513 Bacterial Transposon

7/29/2019 513 Bacterial Transposon

1/9

Bacterial TransposonsCornelis, G .Dep artm ent o f Microbiology. University of Louvain. UCL 30.58.B 1200Brussels. Belgium

A. Bacterial PlasmidsBacterial plasmids have been know n for more than twenty years a nd , duringthis period, on e witnessed an extraordinary spread of those extra-chromo-somal DNA elements.Resistance plasmids were first identified in Enterobacteriaceae but, laterOn. have been found in almo st every patho genic bacterial species, includ ingmore recently Hemophilus influenzae and Neisseria gonorrheae.Plasmids, however. do not only contribute to antibiotic resistance. Theycan code for m any oth er properties, including heavy meta1 resistance, produc-tion of a cholera-like enterotox in, tumorigenicity in plants a nd the capacity tocatabolize various substrates. Some plasmids have been found to code forlactose ferm entation an d this type of plasmid also happe ns to be of medicalconcern. Their presence can indeed obscure the detection of pathogens suchas Salmonella and Yersinia since the capac ity to fermen t lactose is the m ajorkey in the sorting of the Enterobacteriaceae.

Plasmids consist of covalently closed circular DNA and their essentialfeature is a replication region that ensures the propaga tion of the entire struc-ture.Some plasmids also contain an operon promoting the transfer of theplasmid itself from o ne bacteria to ano ther one.

B. Transloca tion of Plasmid G enesIn 1974, Hedg es and Jacob observed that the ampicillin resistance determ i-nan t of a plasmid called RP4 could be translocated to the bacterial chrom o-some and various other plasmids genetically unrelated. From one of thesederivative plasmids, they could subsequently translocate the -lactamasegene to a third p lasm id. After transposition, the increase in mo lecular weightwas similar in every instanc e. These auth ors concluded that a DNA sequence,including the -lactamase gene, had a specific transposition mechanism andthey called it a transposon (now T nl ).Later On, Berg et al. (1975) observed the transposition of the kana my cinresistance genes from two different resistance plasmids to bacteriophage X(Tn5 and Tn6).

Gottesman and Rosner (1975) ma de similar observations with a d eterm i-nant for chloramphenicol resistance: initially detected on a resistance plasmid


2/9

5 14 Corne lis. G.an d, later, transferred to phage P1, it could subsequently be translocated fromP1 to bacte riopha ge h (Tn9j.Still in the Same year, Kleckner et al. (1975) reported that a genetic ele-ment . 8.3 Kb long , carryin g the tetracycline resistance gene was also cap ableof translocation (Tn 10).Recently. we show ed th at genes coding for lactose fermen tation ca n ju m pfrom pGC1, a plasmid that originated in Yersinia enterocolitica, to RPI, aresistance plasmid that originated in Pseudomonas aeruginosa (Cornelis et al.,1978) (Fig. 1). This lactose transposon that alters the biochem ical ph en oty peof its host is 1 6 , 6 Kb long an d has been given the num ber 95 1.Som e transposo ns are listed in Table 1.

host : Yersinia Pseudoronas Escherichiaenterocoli i a aeruginosr col l

s t ruc tu re :

Fig. 1. Schem atic representation of the transposition of Tn95l. carrying a lactose opero n. frompG Cl to RP l. RP1 confers resistance to kanamycine (Km). tetracycline (Tc) and ampicillin (A mp ).The latter gene is part ofa no the r transposon (Tn 1 )

Table 1. Some transposable elements

Transposo n Geneiical inform ation Size References-lactam antib iotics resistance 4.5 KbKan am ycin resistance 3.8 KbTrim ethoprim and 13.5 K bStreptom ycin resistanceCh loram phe nicol resistance 2.6 K bTet racyc line resistance 8.3 K bTrim etho prim resistance 7.5 K bMerc uric ions resistance 7.8 KbLactose ferm entat ion 16.6 Kb

Hedges et al.. M.G.G. 132,31 (1974)Heffron et al.. J. Bact. 122,250 (1975)Berg et al.. P.N.A.S. 72,3628 (1975)Barth et al.. J. Bact. 125,800 (1976)Gottesm an et al.. P.N.A.S. 72,504 (1975)Kleckner et al.. J.M.B. 97, 56 1 (1975)Foster et al., J . Bact. 124, 1153 (197 5)Sha piro et al.. J. Bact. 129, 1632 (197 7)Bennett et al.. M.G.G . 159,101 (1978) -Cornelis et al.. M.G .G. 160,21 5 (1978)


3/9

Bacterial TransposonsC. General Features of T ranspositionAs suggested by Hedges and Jacob (1974). transposons are specific DNAsequen ces of a precise length.The size of any replicon suffering transposition d oes increase a nd the in -crem ent is constan t for a given transposon.Transposition d oes not require extended homology between the two repli-cons exchanging the transposon. This is shown in Table 2 for pGCI (lac+,tra ts. inc unkn ow n) a nd RPI (Am p, kan , tet, tra, inc P), the two plasmid s ex-chang ing Tn95 1. Thus, transposition canno t involve ordinary recom binationoccuring after physical breaking and reciprocal exchange of DNA. Accord-ingly, in som e instances at least, transposition turns o ut to be ind ep en da nt ofth e rec A gene produ ct, the bacterial function involved in such recomb inationprocess (R ube ns et al., 1976).Table 2. DNA -DNA hybridization between pGCI and RPIDNA bound 3H-pGC l DNAto filter hybrid ized

Exp I Exp I1p D G l 64 101 Filters were load ed with 2 mcg of DNA. T hep G C 1 2300 2303 values given ar e norm alized to 10000 cpm in -RP 1 74 112 put. pD GI is an unrelated plasmid taken as aRPI ::Tn951 1490 - negative control. For details. See Cornelis etal.. 1978

Transposition is therefore considered as some kind of "illegitimate" re-combination involving the termini.The known transposons can jum p on a great num ber of targets such asdifferent plasmids. bacteriophages an d bacterial chromoso mes. There seem show ever to be some kind of specificity: Tnl for instance was found by Hedgesand Jacob (1974) to transpose from RP4 to three different plasmids but noton a fourth o ne. Bennett an d Richmond (1976) also observed that the trans -position frequencies of Tn 1 could vary 10 000 times according to the chosentarget. Kretschmer et al. (1977) observed th e sam e phe no me non with Tn3.Inserted transposons can readily be detected on small replicons such asplasm ids a nd b acteriophag es by electron microscopy. When a mixtu re of twodifferent DNAs sharing homologous regions is denatured and allowed toreannea l slowly, a n um ber of hybrid molecules are generated. If such "hetero-duplex" molecules are constructed between a given plasmid and its ownderivative carrying a transposon, the structure observed in electron micro-scopy will be a circular double stranded plasmid carrying a single strandedloop corresponding to the transposon. This kind of analysis clearly demon-strates th at transpo sons are inserted without any loss of DNA from the recip-ient plasmid.Moreover, heteroduplex analysis will indicate whether the insertions ontwo inde pen dan tly isolated molecules occur at the sam e site or a t two differ-ent sites. If the transposon m aps at two different loci on the target plasmid,


4/9

Cornelis. G.

Fig. 2. Heteroduplex m olecules formed be-tween two differentRPl : Tn95 1molecules.The arrows indicate the positions whe re thesingle stranded loops (16.6 K b) emergesfrom the 57 Kb long double stranded cir-cular DN Aheteroduplex molecules will consist of a double stranded circular mole-cule having the length of the parent plasmid and two single stranded loopscorresponding to the transposon (Fig. 2). The distance between the two in-sertion sites can be measured, but this type of analysis however doesn'tallow to localize the insertion sites on the plasmid m ap , at least with out an yrefinements.This kind of information can be inferred from experiments using restric-tion endonucleases. These nucleases cleave DNA at very specific sites andthus g enerate defined fragm ents from a given replicon. These fragm ents aresubseq uently resolved by agaro se gel electrophoresis. Partial digestions an ddou ble d igestions using simultaneously two different nucleases allow to alignthese fragments and to draw a m ap , referred to as the physical m ap . Insertionof a t ranspos on i nto a plasm id will either introduce new cleavage sites, gen er-ating new bands, or specifically increase the length of a given fragment. Ifone know s the physical rnap of the target plasmid, it is possible to rnap thetransposition sites and even to determine the orientation of the transposon,m aking use of the restriction nucleases.Electron microscopy an d restriction nucleases have been used to rnap theinsertion sites of various transposons on a number of targets. Clearly, thenu m be r of insertion sites is usually very high: we map ped eight insertions ofTn95 1 on R Pl an d foun d eight different loci (Fig. 3). Heffron an d CO-workers(1975 a ) could detect at least 19 sites for Tn2 o n a n 8 Kb long target plasmid!Con sidering th at tran sposition consists of the insertion of DNA sequencesof a certain le ngth in to a g enom e, it is not surprising that it sometim es leads tothe inactivatio n of genes. Plasmid pGC 530 is an RP1: Tn95 1 derivative wh erethe insertion was found to rnap at coordinate 13 Kb, i.e. within the


5/9

Bacterial Transposon s

Fig. 3. Physical ma p of R Pl showing the 8 insertion sites ofTn951. pGC3210. pGC1500. pGCl210 .pGC270. pGC530, pGC29 10. pGC9 114 and pGC210 are the 8 RP1: :Tn95 1 derivatives. E = EcoR1 endon uclea se cleavage site: B = Bam H1 end onucle ase cleavage site: P = Pst 1 endon ucleas ecleavage site: H = Hind I11 endonuclease cleavage site: Ap = ampicillin resistance gene: Tc =tetracycline resistance gene: Km = kanamycin resistance gene. Coordinates are in kilobases. Theinner lines refer to distances measured in heteroduplex analysis

tetracycline resistance gene (See Fig. 3). In accordance with our physicalmapping, pGC530 does no more confer resistance to tetracycline. Insertionof a transposon not only abolishes the function of the gene into which theelement lands but it can also be polar, affecting the expression of the geneslocated downstream on the transcriptional wave (Kleckner et al., 1975). Forinstance, Tn2 can land in the Sulfonamide resistance gene of a plasmid andthis inhibits the expression of the neighbouring streptomycin resistance gene.

Since transposons behave like mutagens and since their insertion canreadily be physically mapped, they appear as valuable tools for the geneticalanalysis of plasmids. Tn7 has recently been used to analyse the transfer genesof the plasmid RP4 (Barth et al., 1978).

Transposons can sometimes be excised from a replicon where they havebeen inserted but the excision frequency is usually lower than the insertionfrequency. Moreover, the process is often not precise. In our hands, a straincarrying pGC530 does not revert to tetracycline resistance, indicating thatTn951 does not excise accurately at a detectable frequency.


6/9

518 Cornelis, G.D. Structure of the TransposonsFive years ago , Sh arp a nd CO-workers 1973) discovered inverted rep eats o nplasmid DNA. These inverted repeats consist of identical DNA Segmentsoccuring twice in a genome but in opposite orientation. When a DNA frag-ment containing such inverted repeats or palindromes is submitted to hetero-dup lex analysis, intras trand anne aling of these fragments takes place giving amu shroo m like structure (Fig. 4), consisting of a double s trand ed stem an d asingle stranded loop.Later on, the m ushroo m like structure observed by S harp et al. turned ou tto contain tetracycline resistance genes, in the loop. Moreover, the tetracyclinetranspo son of Kleckner et al. (1975) gave the sam e structure in he terod up lexanalysis. Other transposons were also found to consist of a DNA sequenceflanked by two inverted repeats. For instance, Tnl is flanked by a repeatedsequence abo ut 140 base p airs long (Rubens et al., 1976) and Tn5 is flanked bya repeated seq uen ce ten times longer (Berg et al., 1975). These two inv ertedrepeats do not correspond to any known Insertion Sequence. Tn9, on theothe r hand . is bordered by the well know n sequenc e IS1 (Starlinger an d Sa-edler, 1976) but repeated in tandem. Transposons are thus, often, if not al-ways, flanked by re peated sequences.

Fig. 4. Homoduplex format ion of pG ClDNA. The arrow indicates a small doublestranded DNA region due to reannealing o fsmall inverted repea ts


7/9

Bacterial Transposons 5 19E. Me chanism of TranspositionAlthough a nu m be r of mod els have been propo sed, we have, so far, n o clearunders tanding of the transposition mechanism. Experimental data from Hef-fron et al. (1977) suggest that a central region and the termini of Tn2 arerequired for transposition. The m ost attractive interpretation of their da ta isthat the term inal s equen ce of the transposon serve a structural role while thecentral region encodes an enzyme required for transposition. Apart fromthat. it is not yet clear w heth er the process requires the recogn ition of a specialsequence on the target.

F. Ro le o f the T ransposons in the Evolution of the Bacterial Plasm idsThe discovery of transposab le elements seems to accoun t significantly for thestructural and genetic diversity of nlasmids. It has been known for long in-deed tha t there is no relation between the genes carried by a plasmid an d thetype of plasm id. For instance, the TEM -lactamase can be coded by plasm idsthat belong to, at least, 14 different incompatibility groups (Matthew andHedges. 1976). It is very tem pting to believe that the spre ad of Tn l an d Tn2accoun ts for this situation. In accordance with this hypothesis, Heffron et al.(1975 b) showed that, inde ed, a great variety of different plasm ids en codingthe TEM lactamase contain a 4,5 Kb long sequence in common, while theTEM gene itself is mu ch smaller.

The evolution of plasmids would thus essentially be due to terminus-site-specific recomb ination. Plasmids themselves seem to consist of two Parts: o neis essential for replication and transfe r of the plasm id, while the o ther is a suc-cession of transposo ns (an d defective transposons?) acquired an d lost durin gthe history of the plasmid. In other words. plasmids would be nothing elsethan genetic carriers whose function is to replicate and propagate transpo-sons.

G. Origin of the Transposons Carried GenesSo far, little is know n abo ut the origin of the transposons carried genes. Forinstance, the TEM -lactamase coded by Tn l and Tn2 appea rs different fromall the know n chro mo som al -lactamases.For the lactose fermentation genes, however, we observed th at the Tn95 1coded op ero n is identical to the E. coli chromosom al lactose ope ron . In hetero-duplex analysis. we detected 5,6 Kb homology and this length accounts forthe genes encoding the repressor. the -galactosidase and the permease(Fig. 5) (Corn elis et al.. 1978). This indicates that, at least in som e case, atransposon coded and a chromosom al (to date non transposable) operon mayevolve from a co mm on ancestor.


8/9

Cornelis. G.

Fig. 5. Diagram of an h etero dup lex molecule showing the 5.6 Kb homology between the E. colichromosomal lactose oper on (here carried by X hdlac) and the Tn95 1 lac operon. here carried byp G C l

ReferencesBarth. P.T.. Datta, N., Hedges. R. W.. Grinter. N. J.: Transposition of a deoxyribo nucleic acid en-cod ing trim ethoprim and streptomy cin resistances from R483 to other replicons. J. Bacteriol.125,800-8 10 1 976)Barth, P.T.. Grinter. N. J.. Bradley. D. E.: Conjugal transfer System of plasmid RP4: Analysis bytransposon 7 insertion. J. Bacteriol. 133,43-52 (1978)Benn ett. P. M.. Grin sted. J., Choi. C. L., Richmond, M. H.: Characterization ofTn501. a transposondeter min ing resistance to m ercuric ions. Molec. Gen . Genet. 159, 101-106 ( 1 978)Bennett. P. M., Richmond. M. H.: Translocation of a discrete piece ofdeoxy ribonucleic acid carry-ing an a m p gene between replicons in Escherichia coli. J. Bacteriol. 12 6,1 -6 (1976)Berg. D.N.. Davies. J.. Allet. B., Rochaix. J.-D.: Transposition of R factor genes to bactqrio-phage X . Proc. Na tl. Aca d. Sci. USA7 2,3628-3632 (1975)Cornelis. G.,Ghosal. D.. Saedler. H.: Tn95 1: A new transposon carrying a lactose ope ron. M olec.Ge n. Gene t. 160,215-224 (1978)Foster. T. J.. H owe. T.G . B.. Ric hm ond . K. M.V.: Transloca tion of the tetracyc line resistancede term ina nt from R100-1 to the E. coli K12 chrom osom e. J. Bacteriol. 124,1153-1 158 (1975)Go ttesm an, M . M., Rosner. J. L.: Acquisition of a determinant for chloramphenicol resistanceby coliphage X . Proc. Natl. Acad . Sci. USA72,5041-5045 (1975)Hedges. R. W.. Jacob. A. E.: Transposition of ampicillin resistance from RP4 to other replicons.Molec. Ge n. Gene t. 1 32,3 1-40 ( 1 974)Heffron. F.. Bedinger. P.. Cham pou x, J. J., Falkow. S.: Deletions affecting the transposition of anantib iotic resistance gene . Proc. Natl. Acad. Sci. USA74,702-706 (1977)Hef f ron ,F., Ru ben s, C.. Falkow. S.: Translocation of a plasmid DNA seq uence which mediatesampicillin resistance: Molecular nature and specificity of insertion. Proc. Natl. Acad.Sci.USA 72,3623-3627 (1 975 a )Hef'fron. F.. Sublett, R.. Hedges. R.W.. Jacob. A.. Falkow. S.: Origin of the TEM -lactamasegen e found on plasmids. J . Bacteriol. 122,250-256 (1975 b)ueckner. N.. Chan. R. K.. Tye, B.-K.. Botstein. D.: M utagenesis by insertion of a drug -resis tanceelem ent carrying an inverted repetit ion. J. Mo l. Biol. 97,561-575 (1975)Kretschmer. P.J.. Cohen . S. N.: Selected translocation of plasmid genes: Frequency an d regionalSPecificityof translocation of the Tn3 element. J. Bacteriol. 130,888-899 (1977)

"t thew. M.. Hedges, R . W.: Analytical isoelectric focusing of R factor-determ ined -lactam ases:c ~ r r e l a t i o nwith plasmid c om patib ility. J. Bacteriol. 125,713-718 (1976)


9/9

Bacterial Tran sposo ns 52 1Rubens. C., Heffron. F.. Falkow, S.: Transposition of a plasmid deo xyribonucleic acid sequen cethat mediates ampicillin resistance: Independence from host rec functions and orientationof insertion. J. Bacteriol. 128 ,42 543 4 (1976)Shapiro. J.A..Sporn. P.:Tn402: A new transposable elemen t determining trim ethoprim resistancethat inserts in bacteriophage h .J. Bacteriol. 129,1632-1635 (1977)Sharp, P.A. .Cohen. S.N .. Davidson, N.: Electron microscope heteroduplex studies of sequencerelations a m on g plasmids of E. coli. 11. Structure of dru g resistance (R ) factors and F ftictors.J. Mol. Biol. 75,235-255 (1973)Starlinger. P. . Saedler. H.: 1s-elemen ts in microorganisms. Curr. Top. Microbiol. Imm unol. 75,11 1-152 (197 6)

Documents

513 Bacterial Transposon