Steps Of Cell Cycle

Strain 2 of E. coli (met+ bio+ thr - leu-) requires threonine and leucine for growth

| Growth occurs on minimal medium + threonine and leucine.

Complete medium (many colonies grow).

Complete medium (many colonies grow).

There is no growth on minimal medium.

There is no growth on minimal medium.

|On minimal medium with no supplements, a few colonies of prototrophic bacteria (met+ bio+ thr+ leu+) grow.

Conclusion: The prototrophic colonies growing on minimal medium could have arisen only by genetic recombination between the two different strains.

13.7 Lederberg and Tatum's Experiment After growing together, a mixture of complementary auxotrophic strains of E.coli contained a few cells that gave rise to new prototrophic colonies.This experiment proved that genetic recombination takes place in prokaryotes.

at a rate of approximately one for every 10 million cells originally placed on the plates (1/107).

Where did these prototrophic colonies come from? Lederberg and Tatum were able to rule out mutation, and other investigators ruled out transformation (a process we discussed in Chapter 11 and which we'll look at in more detail below). A third possibility is that the two strains of E. coli had exchanged genetic material, producing some cells containing met+ and bio+ alleles from strain 2 and thr+ and leu+ alleles from strain 1 (see Figure 13.7). Later experiments showed that such an exchange, called conjugation, had indeed occurred. One bacterial cell—the recipient—had received DNA from another cell—the donor—that included the two wild-type (+) alleles that were missing in the recipient. Recombination had then created a genotype with four wild-type alleles.

The physical contact required for conjugation can be observed under the electron microscope (Figure 13.8). It is initiated by a thin projection called a sex pilus. Once the sex pili bring the two cells into proximity, the actual transfer of DNA

occurs by a thin cytoplasmic bridge called a conjugation tube. Since the bacterial chromosome is circular, it must be made linear (cut) so that it can pass through the tube. Contact between the cells is brief—certainly not long enough for the entire donor genome to enter the recipient cell. Therefore, the recipient cell usually receives only a portion of the donor DNA.

Sexpili Konjugation Cke
13.8 Bacterial Conjugation Sex pili draw two bacteria into close contact, and a cytoplasmic conjugation tube forms. DNA is transferred from one cell to the other via the conjugation tube.

DNA (from donor chromosome)

DNA (from donor chromosome)

Plasma Cell Membrane

Chromosome of recipient cell

Sites of crossing over

Chromosome of recipient cell

Sites of crossing over

DNA from a donor is incorporated into the recipient cell's chromosome through crossing over.

The reciprocal A~B~C+ segment, not being linked to an origin of replication, is lost.

The part of the donor chromosome containing the A+ and B+ genes is incorporated through recombination. The sequence A+B+C-becomes a permanent part of the recipient genotype.

DNA from a donor is incorporated into the recipient cell's chromosome through crossing over.

Once the donor DNA fragment is inside the recipient cell, it can recombine with the recipient cell's genome. In much the same way that chromosomes pair up, gene for gene, in prophase I of meiosis, the donor DNA can line up beside its homologous genes in the recipient, and crossing over can occur. Enzymes that can cut and rejoin DNA molecules are active in bacteria, so gene(s) from the donor can become integrated into the genome of the recipient, thus changing the recipient's genetic constitution (Figure 13.9), even though only about half the transferred genes become integrated in this way.

The reciprocal A~B~C+ segment, not being linked to an origin of replication, is lost.

The part of the donor chromosome containing the A+ and B+ genes is incorporated through recombination. The sequence A+B+C-becomes a permanent part of the recipient genotype.

13.9 Recombination Following Conjugation DNA from a donor cell can become incorporated into a recipient cell's chromosome through crossing over. This recombination explains the results of the Lederberg-Tatum experiment shown in Figure 13.7.

13.9 Recombination Following Conjugation DNA from a donor cell can become incorporated into a recipient cell's chromosome through crossing over. This recombination explains the results of the Lederberg-Tatum experiment shown in Figure 13.7.

13.10 Transformation and Transduction After a new DNA fragment enters the host cell, recombination can occur. (a) Transforming DNA can leak from dead bacterial cells and be taken up by a living bacterium, which may incorporate the new genes into its chromosome. (b) In transduction, viruses carry DNA fragments from one cell to another.

In transformation, cells pick up genes from their environment

Frederick Griffith obtained the first evidence for the transfer of prokaryotic genes more than 75 years ago when he discovered the transforming principle (see Figure 11.1). We now know the reason for his results: DNA had leaked from dead cells of virulent pneumococci and was taken up as free DNA by living nonvirulent pneumococci, which became virulent as a result. This phenomenon, called transformation, occurs in nature in some species of bacteria when cells die and their DNA leaks out (Figure 13.10a). Once transforming DNA is inside a host cell, an event very similar to recombination occurs, and new genes can be incorporated into the host chromosome.

In transduction, viruses carry genes from one cell to another

When bacteriophage undergo a lytic cycle, they package their DNA in capsids. These capsids generally form before the

(a) Transformation

A lysed bacterium releases DNA fragments

Recombination occurs between the DNA fragment and host chromosome.

A lysed bacterium releases DNA fragments

13.10 Transformation and Transduction After a new DNA fragment enters the host cell, recombination can occur. (a) Transforming DNA can leak from dead bacterial cells and be taken up by a living bacterium, which may incorporate the new genes into its chromosome. (b) In transduction, viruses carry DNA fragments from one cell to another.

Recombination occurs between the DNA fragment and host chromosome.

Bacterial cell

Bacterial chromosome

Chromosome of recipient cell l

Bacterial cell

Bacterial chromosome

Chromosome of recipient cell l

(b) Transduction

| Bacteriophage DNA is injected to begin a lytic cycle

'1 During the lytic cycle, bacterial DNA fragments

(b) Transduction

| Bacteriophage DNA is injected to begin a lytic cycle

'1 During the lytic cycle, bacterial DNA fragments

Lytic Cycle

Phage DNA (prophage)

Bacterial chromosome

Phage coats

Phage DNA (prophage)

Bacterial chromosome

Phage coats viral DNA is inserted into them. Sometimes, bacterial DNA fragments are inserted into the empty phage capsids instead of the phage DNA. (Figure 13.10fr). Recall that the binding of a phage to its host cell and the insertion of phage DNA are carried out by the capsid. So, when a phage capsid carries a piece of bacterial DNA, the latter is injected into the "infected" bacterium. This mechanism of DNA transfer is called transduction. Needless to say, it does not result in a productive viral infection. Instead, the incoming DNA fragment can recombine with the host chromosome, resulting in the replacement of host cell genes with bacterial genes from the incoming phage particle.

Plasmids are extra chromosomes in bacteria

In addition to their main chromosome, many bacteria harbor additional smaller, circular chromosomes. These chromosomes, called plasmids, usually contain at most a few dozen genes, and, importantly, an origin of replication (the sequence where DNA replication starts), which defines them as chromosomes. Usually plasmids replicate at the same time as the main chromosome, but that is not necessarily the case.

Plasmids are not viruses. They do not take over the cell's molecular machinery or make a protein coat to help them move from cell to cell. Instead, they can move between cells during conjugation, thereby adding some new genes to the recipient bacterium (Figure 13.11). Because plasmids exist independently of the main chromosome (the term episomes is sometimes used for them), they do not need to recombine with the main chromosome to add their genes to the recipient cell's genome.

There are several types of plasmids, classified according to the kinds of genes they carry. Some code for catabolic enzymes, others enable conjugation, while others code for genes that circumvent antibiotic attack.

some plasmids carry genes for unusual metabolic functions.

Some plasmids, called metabolic factors, have genes that allow their recipients to carry out unusual metabolic functions. For example, there are many unusual hydrocarbons in oil spills. Some bacteria can actually thrive on these molecules, using them as a carbon source. The genes for the enzymes involved in breaking down the hydrocarbons are carried on plasmids.

A plasmid has an origin of replication and genes for other functions

A plasmid has an origin of replication and genes for other functions

Bacterium with plasmids

Bacterium without plasmids

Bacterium with plasmids

Bacterial chromosome

Bacterium without plasmids

Bacterial chromosome

Plasmid

\ Conjugation tube f

\ Conjugation tube f

Conjugation Tube

When bacteria conjugate, plasmids can pass through the conjugation tube to the recipient bacterium.

Plasmids replicate as the host cell grows and divides.

13.11 Gene Transfer by Plasmids When plasmids enter a cell via conjugation, their genes can be expressed in the new cell.

When bacteria conjugate, plasmids can pass through the conjugation tube to the recipient bacterium.

Plasmids replicate as the host cell grows and divides.

13.11 Gene Transfer by Plasmids When plasmids enter a cell via conjugation, their genes can be expressed in the new cell.

Sometimes the F factor integrates into the main chromosome (at which point it is no longer a plasmid), and when it does, it can bring along other genes from that chromosome when it moves through the conjugation tube from one cell to another.

some plasmids carry genes for conjugation. Other plasmids, called fertility factors, or F factors for short, encode the genes needed for conjugation. They have approximately 25 genes, including the ones that make both the pilus for attachment and the conjugation tube for DNA transfer. A cell harboring an F factor is referred to as F+. It can transfer a copy of the F factor to an F- cell, making the recipient F+.

some plasmids are resistance factors. Resistance factors, or R factors, may carry genes coding for proteins that destroy or modify antibiotics. Other R factors provide resistance to heavy metals that bacteria encounter in their environment.

R factors first came to the attention of biologists in 1957 during an epidemic of dysentery in Japan, when it was discovered that some strains of the Shigella bacterium, which causes dysentery, were resistant to several antibiotics. Researchers found that resistance to the entire spectrum of antibiotics could be transferred by conjugation even when no genes on the main chromosome were transferred. Eventually it was shown that the genes for antibiotic resistance are carried on plasmids. Each R factor carries one or more genes conferring resistance to particular antibiotics, as well as genes that code for proteins involved in the transfer of DNA to a recipient bacterium. As far as biologists can determine, R factors providing resistance to naturally occurring antibiotics existed long before antibiotics were discovered and used by humans. However, R factors seem to have become more abundant in modern times, possibly because the heavy use of antibiotics in hospitals selects for bacterial strains bearing them.

Antibiotic resistance poses a serious threat to human health, and the inappropriate use of antibiotics contributes to this problem. You probably have gone to see a physician because of a sore throat, which can have either a viral or a bacterial cause. The best way to determine the causative agent is for the doctor to take a small sample from your inflamed throat, culture it, and identify any bacteria that are present. But perhaps you cannot wait another day for the results. Impatient, you ask the doctor to give you something to make you feel better. She prescribes an antibiotic, which you take. The sore throat gradually gets better, and you think that the antibiotic did the job.

But suppose the infection is viral. In that case, the antibiotic does nothing to combat the disease, which just runs its normal course. However, it may do something harmful: By killing many normal bacteria in your body, the antibiotic may select for bacteria harboring R factors. These bacteria may survive and reproduce in the presence of the antibiotic, and may soon become quite numerous. The next time you get a bacterial infection, there may be a ready supply of resistant bacteria in your body, and antibiotics may be ineffective.

Antibiotic resistance in pathogenic bacteria provides an example of evolution in action. In the years after they were first discovered in the twentieth century, antibiotics were very successful in combating diseases that had plagued humans for millennia, such as cholera, tuberculosis, and leprosy. But as time went on, resistant bacteria appeared. This was, and is, classic natural selection: Genetic variation existed among bacteria, and those that survived the onslaught of antibiotics must have had a genetic constitution that allowed them to do so.

Transposable elements move genes among plasmids and chromosomes

As we have seen, plasmids, viruses, and even phage capsids (in the case of transduction) can transport genes from one bacterial cell to another. There is another type of "gene transport" that occurs within the individual cell. It relies on segments of DNA that can be inserted either at a new location on the same chromosome or into another chromosome. These DNA sequences are called transposable elements. Their insertion often produces phenotypic effects by disrupting the genes into which they are inserted (Figure 13.12a).

The first transposable elements to be discovered in prokary-otes were large pieces of DNA, typically 1,000 to 2,000 base pairs long, found at many sites on the E. coli main chromosome. In one mechanism of transposition, the transposable element replicates independently of the rest of the chromosome. The copy then inserts itself at other, seemingly random sites on the chromosome. The genes encoding the enzymes necessary for this insertion are found within the transposable element itself. Other transposable elements are cut from their original sites and inserted elsewhere without replication. Later, many longer transposable elements were discovered (about 5,000 base pairs). These large elements carry one or more additional genes and are called transposons (Figure 13.12fr).

Transposable element ABC

Transposable element ABC

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