-S0966842X15002474-gr1.jpg
2. http://2014.igem.org/wiki/images/e/e8/B._subtilis_dna_uptak.jpg
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC372826/
4. https://en.wikipedia.org/wiki/Transformation_(genetics)
5. https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/bacterial-transformation-selection
6. https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/microbial-genetics-7/genetic-transfer-in-prokaryotes-81/bacterial-transformation-442-6842/
7. https://www.sciencelearn.org.nz/resources/2032-bacterial-transformation
8. https://www.ncbi.nlm.nih.gov/books/NBK21993/
9. https://www.nature.com/subjects/bacterial-transformation
10. http://jb.asm.org/content/196/8/1471.full.pdf
11. https://www.jove.com/science-education/5059/bacterial-transformation-the-heat-shock-method
12. https://www.thermofisher.com/np/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-workflow.html
13. http://microbeonline.com/bacterial-transformation-mechanism/
14. http://study.com/academy/lesson/bacterial-transformation-definition-steps-analysis.html

Bacterial Transformation: Competence in gram positive and gram negative bacteria

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