Last revised: Monday, March 31, 2003
Reading: Ch. 15 in text
Significance of Gene transfer
- What is Horizontal gene transfer?
- What is Vertical gene transfer?
- Evolutionary Significance of Horizontal Gene Transfer
- Results of small ribosomal RNA suggest a single phylogenetic tree. See tree based on 16S RNA phylogeny alone. ("protected" image.) Why is this protected?
- But, when different genes are used to create the same tree, position of some organims seems to move. See tree including other gene markers to establish phylogeny. ("protected" image.) Why is this protected?
- Explanation: over evolutionary time, different genes have been transferred across different groups, even across all 3 domains.
- Can an accurate phylogeny be constructed? Considerable debate, but molecular evolution is still a young field.
- Medical Significance of Horizontal Gene Transfer
- Within the past 50 years, as antibiotics have been introduced widely, have seen considerable evidence of horizontal gene transfer, both within species and across species.
- Example: toxic E. coli strain 0157:H7 was not known until recent decades, appears to have arisen by horizontal gene transfer of toxin genes from Shigella (another enteric bacterium) to E. coli. See gene transfers that have increased virulence. ("protected" image.) Why is this protected?
View diagram of different mechanisms for HGT
Transformation
- Natural transformation = uptake of DNA fragments from medium surrounding cell. Requires specific proteins in cell membrane, energy.
- Only found naturally in certain cells; e.g. Pneumococcus, Hemophilus, etc. Not found in E. coli.
- Why did this evolve? Maybe as way to scrounge DNA from dead cells, save trouble of having to make nucleotides from scratch.
- Cells that can take up DNA are said to be competent. Requires induction of several genes. Typically occurs during exponential growth, shuts off in stationary phase.
- Artificial transformation = laboratory technique to enhance DNA uptake of cells that do not have genetic machinery for transformation.
- Example: treat E. coli with high Ca++ concentrations, then chill. DNA uptake now occurs (though not as well as with naturally competent bacteria).
- Electroporation: pulsed electric fields produce short-lived membrane pores, allows DNA movement (either in or out of cell). Useful way to move small pieces of DNA and plasmids between cells.
- See natural transformation and artificial transformation ("protected" image.) Why is this protected?
- DNA bacterial viruses = bacteriophages normally replicate in cell, produce many copies of phage DNA, many
copies of phage head coat proteins, finally assemble phage heads by packing
phage DNA into new phage heads. Attach tail (if present), open cell, release
progeny.
View animation of phage infection -
Host DNA often degraded. But occasionally, piece of partially degraded
bacterial DNA is correct size to be packed inside phage coat proteins, phage
erroneously packs up a "mistake" = transducing phage.
- This "mistake" phage can't cause infection; but it can be transferred to a different bacterium, get DNA into cell without risk of being degraded in environment
- Even if this occurs, chances are slim that successful expression of DNA will occur --- still needs to undergo recombination. If most cells are killed by phage, not much use.
- E. coli phage P1 makes this DNA packing mistake about 1 in every 1000 phage particles. Astonishingly high error rate (relative to other phages). P1 can be used as very efficient way to move small pieces of DNA from one bacterium (donor) to another (recipient). Maximum size of DNA that can fit in phage head is only about 2% of bacterial chromosome.
- See diagram of transduction ("protected" image.) Why is this protected?
Properties of Plasmids
- Circular DNA elements, always double-stranded DNA, Supercoiled
-
Can occur in as few as 1 copy per cell (single copy plasmids) to as many as
several dozen (multicopy plasmids).
- Variable sizes; small plasmids about 0.1% size of host chromosome, large plasmids can be as much as 10% the size of host chromosome. Smaller plasmids have few genes (30 or less). Size ranges from 1000 bp (1 kbp) to 1000 kbp.
- Ubiquitous; almost all cells isolated in nature carry plasmids, often more than one kind. (In E. coli alone, more than 300 different plasmids isolated.)
- View EM of plasmid DNA
- Have a replicon (origin for DNA replication), number of copies per cell regulated. Large plasmids typically only 1-5 copies/cell (stringent control); small plasmids ~10-50 copies/cell (relaxed control)
- Many plasmids are incompatible; if one is present, cell cannot support another plasmid of same compatibility group.
- Not essential to cell under all circumstances; can be "cured" by agents that impair DNA replication ----> cured cell lacking plasmid. Can be spontaneously lost over time unless some selection makes plasmid valuable to cell.
- Extend range of environments in which a cell can live (e.g., by degrading antibiotics, or providing enzymes for digestion of novel catabolites).
- Antibiotic resistance genes (enzymes that modify or degrade antibiotics) -- plasmids with these genes are called R factors
- Heavy metal resistance (enzymes that detoxify metals by redox reactions)
- Growth on unusual substrates (enzymes for hydrocarbon degradation, etc.)
- Restriction/modification enzymes (protect DNA, degrade unprotected DNA)
- Bacteriocins (proteins toxic to other bacteria lacking the same plasmid)
-
Toxins (proteins toxic to other organisms; e.g. humans) -- called virulence
plasmids. Some Examples:
- Staph aureus virulence factors: coagulase, hemolysin, enterotoxin, others
- pathogenic E. coli strains: hemolysin, enterotoxin
-
Proteins that mediate plasmid transfer to uninfected strains
Conjugation
- Conjugation = plasmid-directed transfer of DNA from one cell to another.
- Self-transmissible plasmids. Some plasmids encode machinery to attach to other cells lacking plasmid, pull them into contact, create a "mating bridge", and replicate a copy of plasmid DNA into recipient cell.
- See beginning of plasmid transfer and completion of plasmid transfer ("protected" image.) Why is this protected?
- Sex pilus: rigid fiber, sticks out from cell wall. Acts as recognition molecule to locate sensitive cell (plasmid minus). When + and - cell are attached by pilus, cells pulled together. (Grappling hook analogy).
-
Plasmid DNA replicates during transfer; one copy remains in donor cell,
identical copy transferred to recipient.
-
Once recipient receives plasmid, it grows pili, becomes a donor. One plasmid
could potentially infect a whole population, convert all cells to
plasmid-containing cells.
- Mobilizable plasmids. Many plasmids are not conjugative, but can use mating bridges created by conjugative plasmids to transfer their DNA. See mobilized plasmid ("protected" image.) Why is this protected?
- Retrotransfer. In some cases, the donor cell can acquire a plasmid from the recipient, again using the mating bridges created by conjugative plasmids. See plasmid retrotransfer. ("protected" image.) Why is this protected?
- Conjugative transposons. Transposons are segments of DNA that normally reside integrated into a larger DNA molecule, but can sometimes move to a different location. Conjugative transposons have the additional property of forming covalently closed circles resembling plasmids (but not replication). These can be transferred to recipient cells. See Fig. 7.7A and Fig. 7.7B. ("protected" image.) Why is this protected?
- Insertion Sequences (IS) and transposons
- IS are small DNA segments (1-2 Kbp) that can move from one region of DNA to another, typically in random fashion. Carry no known genes except those that are required for transposition. Nomenclature: IS-1, IS-2, etc.
- View diagram of transposable element, showing how IS can form a loop.
- IS encodes an enzyme needed to excise DNA and integrate it elsewhere.
- IS typically have promoters pointing towards their edges. This allows them to activate neighboring genes where they are inserted.
- Transposon. When 2 IS regions flank a piece of chromosomal DNA encoding genes in addition to those needed solely for transposition, the result is a transposon, which can move as a single unit. Note: other names include: jumping genes, cassettes, roving genes. Nomenclature: Tn1, Tn2, etc.
- See comparison of IS and transposon. ("protected" image.) Why is this protected?
- Many examples of transposons causing horizontal gene transfer. See evolution of antibiotic resistance in Klebsiella part 1 and part 2, which illustrates how Klebsiella acquired ampillin resistance in recent past. ("protected" image.) Why is this protected?
- Integrons
- Some transposons contain large blocks of genes that move as a single unit, called integron
- Integrons include gene for an enzyme that allows it to "capture" other genes: integrase gene. See diagram of integron. ("protected" image.) Why is this protected?
- Gene disruptions
- Can use a plasmid with antibiotic resistance to selectively inactivate a gene = a type of very specific mutation. See effect of insertion on gene function.
- Transposon Mutagenesis
- Transposons are movable genetic elements, flanked by insertion sequences.
- When transposon moves, can insert itself within a structural gene.
- Example: Like taking
sequence XYYWWLALL and moving in into coherent phrase; FOURSCORE AND SEVEN
.... Result: FOURSCXYYWWLALLORE AND SEVEN .... this is gibberish, destroys sense of a word. Similarly, inserted DNA will be transcribed, and disrupt the normal protein. - Transposon mutagenesis is much more precise than chemical or radiation mutagenesis. Can get a single insertion (rather than a cluster of mutants as in chemical mutagenesis); can actually find site of mutation on gels after digesting DNA with restriction enzymes. Can physically locate the site of the mutation by using DNA probes that bind to sequences within the transposon.
- See selection of transposon-generated mutants. ("protected" image.) Why is this protected?