Page 816 CLINICAL CORRELATION 19.1 Transmissible Multiple Drug Resistances Pathogenic bacteria are becoming increasingly resistant to a large number of antibiotics, which is viewed with alarm by many physicians. Many cases have been documented in which a bacterial strain in a patient being treated with one antibiotic suddenly became resistant to that antibiotic and, simultaneously, to several other antibiotics even though the bacterial strain had never been previously exposed to these other antibiotics. This occurs when the bacteria suddenly acquire from another bacterial strain a plasmid that contains several different transposons, each containing one or more antibioticresistance genes. Examples include the genes encoding b lactamase, which inactivates penicillins and cephalosporins, chloramphenicol acetyltransferase, which inactivates chloramphenicol, and phosphotransferases, which modify aminoglycosides such as neomycin and gentamycin. Neu, H. C. The crisis in antibiotic resistance. Science 257: 1064, 1992. 19.6— Bacterial Transposons Transposons Are Mobile Segments of DNA Figure 19.16 General structure of transposons. Transposons are relatively rare mobile segments of DNA that contain genes coding for their own rearangement and (usually) genes that specify resistance to various antibiotics. So far we have only discussed the regulation of bacterial genes whose locations are fixed in the chromosome. Their positions relative to the neighboring genes do not change. The vast majority of bacterial genes are of this type. In fact, genetic maps of E. coli and Salmonella typhimurium are quite similar, indicating the lack of much evolutionary movement of most genes within the bacterial chromosome. There is a class of bacterial genes, however, in which newly duplicated gene copies ''jump" to another genomic site with a frequency of about 10–7 per generation, the same rate as spontaneous point mutations occur. The mobile segments of DNA containing these genes are called transposable elements or transposons (Figure 19.16). Transposons were first detected as rare insertions of foreign DNA into structural genes of bacterial operons. Usually, these insertions interfere with the expression of the structural gene into which they have inserted and all downstream genes of the operon. This is not surprising since they can potentially destroy the translation reading frame, introduce transcription termination signals, affect the mRNA stability, and so on. Many transposons and the sites into which they insert have been isolated using recombinant DNA techniques and have been extensively characterized. These studies have revealed many interesting features about the mechanisms of transposition and the nature of genes located within transposons. Transposons vary tremendously in length. Some are a few thousand base pairs and contain only two or three genes; others are many thousands of base pairs long, containing several genes. Several small transposons can occur within a large transposon. All active transposons contain at least one gene that codes for a transposase, an enzyme required for the transposition event. Often they contain genes that code for resistance to antibiotics or heavy metals. Most transpositions involve generation of an addition copy of the transposon and insertion of this copy into another location. The original transposon copy is the same after the duplication as before; that is, the donor copy is unaffected by insertion of its duplicate into the recipient site. Transposons contain short inverted terminal repeat sequences that are essential for the insertion mechanism, and in fact these inverted repeats are often used to define the two boundaries of a transposon. The multiple target sites into which most transposons can insert seem to be fairly random in sequence; other transposons have a propensity for insertion at specific "hot spots." The duplicated transposon can be located in a different DNA molecule than its donor. Frequently, transposons are found on plasmids that pass from one bacterial strain to another and are the source of a suddenly acquired resistance to one or more antibiotics by a bacterium (Clin. Corr. 19.1). As with bacterial operons, each transposon or set of transposons has its own distinctive characteristics. The wellcharacterized transposon Tn3 will be discussed as an example of their general properties. Tn3 Transposon Contains Three Structural Genes The transposon Tn3 has been cloned using recombinant DNA techniques and its complete sequence determined. It contains 4957 base pairs including 38 base pairs at one end that occur as an inverted repeat at the other end (Figure 19.17). Three genes are present in Tn3. One gene codes for the enzyme b lactamase, which hydrolyzes ampicillin and renders the cell resistant to this antibiotic. The other two genes, tnpA and tnpR, code for a transposase and a repressor protein, respectively. The transposase has 1021 amino acids and binds to singlestranded DNA. Little else is known about its action, but it is thought Page 817 Figure 19.17 Functional components of the transposon Tn3. Genetic analysis shows there are at least four kinds of regions: the inverted repeat termini; a gene for the enzyme blactamase, which confers resistance to ampicillin and related antibiotics; a gene encoding an enzyme required for transposition (transposase); and a gene for a repressor protein that controls transcription of genes for transposase and for repressor itself. The horizontal arrows indicate direction in which DNA of various regions is transcribed. Redrawn from Cohen, S. N., and Shapiro, J. A. Sci. Am. 242:40, 1980. W. H. Freeman and Company, Copyright © 1980. to recognize the repetitive ends of the transposon and to participate in the cleavage of the recipient site into which the new transposon copy inserts. The tnpR gene product is a protein of 185 amino acids. In its role as a repressor it controls transcription of both the transposase gene and its own gene. The tnpA and tnpR genes are transcribed divergently from a 163 base pair control region located between the two genes that is recognized by the repressor. The tnpR product also participates in the recombination process that results in the insertion of the new transposon. Transcription of the ampicillinresistance gene is not affected by the tnpR gene product. Mutations in the transposase gene generally decrease the frequency of Tn3 transposition, demonstrating its direct role in the transposition process. Mutations that destroy the repressor function of the tnpR product cause an increased frequency of transposition. These mutations derepress the tnpA gene, resulting in more molecules of the transposase, which increases the formation of more transposons. They also derepress the tnpR gene but, since the repressor is inactive, this has no effect on the system. When a transposon, containing its terminal inverted repeats, inserts into a new site, it generates short (5–10 bp) direct repeats of the sequences at the recipient site that flank the new transposon. This is due to the mechanism of recombination that occurs during the insertion process (Figure 19.18). The first step is the generation of staggered nicks at the recipient sequence. These staggered singlestrand, protruding 5 ends then join covalently to the inverted repeat ends of the transposon. The resulting intermediate resembles two replicating forks pointing toward each other and separated by the length of the transposon. The replication machinery of the cell fills in the gaps and continues the divergent elongation of the two primers through the transposon region. This ultimately results in two copies of the transposon sequence. Reciprocal recombination within the two copies regenerates the original transposon copy at its original position and completes the process of forming a new copy at the recipient site that is flanked by direct repeats of the recipient sequence. The practical importance of transposons located on plasmids has taken on increased significance for the use of antibiotics in treatment of bacterial infections. Plasmids that have not been altered for experimental use in the laboratory usually contain genes that facilitate their transfer from one bacterium to another. As the plasmids transfer (e.g., between different infecting bacterial strains), their transposons containing antibioticresistance genes are moved into new bacterial strains. Once inside a new bacterium, the transposon can be duplicated onto the chromosome and become permanently established in that cell's lineage.