The following points highlight the seven important transposable elements found in bacteria. The transposable elements are: 1. Insertion Sequences 2. Transposons 3. Cointergration Model of Transposition 4. Conjugative Transposons 5. Integrons 6. Transposon Mutagenesis 7. Transposing Bacteriophage Mu.
Transposable Element # 1. Insertion Sequences (IS-Elements):
The insertion sequences are the simplest form of transposable elements found in prokaryotes. They were first discovered in connection with genes controlling galactose utilization in E. coli. The IS-elements are normal constituents of the bacterial genome. They may be present in the chromosome or extra-chromosomal genetic elements, called plasmids.
For example, IS-elements present in the F-plasmid of E. coli are involved in the integration of the F-plasmid with the bacterial chromosome producing Hfr strains. During the process, genetic exchange between IS-elements of the plasmid and the chromosome takes place.
There are many IS-elements in bacteria which differ in their nucleotide sequence. But all such elements have some common features in their structure. One important characteristic of all IS-elements is the presence of 9 to 41 nucleotide-long sequences at each end repeated nearly identically, but oriented in opposite directions. These sequences are called inverted terminal repeats. In between the terminal repeats is included a long stretch of DNA double helix containing several hundred to more than a thousand base pairs.
A generalized picture of an IS-element is shown diagrammatically in Fig. 9.78:
The central segment of prokaryotic IS-elements carries genetic information for synthesizing only one or two enzymes which are required for insertion of the element in its target site. One of these enzymes is transposase. Another enzyme, resolves may also be produced by some IS-elements. It is a repressor.
The IS-elements may be present in multiple copies in bacterial cells i.e. there may be more than one copy of the same IS-element in each cell. Altogether IS-elements may account for about 0.3% of the total bacterial genome. Some of the IS-elements of E. coli that have been identified and sequenced are IS-1, IS-2, IS-4 and IS-5.
The characteristics of these elements are shown in Table 9.5:
The inverted repeats of IS-I and its central segment are shown in Fig 9.79:
Mechanism of Transposition of IS-Elements:
It might appear during transposition a segment of DNA leaves its original site on the chromosome and gets inserted at a new site — the target site. However, transposition does not happen in this way.
In transposition of an IS-element or a transposon, the transposable element is precisely duplicated and one copy is inserted in the target site and the other copy remains at the original site. Thus, with each transposition event, the number of copies of the element doubles.
As transposition occurs at random points along the chromosome, the base sequence of the transposable element and that of the target site do not bear any homology. In this respect, transposition differs basically from homologous recombination which occurs during genetic recombination. However, transposition often results in mutations by disrupting the coding sequence of the gene in which an IS-element is inserted.
An important feature of transposition of an IS-element is that insertion results in the duplication of the target site. This site consists of a short DNA segment having 3 to 12 nucleotide pairs. For example, the target site of IS-1 has 9 base pairs and that of IS-2 has 5 base pairs.
The IS-elements are inserted between the two duplicated copies of the target site. Thus, with each transposition event, the target site is also duplicated. The sequence of bases in the target site may vary for the same transposable element at different target sites, but the number of base-pairs remains constant for a particular element.
The process of insertion of an IS-element involves a pair of staggered nicks on the two opposite sides of the strands of the target DNA producing two free single-stranded ends to which the copy of the IS-element is joined.
As a result two gaps are created in opposite strands of the recipient DNA. The gaps are filled up by DNA polymerase and ligase. Thereby, an integrated IS-element with its terminal indirect repeats is flanked on either side by two short direct repeats originating from the duplicated target sequence (Fig. 9.80).
Transposable Element # 2. Transposons (Tn-Elements):
Transposons are larger mobile elements and are more complex in structure than IS-elements. They contain functional genes in addition to those required for transposition. Such genes generally confer resistance to antibiotics.
Transposons are distinguished into two main types:
Composite transposons and
The composite transposons have a central segment containing genes for drug resistance and are flanked on both ends by IS-elements. The IS-elements are of the same type in a particular transposon and they are designated as IS-L (left) and IS-R (right). The IS-elements may be oriented in the same direction or in opposite direction. The total length of the transposon may be several thousand nucleotide long.
The non-composite transposons are without IS-elements, but they do have inverted repeats at both ends, like the IS-elements. They differ from IS-elements in being longer and having genes for drug resistance. Characteristics of some transposons are shown in Table 9.6 and two representative transposons, One composite (Tn-5) and one non-composite (Tn-3) are diagrammatically represented in Fig. 9.81.
A composite transposon may be considered as a segment of chromosomal DNA containing one or more bacterial genes which has acquired two IS-elements on either side and can move as a single unit from one location of the chromosome to another with the help of the IS-elements.
On the other hand, the non-composite transposons which do not have the IS-elements have the genes for transposition in their central segment as shown in Tn-3 in above figure. Both types of transposons cause duplication of the target site like the IS-elements.
Target site duplication and integration of Tn-3 is shown in Fig. 9.82:
Transposable Element # 3. Cointergration Model of Transposition:
Transposition of Tn-3 type transposons from a plasmid to a bacterial chromosome, or vice versa, occurs through the formation of a co-integrate followed by its resolution. In the process, the transposon is duplicated and the recipient DNA receives a copy of the transposon. The target site in the recipient is, as usual, duplicated and the transposon is integrated in between them.
The process starts with the formation of two single-stranded nicks on either side of the transposable element, as well as on either side of the target site. Then the nicked ends of the transposon are joined to the nicked ends of the target sites, creating two replication forks. DNA synthesis from the open ends by DNA polymerase and ligation result in the formation of a co-integrate.
The co-integrate now contains two copies of the transposon and two copies of the target sites. This is followed by recombination between the two transposon strands and the co-integrate is resolved to yield the original transposon containing plasmid and a recipient DNA containing a copy of the transposon flanked by duplicated target sites. The process has been shown in a simplified manner in Fig. 9.82.
Transposable Element # 4. Conjugative Transposons:
Conjugative transposons are genetic elements resembling the self-transmissible plasmids which, like the F-plasmid, can integrate into the bacterial chromosome, or may remain free in the cell. They have also the ability to mobilize non-transmissible plasmids from cell to cell when they fuse with such plasmids.
Normally, the conjugative transposons remain integrated with the bacterial chromosome. Occasionally, they are excised from the chromosome to form a circular intermediate, a copy of which is transferred to a recipient cell, presumably through a mating bridge, just like a self-transmissible plasmid.
The copy transferred to the recipient cell is eventually integrated into the chromosome. A conjugative transposon possesses an attachment site with which it is inserted into the chromosome. Replication of the circularized free transposon occurs by rolling circle model originating from a site, OriT, as in case of a self-transmissible plasmid, like F.
Conjugative transposons have been found in several bacteria where they act as potential carriers of bacterial genes. The transmission of a conjugative transposon is schematically shown in Fig. 9.84.
Transposable Element # 5. Integrons:
Integrons represent a type of mobile elements that possesses the ability to accumulate a cluster of genes (gene cassette) which can move as a single unit and have a single promoter. An integron has also a gene producing integrase which catalyses site-specific integration of the gene cluster into the integron.
Another gene codes for a transposase controlling transposition which mediates movement of an integron from one site to its target site. Most of the other genes of the cluster confer resistance to different antibiotics; As all these resistance genes are controlled by the same promoter, they form an operon.
A schematic representation of an integron is shown in Fig. 9.85:
Integrons containing drug-resistance genes pose a great threat for human health. When present in a pathogenic organism, an integron not only makes the host resistant to the antibiotics for which the resistance genes are already accumulated in the integron, but it also possesses the potentiality to expand the resistance spectrum by adding new resistant genes.
Integrons are even more ominous, because they have the ability to move into self-transmissible plasmids or conjugative transposons and then can be transferred to another potential pathogen making it resistant to all the antibiotics for which resistance genes are present in the integron.
There is another important aspect of an integron carrying a number of drug-resistant genes. Because all such genes are regulated by the same promoter, the expression of the first resistance gene of the cassette leads to expression of all other resistance genes.
For example, if the first resistance gene is that of ampicillin resistance, it is expressed when there is ampicillin present in the environment in a concentration which inhibits the ampicillin-sensitive bacteria. Simultaneously, all the resistance gene are expressed, even though the other antibiotics are absent in the environment. In case such an integron is transferred to a self-transmissible plasmid like R-plasmid, it creates a great clinical problem.
Transposable Element # 6. Transposon Mutagenesis:
We know that insertion sequences or transposons may result in mutation when they are integrated into a target site located within a gene, or in the regulatory sequences of DNA controlling the gene. When a transposable element is inserted into a gene, the reading frame is disrupted. As a result, transcription of the gene produces a wrong messenger and the protein produced from such a messenger becomes non-functional.
Transposon mutation in an organism can be identified if the transposon carries a marker, like that of antibiotic resistance, and if the gene in which the transposon has been inserted has a known function. For inducing transposon mutation, the transposon is at first introduced into a plasmid.
The plasmid containing the integrated transposon is next transferred into the host bacterium by transformation. Now, if the transposon is inserted in the host chromosome at the site where the gene of choice is located, the gene loses its normal function.
The mutation so caused by the transposon (transposon mutation) can be selected from a large population with the help of the antibiotic resistance marker carried by the transposon. An example can clarify the selection of such mutants.
A bacterial strain is able to utilize both glucose and sucrose as carbon sources. When it grows on sucrose, it hydrolyses the substrate with the enzyme sucrase (invertase) into glucose and fructose. The gene of choice here is that coding for this enzyme which, when mutated, makes the organism incapable of utilizing sucrose, though it can still grow on glucose.
Now, if a transposon carrying a marker of tetracycline resistance is inserted into the gene controlling sucrase synthesis, the mutant becomes tetracycline-resistant, and it is also rendered incapable of utilizing sucrose. From a large population, such sucrase-negative mutants can be identified by comparing the plates of sucrose medium containing tetracycline and of glucose medium containing tetracycline. The sucrase-negative colonies would be absent in sucrose-plates, but present in glucose-plates.
The selection of such transposon-induced mutants is schematically shown in Fig. 9.86:
Transposable Element # 7. Transposing Bacteriophage Mu:
Bacteriophage Mu is a temperate phage of E. coli having a double-stranded 38 kb linear DNA genome. Like other temperate phages, it can either have a lytic cycle or a lysogenic pathway. The phage possesses the ability to integrate its genome into the bacterial chromosome without any site-specificity. Such random insertion of the phage genome into the bacterial chromosome causes mutation and, hence, the name of the phage (Mu stands for mutator). The phage Mu is, therefore, considered as a transposable agent.
It may be remembered that a temperate phage, on infecting a host bacterium, can integrate its genome into the chromosome of the host cell making the host lysogenic. The phage genome may persist as a prophage in the host chromosome for many generations.
On induction, either spontaneously or by an external agent like UV, the prophage is excised and begins the lytic cycle by production of phage genome and phage proteins. In case of phage Mu, induction does not result in excision of the prophage.
When the phage Mu infects an E. coli cell, the phage DNA is integrated into the host DNA randomly, but with a low frequency. However, when the prophage is induced to enter into a lytic cycle, the frequency of integration into the host DNA increases significantly. This has been designated as replicative transposition.
It appears that copies of phage DNA produced by replication can integrate with higher efficiency into the host chromosome at multiple sites resulting in mutation of host genes. Eventually, phage-coded proteins are synthesized and new phage particles are assembled and released by lysis of the host cells to complete the lytic cycle.
The exact mechanism of transposition by phage Mu is not yet well understood. Transposition- induced mutations by Mu can be of different types including deletions and inversions of DNA segments of the host chromosome.
The structure of bacteriophage Mu is shown in Fig. 9.87:
Deletions or inversions of the host chromosome occur when two Mu genomes are integrated into the same chromosome and homologous recombination between the Mu genomes takes place.
These are diagrammatically shown in Fig. 9.88: