Transposable Genetic Elements in Prokaryotes

This article throws light upon the four transposable genetic elements in prokaryotes.

The four transposable genetic elements in prokaryotes are: (1) Bacterial Insertion Sequences (2) Prokaryotic Transposons (3) Insertion-Sequence Elements and Transposons in Plasmids and (4) Phage mu.

(1) Bacterial Insertion Sequences:

Insertion Sequences or Insertion-Sequence (IS) Elements:

Insertion sequences, or insertion-sequence (IS) elements, are now known to be segments of bacterial DNA that can move from one position on a chromosome to a different position on the same chromosome or on a different chromosome. An IS element contains only genes required for mobilizing the element and inserting the element into a chromosome at a new location. Is elements are normal constituents of bacterial chromosome and plasmids.

When IS elements appear in the middle of genes, they interrupt the coding sequence and inactivate the expression of that gene. Owing to their size and in some cases the presence of transcription and translation termination signals, IS elements can also block the expression of other genes in the same operon if those genes are downstream from the promoter of the operon.

IS elements were first found in E. coli as a result of their affects on the expression of a set of three genes whose products are needed to metabolize the sugar galactose as a carbon source. Careful investigations showed that the mutant phenotypes resulted from the insertion of an approximately 800 base pairs (bp) DNA segment into a gene. This particular DNA segment is now called insertion sequencel (IS1).

Properties of IS Elements:

Is1 is the genetic element capable of moving around the genome. It integrates into the chromosome at locations with which it has no homology, thereby distinguishing it from recombination. This event is an example of transposition event. There are number of IS elements that have been identified in E. coli, including IS1, IS2, and IS 10, each present in 0 to 30 copies per genome, and each with a characteristic length and unique nucleotide sequence.

IS 1 is 768 bp long, and is present in 4 to 19 copies on the E. coli chromosomes. IS2 is present in 0 to 12 copies on the E. coli chromosome and in one copy on the F plasmid, and IS 10 is found in a class of plasmids called R plasmid that can replicate in E. coli (Fig. 12.1).

Among prokaryotes, the IS elements are normal cell constituents, that is, they are found in most cells. Altogether, IS elements constitute approx. 0.3% of the cell’s genome. All IS elements that have been sequenced, end with perfect or nearly perfect inverted terminal repeats (IRs) of between 9 and 41 bp. This means that essentially the same sequence is found at each end of an IS but in opposite orientations.

IS Transposition:

When transposition of an IS element takes place, a copy of the IS element inserts into a new chromosome location while the original IS elements remains in place. That is, transposition requires the precise replication of the original IS element, using the replication enzymes of the host cell. The actual transposition also requires an enzyme encoded by the Is element called transposase.

The IR sequences are essential for the transposition process, that is, those sequences are recognized by transposase to initiate transposition. Is elements insert into the chromosomes at sites with which they have no sequence homology?

Genetic recombination between non-homologous sequences is called illegitimate recombination. The sites into which IS elements insert are called target sites. The process of IS insertion into a chromosome is shown in Figure 12.2. Firstly, a taggered cut is made in the target site and the IS element is then inserted, becoming joined to the jingle-stranded ends.

The gaps are filled in by DNA polymerase and DNA ligase, producing an integrated IS element with two direct repeats of the target site sequence flanking the IS element. ‘Direct’ in this case means that the two sequences are repeated in the same orientation. The direct repeats are called target site duplications. The sizes of target site duplication vary with the IS elements, but tend to be small. Integration of some IS elements show preference for certain regions, while others integrate only at particular sequences.

All copies of a given IS element have the same sequence, including that of the inverted terminal repeats. Mutations that affect the inverted terminal repeat sequence of IS elements affect transposition, indicating that the inverted terminal repeat sequences are the key sequences recognized by transposase during a transposition event.

(2) Prokaryotic Transposons:

A transposon (Tn) is more complex than an IS elements. A transposon is a mobile DNA segment that, like an IS element, contains genes for the insertion of the DNA segment into the chromosome and for the mobilization of the element to other locations on the chromosome. There are two types of prokaryotic transposons: composite transposons and non-composite transposons.

(1) Composite Transposons:

They are complex transposons with a central region containing genes, e.g., drug resistance genes, flanked on both sides by IS elements (also called IS modules). Composite transposons may be thousands of base pairs long. The IS elements are both of the same types and are called IS-L (for “left”) and IS-R (for “right”). Depending upon the transposon, IS-L and IS-R may be in the same or inverted orientation relative to each other. Because the ISs themselves have terminal inverted repeats, the composite transposons also have terminal inverted repeats.

Figure 12.3 shows the structure of the composite transposon Tn 10 to illustrate the general features of such transposons. The Tn 10 transposon is 9,300 bp long and consists of 6,500 bp of central, nonrepeating DNA containing the tetracycline resistance gene flanked at each end with a 1,400-bp IS element. These IS elements are designated IS10L and IS10R and are arranged in an inverted orientation. Cells containing Tn 10 are resistant to tetracycline resistance gene contained within the central DNA sequence.

Transposition of composite transposon occurs because of the function of the IS elements they contain. One or both IS element supplies the transposase. The inverted repeats of the IS elements at the two ends of the transposon are recognized by transposase to initiate transposition (as with transposition of IS elements).

Transposition of Tn 10 is rare, occurring once in 10 cell generations. This is the case because less than one transposase molecule per cell generation is made by Tn 10. Like IS elements, composite transposons produce target site duplications after transposition.

(2) Non-composite Transposons:

They like composite transposons, contain genes such as those for drug resistance. Unlike composite transposons, they do not terminate with IS elements. However, they do have the repeated sequences at their ends that are required for transposition. Tn3 is a non-composite transposon.

Tn3 has 38 bp inverted terminal repeats and contains three genes in its central region. One of those genes, bla, encodes β-lactamase which breaks down ampicillin and therefore makes cells containing Tn3 resistant to ampicillin. The other two genes, tnpA and tnpB, encode the enzymes transposase and resolvase that are needed for transposition of Tn3 (Fig. 12.4). Transposase catalyzes insertion of the Tn into new sites, and resolvase is an enzyme involved in the particular re-combinational events associated with transposition.

Resolvase is not found in all transposons. The genes for transposition are in the central region for non-composite transposons, while they are in the terminal IS elements for composite transposons. Non composite transposons also cause target site duplications when they move.

(3) Mechanism of Transposition in Prokaryotes:

Several different mechanisms of transposition are employed by prokaryotic transposable elements. And, as we shall see later, eukaryotic elements exhibit still additional mechanisms of transposition. In E. coli, we can identify replicative and conservative (non-replicative) modes of transposition. In the replicative pathway, a new copy of the transposable element is generated in the transposition event. The results of the transposition are that one copy appears at the new site and one copy remains at the old site. In the conservative pathway, there is no replication. Instead, the element is excised from the chromosome or plasmid and is integrated into the new site (Fig. 12.5).

Replicative Transposition:

The transposition of Tn3 occurs in two stages. Firstly, the transposase mediates the fusion of two molecules, forming a structure called a cointegrate. During this process, the transposon is replicated, and one copy is inserted at each junction in the cointegrate. The two Tn3 are oriented in the same direction. In the second stage of transposition, the tnpR-encoded resolvase mediates a site-specific recombination event between the two Tn3 elements. This event occurs at a sequence in Tn3 called res, the resolution site, and generates two molecules, each with a copy of the transposon.

The tnpR gene-product also has another function, namely, to repress the synthesis of both the transposase and resolvase proteins. This repression occurs because the res site is located in between the tnpA and tnpR genes. By binding to this site, the tnpR protein interferes with the synthesis of both gene-products, leaving them in chronic short supply. Consequently, the Tn3 element tends to remain immobile (Fig. 12.6).

Conservative transposition:

Some transposons, such as Tn10, excise from the chromosome and integrate into the target DNA. In these cases, DNA replication of the element does not occur, and the element is lost from the site of the original chromosome. This mechanism is called conservative (non replicative) transposition or simple insertion. Tn 10, e.g., transposes by conservative transposition.

Insertion of a transposon into the reading frame of a gene will disrupt it, causing a loss of function of that gene. Insertion into gene’s controlling region can cause changes in the level of expression of the gene. Deletion and insertion events also occur as a result of activities of the transposons, and from crossing-over between duplicated transposons in the genome.

(3) IS Elements and Transposons in Plasmids:

The transfer of genetic material between conjugating E. coli is the result of the function of the fertility factor F. The F factor, a circular double stranded DNA molecule, is one of the example of bacterial plasmid. Plasmids such as F that are also capable of integrating into the bacterial chromosomes are called episomes. F factor consists of 94,500 bp of DNA that code for a variety of proteins.

The important elements are:

(i) Transfer gene (tra) required for the conjugation transfer of the DNA.

(ii) Genes that encode proteins required for the plasmid’s replication,

(iii) Four IS elements, two copies of IS3, one of IS2, and one of an insertion sequence element called gamma- delta.

It is because the E. coli chromosome has copies of these four insertion sequence at various positions that the F factor can integrate into the E. coli chromosome at different sites and in different orientations with homologous sequence of the insertion elements.

Another class of plasmids that has medical significance is the R plasmid group, which was discovered in Japan in the 1950s, during the cure for dysentery. The disease is the result of infection by the pathogenic bacterium Shigella. Shigella was found to be resistant to most of the commonly used antibiotics.

Subsequently, they found that the genes responsible for the drug resistances were carried on R plasmids, which can promote the transfer of genes between bacteria by conjugation, just as the F factor. One segment of an R plasmid that is homologous to a segment in the F factor is the part needed for the conjugal transfer of genes.

That segment and the plasmid-specific genes for DNA replication constitute what is called the RTF (resistance transfer factor) region (Fig. 12.7). The rest of the R plasmid differs from type to type and includes the antibiotic-resistance genes or other types of genes of medical significance, such as resistance to heavy metal ions.

The resistance genes in R plasmid are, in fact, transposons, that is each resistance gene is located between flanking, directly repeated segments such as one of the IS modules (Fig. 12.8). Thus, each transposon with its resistance gene in the R plasmid can be inserted into new location on other plasmids or on the bacterial chromosome, while at the same time leaving behind a copy of itself in the original position.

(4) Phage mu:

Phage mu is a normal-appearing phage. We consider it here because, although it is a true virus, it has many features in common with IS elements. The DNA double helix of this phage is 36,000 nucleotides long-much larger than an IS element. However, it does appear to be able to insert itself anywhere in a bacterial or plasmid genome in either orientation. Once inserted, it causes mutation at the locus of insertion-again like an IS element. (The phage was named for this ability: mu stands for “mutator.”)

Normally, these mutations cannot be reverted, but reversion can be produced by certain kinds of genetic manipulation. When this reversion is produced, the phages that can be recovered showing no deletion, proving that excision is exact and that the insertion of the phage therefore does not involve any loss of phage material either. Each mature phage particle has on each end a piece of flanking DNA from its previous host (Fig. 12.9). However, this DNA is not inserted anew into the next host. Its function is unclear. Phage mu also has an IR sequence, but neither of the repeated elements is at a terminus.

Mu can also act like a genetic snap fastener, mobilizing any kind of DNA and transposing it anywhere in a genome. For example, it can mobilize another phage (such as λ) or the F factor. In such situations, the inserted DNA is flanked by two mu genomes (Fig. 12.10).