Updated:12/16/99 Updated:12/16/99
MICROBIOLOGY | GENETICS | MEDICAL
Bacteria can exchange or transfer DNA between other bacteria in three different ways. In every case the source cells of the DNA are called the DONORS and the cells that receive the DNA are called the RECIPIENTS. In each case the donor DNA is incorporated into the recipients cell's DNA by recombination exchange (Fig. 1). If the exchange involves an allele of the recipient's gene, the recipient's genome and phenotype will have changed. The three forms of bacterial DNA exchange are (1) TRANSFORMATION, (2) CONJUGATION and (3) TRANSDUCTION.
Before DNA exchange can be discussed it is necessary to understand what PLASMIDS are. Plasmids are best thought of as
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MINI-CHROMOSOMES. Plasmids are composed of DNA which usually exists as a CIRCULAR MOLECULE, only much SMALLER than the genomic DNA (Fig. 2). Click here for a discussion of plasmid vectors and their component parts.
Plasmids vary in size, but most are between 1,000 to 25,000 base pairs vs. 4,000,000 bp in the genome. Plasmids REPLICATE AUTONOMOUSLY from the genomic chromosome. Often there are MANY PLASMID COPIES present in one cell. Further, a cell may contain SEVERAL DIFFERENT PLASMIDS or it may contain NO PLASMIDS at all. Plasmids generally carry genes that are NOT
ESSENTIAL for a cell's survival except under special circumstances. For example, many plasmids carry genes for ANTIBIOTIC RESISTANCE. When these plasmids are present in a cell, it is unaffected by the appropriate antibiotic, but if the plasmid and its antibiotic resistant gene is lost, the host cell becomes sensitive to a given antibiotic. Some plasmids carry resistance genes to several antibiotics, making them very dangerous pathogens. In other cases plasmids, called VIRULENCE-PLASMIDS, carry #VIRULENCE GENES that enhance a host's ability to cause a disease. That is, a bacterium carrying a plasmid containing the virulence gene is able to CAUSE A DISEASE, but when the plasmid is missing that same bacterium is unable to produce that disease. One such plasmid-based disease of recent concern is the strain of E. coli #O157:H7 that produces a severe food-borne disease. We will learn more about this organism in the section on FOOD MICROBIOLOGY. Other plasmids carry genes for protecting a cell against DELETERIOUS substances like mercury, copper or they may carry genes that make it possible for a cell to metabolize an UNUSUAL SUBSTRATE, such as gasoline, as a nutrient or energy source.
The question naturally arises as to the PURPOSE of these plasmids in the evolutionary scheme. The current explanation is that plasmids constitute an EXTRA POOL OF GENE ALLELES and thus enlarge the effective gene pool of the population. Remember that the genome of prokaryotes carries only enough information for between 1,000 to 5,000 genes. But, as we've already learned, the more variety the better a species' chances of #survival are in a fickle universe. The phenomenon of ANTIBIOTIC
This would be like someone afraid of being robbed carrying around an AK-47, a rocket launcher and a small cannon; they might be safe from thieves, but all that bulk and weight is going to seriously interfere with their everyday lives--like getting dates. |
RESISTANCE is a case in point. Antibiotics, being natural products of certain organisms, are never-the-less unlikely to be encountered very often in quantities that endanger susceptible sensitive strains, so there is no need to carry resistance genes against the hundreds of antibiotics that lurk in the nooks 'n crannies of the environment. Indeed, to do so would likely tie up all your genes just for this one purpose; clearly not a survival plus.
However, random mutation has produced antibiotic resistance genes that clearly can prove useful under the RIGHT CIRCUMSTANCES, but how do they remain available, without tying up huge quantities of LIMITED RESOURCES? The answer is PLASMIDS, of course (bet you saw that coming didn't you?). A RARE PLASMID, randomly carrying a RARE ANTIBIOTIC RESISTANT GENE to, for example, penicillin, happens to be in a patient suffering from an infection (e. g. #clap) which is treated by a shot in the you-bloody-well-know-where. All the resistant bacteria's mates, lacking the resistance plasmid, are quickly killed, but the lucky bacterium with its penicillin-resistant-plasmid survives and reproduces while swimming in a sea of penicillin. Naturally, all the subsequent daughter cells carry the resistance plasmid, because if they didn't they'd die very quickly. This is a classical example of SURVIVAL OF THE FITTEST & of #evolution in action.
In the modern world we produce huge quantities of antibiotics, so the selective pressure on bacteria containing plasmids carrying antibiotic resistant genes is intense, particularly in places like hospitals. As a consequence of this evolutionary process, current antibiotics are losing their effectiveness. To compound the problem, most of the plasmids carrying the antibiotic resistant genes have the ability to move from one bacteria to another by conjugation. In effect, a single cell carrying an antibiotic- resistant plasmid can "INFECT" many other cells with this plasmid thereby spreading the resistance plasmid rapidly THROUGHOUT a bacterial population (sort of like us getting a flu shot). The survival logic of this ability is obvious, at least as far as the bacteria are concerned.
Plasmids have one other very significant role to play in this story. They serve as the VEHICLES for carrying genes between cells in the #genetic engineering revolution. You will learn more of this in Chapter X. This site contains tutorials with Quicktime movies.
The discovery of transformation was previous #described. Since its initial discovery transformation has been shown to occur throughout the bacterial world and it has become the most commonly used artificial way of moving genes from one bacterium to another. The basic procedure involves:
Breaking open the donor cells and
removing DNA from them so as to obtain a CELL-FREE, usually purified, form of DNA (NAKED
DNA).
Figure 5. Isolation of CELL-FREE or NAKED DNA. The
cells are broken and the DNA released. The cell-free DNA is subsequently isolated and
collected.
Adding the naked DNA
to recipient cells in a state, called the COMPETENT state, capable of binding the DNA.
Figure 6. Mixing of Donor DNA with Recipient Competent
Cells. The naked donor DNA is incubated with the competent recipient cells to
which it binds.
The recipient cells take the donor DNA
into their cytoplasm where it may EXCHANGE into the recipient's DNA or if it is a plasmid, it will replicate.
Figure 7. Uptake and Recombination of Donor DNA.
Donor DNA binds to competent recipient cells, following which it enters the recipient
cells. Portions of the donor DNA align, at random, with genes on the recipient DNA
and segments of the two DNA's are exchanged. The exchange inserts Donor genes into the
recipient cell"s DNA.
Transformation is used to move DNA between bacteria, plants and animals. In each case the methods used to get the DNA into the recipient cells are slightly different. In bacteria COMPETENCY is an empirical matter; that is it can not be predicted what conditions will produce competency in a given strain of bacteria. However, the following treatment often induces competency in G- bacteria:
Young cells are incubated with a CALCIUM CHLORIDE SOLUTION for
approximately 30 min on ice. In some cases magnesium is also present.
The cells are concentrated and suspended
as a thick suspension in the calcium solution. The cells may be mixed with reagents like
glycerol and stored at -80oC for later use or they may be used immediately.
Cell-free DNA is then mixed with these
competent cells on ice for approximately 30 min followed by a brief mild heating.
The transformed cells are incubated in a
rich medium for approximately 1 to 1.5 hr. and then plated on medium containing materials
that will detect the presence of the transformed genes.
A variety of other transformation techniques are used for eukaryotic cells. These include mixing certain salts with DNA. These salts bind the DNA and the salt-DNA-complex is then taken into the eukaryotic cells where the DNA is subsequently incorporated into the recipient cell's DNA. Plant cells are often covered with a thick cell wall that is difficult to penetrate. To get DNA into these cells tiny metal beads coated with the donor DNA are "shot" into the cytoplasm of the recipient cells using a "gas gun". A strong jolt of electricity is also used to drive the DNA into recipient cells. Because of the similar chemical nature of DNA, DNA from any living form can, in theory, function in any other life form. Animals or plants that have been transformed with DNA from other species are called TRANSGENIC organisms. For example, we have transgenic pigs and cows containing functional "human genes". Transgenic plants containing "bacterial genes" that make a protein toxic to certain insect pathogens are currently growing around the world.
Extra Credit
Commentary 9A:
INSTRUCTOR'S CRITICAL THINKING QUESTIONS ON 9A: |
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The discovery of CONJUGATION, the ability of bacterial cells to transfer DNA between cells that are in physical contact, as a form of DNA exchange between bacteria in the 1950s stunned scientists and lay people alike. Its obvious anthropomorphic similarly to mammalian gene exchange amused some and shocked others. Since its discovery, conjugational exchange of DNA has been shown to be more common and promiscuous than first thought possible. Initially, conjugation was thought to occur only between the SAME or CLOSELY RELATED SPECIES, but data has accumulated which shows that conjugation between bacteria crosses prokaryotic species lines and even occurs between bacteria and some eukaryotic cells. How pervasive this latter situation is remains to be determined.
The basic conditions for conjugation are:
Donor
cells carry a unique plasmid that contain a set of genes that make conjugation possible.
These PLASMIDS are called FERTILITY or SEX PLASMIDS. Cells that
contain the fertility plasmids are called F+ or MALE cells, whereas cells, lacking
the sex plasmids are said to be FEMALE or F-.
The sex plasmid genes are responsible
for the synthesis of special pili called #SEX PILI. Sex pili
are thin long, hollow protein tubes that have "sticky" RECEPTORS on their ends that bind
firmly to molecules (ligands) on recipient cell walls.
Following the ATTACHMENT of the two cells by the
pili, they become united through a "CONJUGATION
BRIDGE". The nature of this union is unclear as is the
exact role the sex pili plays in its formation. But what is clear is that a GATE IS OPENED
between the donor and recipient cells through which DNA can pass.
A special enzyme CUTS one strand of the donor's DNA
at a unique site and a newly synthesized stand of DNA passes through the conjugation
bridge INTO THE RECIPIENT CELL.
This newly synthesized strand of
donor DNA is converted to a DOUBLE STRANDED form which is able to EXCHANGE into the host's DNA by #recombination.
The most common form of conjugation involves the TRANSFER OF PLASMIDS from one cell to another. This process is very efficient, as the recipient cells that receive the F-plasmids, themselves BECOME F+ (they literally change SEX) and quickly begin to mate with F- cells. Certain F+ plasmids are able to FUSE WITH THE GENOMIC DNA of the host cell and upon mating the ENTIRE donor's genome can be transferred into a recipient cell. Under these conditions any of the donor's genes can end up exchanged into the recipient's genome.
FAQ: "Why doesn't every bacterial cell in the universe have all the possible plasmids, if the darn procedure is so bloody efficient?"
ANSWER: Again, the principle of #SURVIVAL EFFICIENCY applies. In a competitive world, it is a losing proposition if a cell wastes precious energy carrying around EXTRA BAGGAGE. Therefore, it makes SURVIVAL sense to bacteria to "DUMP" any EXCESS plasmids as soon as they can. Consider the decisions that one makes in loading a backpack for a trip to the mountains; every item that is not absolutely essential to survival is JETTISONED to make the pack as light as possible. Remember the Indiana Jones movie, Temple of Doom, where they tossed everything out of the airplane to stay up longer? Well that's analogous to bacteria carrying extra plasmids around when they're not absolutely required.
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The conjugative transfer of antibiotic resistant plasmids between bacteria is a major problem facing the medical profession today (that means you and me folks!). In the case of some important pathogens we currently have only ONE EFFECTIVE ANTIBIOTIC (Vancomycin) remaining to use against them and resistant strains against that one are being reported around the world. That is, these SUPERBACTERIA contain plasmids carrying resistant genes to ALL of the other available antibiotics. One of the major foci of transfer of antibiotic resistant plasmids is in hospitals (have I got the attention of the ?). This is a further example of evolution-in-action. Since people infected with pathogens are CONCENTRATED in hospitals and since antibiotics of all kinds are extensively used to treat these infections, the chances are high that antibiotic resistant plasmids will be selected and that they will be passed on to other bacteria. Further, the odds that a bacterium receiving a resistance plasmid will already have other antibiotic resistant plasmids will be statistically high. Other activities like the feeding of antibiotics to farm animals to increase their weight gain, or the rinsing of chickens in antibiotics to increase their shelf-life in stores contribute to the selection and subsequent spread of antibiotic resistant plasmids. The increase of resistant bacteria over time is shown in the figure 5 below.
The third way of transporting DNA between organisms involves the mediation of viruses. As you will learn when #viruses are covered, bacteria also have viruses that attack them. Bacterial viruses are called BACTERIOPHAGE or just plain PHAGE by most microbiologists. The details of phage morphology, life cycle and biochemistry are discussed in Chapter 11.
Two scientists, F. Twort and F. d'Herrelle discovered bacteriophage independently in 1915 & 1917. The term "phage" means "eater". Phage show themselves in two ways. On agar plates holes lacking any bacteria can be seen on a bacterial "lawn"(Fig. 6). These holes, called PLAQUES, vary in size from 1 to 3 mm in diameter, depending on the phage and the growth conditions, but under standard conditions the PLAQUE SIZE is a DEFINING CHARACTERISTIC of each phage.
In liquid cultures the addition of phage often causes the bacteria to DISAPPEAR or "CLEAR" within a few minutes and appear as if it was a sterile, tube of medium. The killing of bacteria suggested to many that they might be used to treat bacterial infections, however this turned out to be ineffective because of the immune response of the body to the phage--more about this in the section on IMMUNITY. However, during the early attempts to use phage therapeutically, it was discovered that they are VERY SPECIFIC as to the bacterial hosts they will attack and kill. In fact they are so specific, that phage are used to IDENTIFY strains of bacteria that cause infections so that the source of the infection can be accurately traced.
The ability of phage to carry bacterial DNA between bacteria was discovered in 1952. Briefly scientists, who were studying conjugation found that if they separated two genetically unique bacteria by a membrane that PREVENTED CONTACT between them they could still detect GENE TRANSFER between them. As this transfer was not prevented by DNase, it was not #TRANSFORMATION. They reasoned that the DNA must be protected from the DNase by something. This "something" turned out to a bacteriophage infecting one of the strains being used in the experiment. Phage are composed of genetic material surrounded by a protein cover that does indeed protect them from DNase. This process of phage-mediated DNA transfer is known as TRANSDUCTION.
Transduction works as follows:
For example, if transducing phage are first grown on a bacterial host that carries a gene CONFERRING RESISTANCE TO THE ANTIBIOTIC PENICILLIN, a few rare DEFECTIVE phage will INCORPORATE the penicillin resistance gene. If the phage from this culture are subsequently used to infect a PENICILLIN-SENSITIVE host and the survivors are plated on medium CONTAINING PENICILLIN, only those rare bacteria which have BOTH received the penicillin-resistant gene from a defective phage and EXCHANGED it into their genome, will produce colonies on the medium.
FAQ: "If the event is so rare, how do we ever see it occurring?".
ANSWER: The answer lies in the prodigious numbers of phage and bacteria one is able to work with. A phage stock may contain 1010 phage per ml and a bacterial culture 2 x 109 per ml, so a 1 in even 108 event becomes very REASONABLE .
Since phage are easy to grow and once obtained a stock of phage can be used for a long time, transduction is an efficient way to pull out all sorts of genes from bacteria for investigation.
Copyright © Dr. R. E. Hurlbert, 1999.
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