Tn5 transposase: The enzyme that helps bacteria
to share antibiotic resistance genes
Some DNA fragments can change their own location in the genome. Such fragments are called transposable elements (TEs) or transposons. Transposable elements are found in almost every organism — from bacteria to higher eukaryotes, including humans — and they play a crucial role in evolution because they are a powerful source of genetic variability. Transpositions and rearrangements of genetic material are normal and often beneficial for the species, but in many cases they may cause harm or death to an organism.
Most TEs carry one or more genes encoding the proteins necessary for transposition. Transposases are among such proteins. The transposase enzyme shown here is encoded by the Tn5 transposon, which is found in bacteria.
Tn5 is among the first described transposons [1]. It was discovered in Escherichia coli during studies of bacterial resistance to the antibiotic kanamycin.
Antibiotics are chemical compounds that kill bacteria. Different antibiotics act using different pathways; an antibiotic can destroy a cell wall or membrane, block crucial enzymes, or interfere with bacterial ribosomes. Kanamycin blocks bacterial protein synthesis by binding the small ribosomal subunit. However, some bacteria carry the gene that encodes a protein capable of inactivating kanamycin. This kind of genes can sometimes travel from one bacterium to another. Antibiotic resistance is developing very quickly in bacterial evolution. It is a large and growing problem in pharmacology and drug design. The Tn5 transposon was discovered in 1974 by Douglas Berg, Julian Davies, Bernard Allet, and Jean-David Rochaix of the University of Geneva [2]. They demonstrated that kanamycin resistance can travel by means of transposition between a bacterial plasmid (circular, extrachromosomal DNA), the genome of a bacteriophage that infects E. coli, and the bacterial genome. One bacterial cell can have several plasmids, which often carry some antibiotic resistance genes. Plasmids can be passed from one cell to another in a genetic transfer called transformation. Viral phages can also be vectors of horizontal gene transfer, in which case the process is called transduction. In fact, some phages can reside in the bacterial genome for many generations. The most interesting facet of the discovery made by Berg and his coauthors was that genetic material can be transferred from a bacterial plasmid to a resident phage.
The Tn5 transposon appeared to be the vector of bacterial kanamycin resistance, and, subsequently, this TE became one of the most popular topics in the area of transposition mechanism research [3].
Dr. Douglas Berg, Professor in Washington University School of Medicine. St. Louis, Missouri, member of the team that discovered the Tn5:
«We discovered Tn5 in Geneva in late 1974. I needed a lambda phage marked with drug resistance, and anticipated being able to make one using Shimada and Weisberg’s protocol [4] of lambda integration into secondary attachment sites and then prophage induction, using E. coli strains that carried natural R factor plasmids. Julian Davies provided R factor plasmids whose resistances were very interesting enzymatically. Jean-David Rochaix’s and Bernard Allet’s electron microscope heteroduplex and DNA restriction analyses showed that lambda kanR phages contained new ~5.6 kb segments with distinctive 1.5 kb terminal inverted repeats, each inserted at a different site in lambda DNA. None contained substitutions next to the lambda attachment site, as predicted by the Shimada-Weisberg model. We quickly realized, with great excitement, that this „kanR“ DNA segment could be like the mysterious transposable controlling elements of maize, discovered by Barbara McClintock some 25 years earlier, but in a system amenable to powerful microbial molecular genetic approaches. Also intriguing were ideas about mobile DNAs as agents that would help spread drug resistance and speed bacterial evolution; and, upon finding Tn5-induced mutations in bacterial genes (after transposition from phage lambda), the possibility of harnessing this transposon as a tool for bacterial genetics.»
Detailed analyses of the Tn5 transposon have demonstrated that it contains genes for resistance to three antibiotics — kanamycin, bleomycin and streptomycin [5,6]. This resistance gene cluster is bracketed by similar sequences, IS50L and IS50R, which are each also transposable elements; the latter sequence, IS50R, encodes the transposase, while IS50L encodes an inactive, truncated version of the protein [7,8].
A transposase recognizes short sequences at the ends of a transposon, makes breaks at those locations (excision), and reinserts the element at another location. The illustration depicts the complex of two transposase molecules bound to the Tn5 DNA ends [9]. The structure of this complex was described by researchers at the University of Wisconsin in 2000 [9].
Dr. William Reznikoff, Professor Emeritus, Department of Biochemistry, University of Wisconsin-Madison, member of the team that solved the transposase structure:
«My interest in Tn5 transposase resulted from a long term interest in genome evolution and chromosome rearrangements in particular and also from fortuitous scientific interactions with Jim Shapiro as a postdoctoral fellow and Julian Davies and Douglas Berg as a new faculty member. The latter interaction led to my laboratory choosing Tn5 as a model system. Following many years of studying Tn5 transposition using genetics and biochemistry, the logical and essential step is to understand the structural basis of the macromolecule catalyzing the transposition; that was the transposase preferably bound to the transposon end sequences.» Transposases are related to retroviral integrases — enzymes that mediate the integration of retroviruses (such as HIV) into the host genome [10]: they contain domains with similar structure and belong to the same protein family of polynucleotidyl transferases. In the words of Reznikoff, «In addition to helping us elucidate the molecular mechanism of Tn5 transposition, the structural determination is of interest as a model for understanding HIV-1 integrase action and that of other DNA rearranging processes.»
DNA cutting and rearrangement underlies many crucial functions of a wide variety of organisms. For example, it is integral to processes as basic as homologous recombination (crossing over), the maturation of immunoglobulin genes, and the maintenance of the chromosomal ends (telomeres) length.
Cast
- Modeller, 3D-visualizator:
- Ivan Konstantinov
- Scientific advisor
- Yury Stefanov
- Acknowledgements
- We want to thank Dr. Berg and Dr. Reznikoff for useful comments
Date: Jun 05, 2012
References
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- , Mol Microbiol. 2003 Mar;47(5):1199-206.
- , J Mol Biol. 1972 Feb 14;63(3):483-503.
- , Cold Spring Harb Symp Quant Biol. 1981;45 Pt 1:107-13.
- , Nucleic Acids Res. 1985 Jan 11;13(1):195-205.
- , Cell. 1980 Mar;19(3):795-805.
- , J Biol Chem. 1998 Mar 27;273(13):7367-74.
- , Science. 2000 Jul 7;289(5476):77-85.
- , Nat Struct Biol. 2001 Apr;8(4):302-7.
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