Gene Transfer

Whats horizontal gene transfer?

• Movement of genes between cells that aren’t direct descendants of another as opposed to vertical transmission (mother to daughter cell)

• Allows cell to quickly acquire new characteristics and fuels metabolic diversity → aids in evolution and adaptability of a bacterium

• Useful molecular tools in bacterial genetics

What are the 3 mechanisms of genetic exchange known in bacteria?

1. Transformation

2. Transduction

3. Conjugation

How many directions does DNA transfer occur in?

• Typically occurs in one direction from donor to recipient

Whats transformation?

• Free DNA released from one cell is taken up by another

• There are 2 versions of transformation

  • Natural

  • Artificial / chemical

Whats transduction?

• DNA transfer mediated by a virus/ a phage

  • Bacterial DNA is mistakenly packaged into a phage capsid (instead of viral DNA)

  • Defective phage then released to infect another cell injecting bacterial DNA

Whats conjugation?

• DNA transfer requires cell-to-cell contact and a conjugation plasmid in the donor cell

• Donor DNA is transferred through a piles

Who did the discovery of transformation and why was it important?

• Griffith did in 1928 with his experiment using Streptococcus pneumoniae

  • Certain strains produced a capsule

• It was important because it provided key evidence that DNA was genetic material

What’re capsules and explain their qualities?

• Capsule is composed of sugar units linked to form a polysaccharide material

• Different forms of S. Pneumoniae affect virulence (I.e. ability to cause disease)

  • Capsule allows bacteria to evade attack by immune system

  • Bacteria gets destroyed by immune system if no capsule

• Chemical composition of capsules can vary slightly between different smooth strains of S. Pneumoniae

  • Ex: type II different from type III depending on type of sugar in capsule

• Can have reversion between smooth (S) and rough (R) but only within a type

  • Ex: type IIS ←> type IIR but not type IIS ←> IIIS

Explain what occurred in Griffiths experiment

• He used type IIR cells and type IS cells

• R killed mice, S didn’t kill mice, R transformed to S killed mice

• IIR cells were transformed into IS cells

• Later experiments showed that:

  • Living S cells could be produced in liquid culture of a mixture of R and heat-killed S cells (I.e. no host required)

  • Transforming activity was retained in filtered extract of dead S cells (I.e small molecule)

What did Griffith hypothesize?

• Griffith theorized that specific protein structure was responsible for transformation

What did Avery, McCloud and McCarty (1944) do?

• Proved that DNA was the agent behind transforming activity

• Reproduced Griffith’s experiment, cell extract was treated with protease, DNAse and RNAse

• Proved that transforming principle was DNA

• DNAse = doesn’t kill mouse, everything else kills mouse

What is natural transformation?

• Capacity to take up free DNA is genetically determined (e.g. Vibrio, Neisseria, Bacillus, Streptococcus)

• When a cell lyses, chromosomal DNA leak out of the cell and due to its large size, fragments (~10-15 kbp)

What does natural transformation require?

1. DNA from donor cells must be fragmented linear dsDNA

   • Does not work with ssDNA, only has to be linear dsDNA

2. Recipient cells must be competent and take up the dsDNA fragments

   • Should be able to make competence factors:

   • Excreted from cell - reaches effective concentration at high cell density

   • Binds to surface receptors - induces synthesis of other proteins involved in the transformation

   • Occurs during late log phase

Explain how competence is directly linked to pili?

• Pili binds and facilitates uptake of DNA into cell by retraction

• Mechanisms exist to account for differences in cell envelope structure for gram negative and gram positive bacteria

What’re the steps of transformation?

1. Transformation complex binds exogenous (donor) dsDNA

2. Uptake of DNA. As dsDNA is brought into the transformation complex, one strand is degraded by nucleases and ssDNA enters into the cell. Need dsDNA to function at all, as it enters the cell, only ssDNA enters cell

3. Homologous recombination

   • The ssDNA fragment will be paired with a homologous region of the chromosome

   • Pairing is mediated by RecA

   • Strand exchange will occur

4. Transformed cell

   • Donor DNA is integrated into chromosome of recipient cell

   • Successful recombination usually by a change in phenotype

   • Ex: donor DNA from Trp+ cells taken up by competent Trp- cells

   • dsDNA converted into ssDNA, recombination, change genotype and hence phenotype

Whats something that happens in natural transformation?

• ssDNA is bound by SSB

• RecA displaces SSB and promotes strand invasion of chromosomal DNA

• Double cross-over event with a ssDNA fragment and chromosomal DNA

• Recombination with heteroduplex region resolved by either DNA repair or replication

What happens in the homologous recombination model?

• In E.coli, the RecBCD pathway will mediate homologous recombination between dsDNA linear fragment and chromosomal DNA

  • RecBCD is a complex that has endonuclease and helicase activity

  • Displaced ssDNA is coated with SSB

  • RecA will displace the SSB protein binding the ssDNA region

  • RecA will mediate strand invasion of the double stranded chromosomal DNA

  • The cross-strand complex is not static; complex will move until strand homology is found (region of 100s to 1000s of bases)

  • Cross-strand complex will then be cleaved on a horizontal or vertical plane - a random event done by a resolvable enzyme

What does resolution of cross strand complex result in?

Two products

1. Patches - no recombination (no exchange of markers) with short heteroduplex regions

2. Splices - recombination has occurred (exchange of markers) with short heteroduplex regions

Heteroduplex regions are mismatched regions which resolved by DNA repair or by DNA replication (2 molecules each with slightly different sequence)

Are all bacteria transformable?

• No, E.coli isn’t naturally transformable

Does the process of transformation differ from groups of bacteria, if so how?

• Yes

• Some bacteria have specific sequences within the DNA to recognize “self” → protective mechanism against incorporation of foreign DNA into its chromosome compromising genetic integrity

  • Ex: Haemophilus influenza

    • Doesn’t require competence factor

    • Receptors on cell surface to recognize a specific 11-bop DNA sequence (occurs ~600 times within chromosome)

    • Only linear dsDNA with this sequence will be taken up by cell

What happens to bacteria that’s not naturally transformable?

They can be made transformable artificially by chemical treatment or exposure to electric current

• Done to uptake plasmids (circular dsDNA)

• Plasmids will not recombine with the chromosomes unless specifically designed to do so

• Plasmids will replicate independently and be passed onto progeny cells

Linear dsDNA fragments introduced through the pores will be degraded by host defense systems that protect against incorporation of foreign DNA

Why is transduction known as a universal mode of gene transfer?

• Because its theoretically possible for all bacteria as long as there’s a compatible phage

Whats a transducing particle?

• Host-derived DNA carried from one cell to another inside a phage capsid

• Phage that has picked up bacterial DNA (carry 1 -2 % of bacterial genome; 1 fragment is ~35-80 genes)

What are the 2 types of transduction?

1. Generalized transduction

2. Specialized transduction

What happens in generalized transduction?

• Only bacterial DNA is carried

• Any gene can be transferred from the donor cell

What happens in specialized transduction?

• Some bacterial DNA and some phage DNA is carried

• Only specific genes can be transferred from the donor

What happens when a transducing particle infects a recipient cell?

• Only steps 1 and 2 are followed as this is mediated by capsid proteins

• Lytic or lytic/ lysogenic cycles aren’t followed due to lack of phage genes

Whats a transducing phage?

• Phages capable of transduction (not all phages can do this)

Whats a donor strain?

• Original bacteria strain in which transducing particle had multiplied and picked up host bacterial DNA

Whats a transductant?

• A cell that has been transduced (a.ka. Recipient strain)

What must a phage be to be a generalized temperate phage?

• Can be temperate or lytic

• Must not completely degrade host DNA because there will be no DNA to package into capsid if it degrades

  • T4 phage is not a good choice (obliterates host DNA)

• Need to have not too specific pac or cos sites for packaging of DNA otherwise host DNA will not be packaged

• Also broad host range of adsorption for possibility on wide range of bacterial species

What’re the 3 possible fates for injected DNA in generalized transduction?

1. Host restriction - degraded by host enzyme system (i.e restriction enzymes)

2. Abortive transduction - not degraded, but fails to recombine

   • Diluted out of population by successive cell divisions

3. Stable gene transfer - recombined into the chromosome by:

   • Pairing of exogenous dsDNA with homologous region in chromosome

   • Unlike transformation, both strands are replaced by homologous reciprocal recombination

What’re the steps of generalized transduction using E.coli?

Trp gene as marker, Donor strain is Trp+ and recipient strain is Trp- (can’t synthesize tryptophan so autotrophic for tryptophan)

1. Infect donor strain (Trp+) with P1 linear dsDNA phage under conditions that will promote lysogeny

2. Expose lysogeni culture to UV light to induce lytic cycle

   • P1 reproduces

   • Host cell lyse, releasing progeny and lysate which is just the debris present after lysis

   • Most are normal phages but some are transducing particles with ~1 in 10000000 carrying the Trp+ gene

3. Briefly expose lysate to UV light

   • Inactivates normal P1 phages by reducing its ability to reproduce but has little effect on the transducing particle

4. Mix phage lysate with recipient (Trp-) strain at MOI slightly less than 1

   • Each cell should be infected by no more than 1 phage or particle

   • Reduce the likelihood of a cell infected by both a transducing particle and a normal phage

5. Selecting transducants (cells which have been transduced)

   • Plate infection mixture on a selective medium → minimal medium without tryptophan

   • Trp- recipient cells fails to grow

   • Only cells that recieved Trp+ will grow (transducants)

What is the frequency of transduction?

• Ratio of successful transductants/ phage infected cells

Whats the frequency of transduction?

What causes pinpoint colonies on the same plate?

• Abortive transduction

• Ex: Trp+ gene has been transferred, but fails to recombine

  • When the merodiploid (2 copies of gene Trp+ and Trp-) divides only one cell receives Trp+

  • Extra chromosomal dsDNA fragment eventually degraded

What is co-transduction frequency?

• Transfer of 2 markers on same fragment of transducing DNA

• Frequency depends on distance between 2 genes

  • Closer together - more likely they will be co-transduced

• Historical method of mapping genes

Whats an example of co-transduction frequency?

• Donor is bio+ and gal- and recipient is bio- and gal+

• Perform transduction experiment

• Select only for bio+ transductants → 100 bio+ colonies are counted of which each may be Gal+ or Gal-

  • Bio is the selected marker, gal is unselected marker

What is specialized transduction?

• Transfer of specific genes

• Uses temperate phage that have specific insertion sites in the host chromosome

  • Only genes close to the prophage insertion sites are transduced

  • Due to aberrant (incorrect) excision of the prophage

In specialized transduction what happens when lysogenized E.coli is exposed to UV?

• Expose lysogenized E.coli briefly to UV → induction → collect lysate

• Phage progeny are mostly normal lambda phage

• Specialized transducing particles arise at low frequency (~1 in 10^6 phages) due to aberrant excision

• Recombination doesn’t occur at normal recombination sites

What does an excised prophage include?

• Some bacterial genes, but excludes some phage genes; limited by phage head sizes

• Cell still contains all phage genes - phage reproduction can complete

What is specialized transduction dependent on?

• Dependent on which side cross-over happens

  • Gain gal genes but loses head/ tail genes

    • Can’t complete virion assembly

    • Follows lysogenic pathway

    Or

  • Gain bio genes but loses xis and int

    • Can be assembled and released

    • Can’t become lysogenic

Whats a gene transfer agents?

• They resemble tiny tailed bacteriophages and contain random small pieces of host DNA

• They are NOT considered true bacteriophages as they don’t contain genes encoding their own production and do not produce characteristic viral plaques

• Genes encoding GTA’s lie within the genome of the cell that produces them, while other regions of the genome are packed within the agents

• Common amongst marine bacteria

• Seems to have evolved as a mechanism for a subpopulation of cells to sacrifice themselves in order to dispose genes in a protected manner

What is conjugation?

• Form of horizontal gene transfer that requires cell-to-cell contact

• Plasmid-encoded conjugation mechanism that can mediate DNA transfer between unrelated cells, even between different genera

  • Transfer copies of themselves and the genes they encode (eg: antibiotic resistance) to new host cells

  • Conjugation plasmid

What does conjugation require?

• Process requires a donor cell (containing the conjugative plasmid) and the recipient cell (doesn’t contain conjugative plasmid)

• Transfer mechanisms may differ depending on the participating plasmid, but most plasmid in gram-negative bacteria employ similar mechanism

• Conjugation has also been demonstrated in a few gram-positive bacteria (streptococcus)

When was conjugation discovered?

• Initially discovered by Lederberg and Tatum (1946)

• Mixed 2 different auxotrophic strains, incubated for several hours and plated on minimal medium

• Growth of several prototrophic colonies

• Genetic exchange occurred

  • Due to Hfr strains

What did Davis discover in 1950?

• Determined that physical contact is necessary for genetic exchange

  • Separated 2 auxotrophic strains by a fine filter which caused genetic exchange to not take place

Which bacteria can conjugate and which ones can act as donors?

• Many but not all bacterial species can conjugate

• Only certain bacterial strains can act as donors

What is the F factor?

Conversion of non donor strains to donor strains

F- to F+

What does F plasmid encode?

It encodes a pious

• F pious of the donor (F+) makes contact with specific receptors on cell surface of F-

  • Pious contracts bringing the cells together

  • Forms a conjugation bridge

What is the F plasmid?

• Fertility plasmid, self replicating circular dsDNA

• ~100 kbp long

• Primary function is to self transfer to F- cells

• F- cells don’t have plasmid, F+ transfers plasmid to F- cells to make them all F+ cells, happens in ~60 min

What are the features of the F plasmid?

• tRNA region: ~21 genes - encode proteins for F-pious and transfer of plasmid

• OriV: origin of replication

• Or IT: origin of transfer

• Insertion sequence elements IS2 and IS3, and transposing Tn1000

  • ~1kbp in size

  • Also present on bacterial chromosome

Whats the conjugation mechanism for the F pilus?

1. Pilus is assembled and establishes cell contact

2. One strand of the F plasmid is nicked at orbit

   • One strand becomes linear with 5’ and 3’ ends

3. The 5’ end is displaced and enters conjugation bridge

   • Transferred to F- as ssDNA

4. The 3’ end is extended using intact circular strand as template

   • Rolling circle replication

   • After 1 complete replication, linear strand is cut

5. Inside the recipient cell: ssDNA is replicated to make dsDNA

   • Replication proceeds 5’ to 3’

   • Okazaki fragments

   • dsDNA is formed

   • Double stranded ends joined to make circular F-plasmid

When does the Hfr strain occurs?

• Occurs when F plasmid can integrate into the host chromosome, this is called an episome

• Episome = plasmid that can integrate itself into host chromosome

• IS2, IS3 and Tn1000 can act as integration sites

• F+ cell is converted into Hfr (high frequency of recombination) cell

• Can be isolated as pure culture

• F plasmid can mediate transfer of chromosomal genes

• F factor genes are expressed producing F pili therefore able to conjugate with F- cells

What happens when conjugation begins in Hfr strain?

• Transfer begins at orbit

• Located in the middle of the F factor

• Only about half of the F factor is transferred

• Followed by chromosomal genes

• To transfer the 2nd half of the F factor, the entire chromosome of the donor would need to be transferred to recipient

How long does the complete transfer of Hfr strain take?

• Takes ~100 minutes

• Very rarely happens as conjugation bridge must remain in place

• Thus, in an Hfr x F- cross, the recipient cell always remains F-

Whats the mechanism of transfer in Hfr strain?

1. One strand is nicked at oriT

   • oriT is at center of F plasmid

   • F factor genes constitute both the 3’ and 5’ ends

2. 5’ end of the linear strand is transferred to the recipient

   • 3’ end serves as primer for DNA synthesis to replace transferred strand

3. Transferred as ssDNA and them converted to dsDNA in recipient

   • Half of F factor

   • Followed by chromosomal genes

4. But conjugation bridge often breaks which results in a fragment of transferred DNA

5. To become a stable genetic element, it must be recombined into a chromosome

How were Hfr strains used to map the E.coli chromosome?

There are several possible insertion sites on E. Coli chromosome

• Location of the F factor is different for each Hfr strain

• Can be located on either strand

  • Transferred in either direction

• Each Hfr strain can be used to map about 25-40% of the chromosome by controlling the breaking of the conjugation pilus at timed intervals

LOOK AT TIME MAPPING SLIDES

What happens when overlapping gene orders are placed together?

• Overlapping gene orders can be placed together to form a composite map

  • Distance on map given in units of time (ie. min)

  • Total length of E. Colin’s chromosome is 100 min

  • If chromosome is 4500 kbp long (so each min = 45 kbp)

How many cells in a Hfr strain will be F+?

• A very small number of cells in any Hfr strain will be F+ (complete transfer is rare), but excision of F (factor) plasmid can occur

Can F factor be excised incorrectly, if so how?

Yes

• Some chromosomal genes are included on the plasmid

• Some F plasmid genes are left behind

  • These cells are known as primary F’ donors

  • Can transfer specific genes at a very high frequency

How does gene transfer by F’ donors occur?

• Primary F’ donors can form a pilus

• All F factor genes are present

  • F’ x F- conjugation

Whats the result of transfer by F’ donors?

• Plasmid (and any chromosomal genes on it) will be transferred at very high frequency

• Recipient gains the entire plasmid and becomes a secondary F’ cell

  • Stable partial diploid or merodiploid (2 copies of a gene)

  • Can be used for complementation tests to see if strain with deletion has impact on phenotype

What are F’ factors containing bits of lac operon used for?

• They were used to derive function of lac promoter, operator and genes

• Make chromosomal mutations in lac operon, and complement function by conjugating F’ factor containing normal copies of lac genetic elements into mutant strains

• Derived gene function by rescued phenotype

This was how the lac operon was studied in the 1950s and 1960s

Where is mobile DNA: transposable elements found?

• Commonly found in chromosomes, plasmids, and viral genomes

What are mobile DNA: transposable elements?

Pieces of DNA that can move from place to place in the genome

• Move by a process called transposition

• Frequency ranges from 1 in 10³ to 1 in 10^7 pre element/ per cell generation

• Important both in natural genome rearrangement and in genetic analysis

What are the qualities of mobile DNA: transposable elements?

• Do not require homology with the destination site

• Do not possess their own origin of replication

  • Replicated when the host DNA molecules into which they are inserted is replicated

What are the 2 types of transposable elements in bacteria?

1. Insertion sequences (IS/ transpons)

2. Composite transpons

Explain what Insertion sequences (IS/ transpons) are

• Short DNA segments of ~1000 nucleotides long

• Typically contain inverted repeats of 10-50 base pairs

• Encode only 1 protein → transposable (helps it move from one place to another)

Explain what composite transposons are

• 2 IS elements flanking antibiotic genes of other genes providing beneficial attributes

• Has additional genes encoded within between IS elements (eg: antibiotic resistance)

• Often the transposable in one of the IS elements is dysfunctional to allow movement of the whole as a unit

Explain the movement of IS elements and composite transposons

• Transposase recognizes inverted repeats and moves transposable elements from one site to another

What are the 2 mechanisms of movement of IS elements and composite transposons?

1. Conservative transposition

2. Replicative transposition

Explain conservative transposition

• Example: Tn5

• Transposase recognizes inverted repeats and cuts on either side

  • Transposon is removed from original DNA molecule

  • Carried to a target site 5-9 bp sequence randomly selected

  • Cuts the target DNA by making staggered nicks

  • Inserts the transposon

  • Staggered cut results in gaps on either side

  • Gaps are filled by DNA pol 1 and DNA ligase

• Results in direct repeats flanking the transposon

Is direct repeats a part of the transposon?

• NO, direct repeats is not a part of a transposon but a result of the transposition event

What is the idea behind replicative transposition?

• Transposon that carries an additional gene encoding enzyme resolvable

• Transposon is replicated as it jumps, so that after transposition, both donor and target DNA molecule have a copy of the transposon

What are the steps of replicative transposition?

Example in Tn3

• Transposase cuts staggered nicks on either side of transposon

• Also cuts staggered nicks on either side of target site

• Strands are exchanged and lighted

  • 2 DNA molecules joined together With gaps corresponding to the target site and the transposon

• The gaps are filled in by DNA polymerase and sealed ligase

• Resolvable catalyzes recombination

  • Resolved into 2 separate DNA molecules, each with a copy of the transposon