Plasmid R1drd-19 Research Articles

The R1 Conjugative Plasmid Increases Escherichia coli Biofilm Formation through an Envelope Stress Response

Differential gene expression in biofilm cells suggests that adding the derepressed conjugative plasmid R1drd19 increases biofilm formation by affecting genes related to envelope stress (rseA and cpxAR), biofilm formation (bssR and cstA), energy production (glpDFK), acid resistance (gadABCEX and hdeABD), and cell motility (csgBEFG, yehCD, yadC, and yfcV); genes encoding outer membrane proteins (ompACF), phage shock proteins (pspABCDE), and cold shock proteins (cspACDEG); and phage-related genes. To investigate the link between the identified genes and biofilm formation upon the addition of R1drd19, 40 isogenic mutants were classified according to their different biofilm formation phenotypes. Cells with class I mutations (those in rseA, bssR, cpxA, and ompA) exhibited no difference from the wild-type strain in biofilm formation and no increase in biofilm formation upon the addition of R1drd19. Cells with class II mutations (those in gatC, yagI, ompC, cspA, pspD, pspB, ymgB, gadC, pspC, ymgA, slp, cpxP, cpxR, cstA, rseC, ompF, and yqjD) displayed increased biofilm formation compared to the wild-type strain but decreased biofilm formation upon the addition of R1drd19. Class III mutants showed increased biofilm formation compared to the wild-type strain and increased biofilm formation upon the addition of R1drd19. Cells with class IV mutations displayed increased biofilm formation compared to the wild-type strain but little difference upon the addition of R1drd19, and class V mutants exhibited no difference from the wild-type strain but increased biofilm formation upon the addition of R1drd19. Therefore, proteins encoded by the genes corresponding to the class I mutant phenotype are involved in R1drd19-promoted biofilm formation, primarily through their impact on cell motility. We hypothesize that the pili formed upon the addition of the conjugative plasmid disrupt the membrane (induce ompA) and activate the two-component system CpxAR as well as the other envelope stress response system, RseA-sigma(E), both of which, along with BssR, play a key role in bacterial biofilm formation.

Hha, YbaJ, and OmpA regulate Escherichia coli K12 biofilm formation and conjugation plasmids abolish motility

Escherichia coli Hha is an environmental-response regulator of the pathogenic hemolysin operon, and Hha and the contiguous YbaJ are both induced 30-fold in E. coli biofilms (Appl. Microbiol. Biotechnol. 64:515, 2004). Here it is shown that Hha and YbaJ regulate biofilm formation since the hha/ybaJ deletion reduces biofilm mass in microtitre plates (81% in minimal medium, 50% in complex medium) and in flow cells (1,000-fold less surface coverage in minimal medium). The addition of the derepressed conjugative plasmid R1drd19, which increases significantly biofilm formation, eliminated motility completely in wild-type E. coli K12, promoted cell aggregation 27.18 +/- 0.05-fold, and produced a flatter biofilm. Deletion of hha/ybaJ or ybaJ restored motility (this motility phenotype may be complemented by providing hha(+)/ybaJ(+) or ybaJ(+) in trans) and reduced cell aggregation to that of the wild-type strain that lacks the conjugation plasmid. This increase in motility due to deleting hha/ybaJ was found to be due to 8-fold induction of fliA transcription. In addition, deletion of ompA reduced biofilm mass by 80% in both LB medium and LB medium with glucose. Also, Hha/YbaJ promotes conjugation since there was five-fold less conjugation in the hha/ybaJ mutant. It appears that conjugation plasmids promote biofilm formation by promoting cell aggregation, and that Hha and YbaJ increase biofilm formation by increasing conjugation and by decreasing motility when a conjugative plasmid (R1drd19) is present (YbaJ plays the most important role in this regulation of motility). When hha/ybaJ are deleted, there is less conjugation, less aggregation, more motility, and less biofilm.

Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage λ recombination functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1 drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved that in the presence of additional homologous regions in the targeting DNA, strand exchanges occurred exclusively within the longest regions of homology. A versatile panel of vectors was created to facilitate convenient PCR amplification of targeting DNAs containing various combinations of different antibiotic resistance genes and fluorescent markers. The choice of 5′ non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E. coli and is not restricted by plasmid size.

Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved that in the presence of additional homologous regions in the targeting DNA, strand exchanges occurred exclusively within the longest regions of homology. A versatile panel of vectors was created to facilitate convenient PCR amplification of targeting DNAs containing various combinations of different antibiotic resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E. coli and is not restricted by plasmid size.

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1 drd-19

Plasmid R1 drd-19 markedly improves the recombination deficiency of recB and recB recC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1 drd-19 cells. This paper further investigates the suppressive effect of plasmid R1 drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1 drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of superoligomerization of the tester pACYC184 plasmid in recB/R1 drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by λ Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recB recC recD genes and the EcoRI-C fragment of R1 drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.

Maintenance of bacterial plasmids: Comparison of theoretical calculations and experiments with plasmid R1 in Escherichia coli

Plasmid R1 was tested for stable maintenance in Escherichia coli K12. Populations carrying a transfer-negative derivative of plasmid R1drd-19 were grown exponentially for 72 generations in LB medium. Out of nearly 5000 cells none had lost the plasmid; hence the rate of loss of the plasmid is less than 3 X 10(-6) per cell and cell generation. Other experiments showed that the loss rate was less than or equal to 10(-7) per cell and cell generation. Plasmid R1 replication is controlled so that, on average, n copies are synthesized per cell and cell generation irrespective of the copy number (as long as it is at least one). A theoretical analysis was performed, based on the assumption that there is a Poissonian spread around n in each class of cells (each class defined by the number of R1 copies at birth). Two models for partitioning were analyzed, Equi-partitioning and Pair Site Partitioning; in the latter, each daughter is guaranteed one plasmid copy, whereas the rest of the copies are assumed to be randomly distributed. The copy number distribution was found to be fairly narrow in both cases, with at least 90% of the population in the range n/2-3n/2 for baby cells and in the range n-3n for cells just before cell division. Plasmid-free cells were estimated to appear at a frequency which for the copy number of R1 (n = 3-4) was 1.5 X 10(-3) - 8 X 10(-5) for equipartitioning and 6 X 10(-3) - 6 X 10(-4) for pair site partitioning, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteus mirabilis chromosome mobilization by plasmid D: a physical characterization.

Plasmid D, a hybrid of plasmids P-lac and R1 drd19, mediates polarized chromosome mobilization from one origin in Proteus mirabilis strain PM5006, while the parental plasmids neither individually nor combined mobilize this chromosome. To elucidate its acquired mobilizing ability plasmid D was characterized physically in relation to P-lac and R1 drd19. Restriction patterns of these plasmids were compared and it was shown that D consists of P-lac and only the r-determinant (r-det) of R1 drd19. A mechanism for the formation of plasmid D, via transduction of the r-det and subsequent transposon-like integration into P-lac, involving insertion sequence IS1, was suggested. Evidence for aberration in plasmid D DNA as a result of r-det integration into P-lac was attributed to IS1 elements which flank the r-det. Recombination regions of parental plasmid DNA were located on HindIII fragments alpha and beta of plasmid D and were subsequently inserted in vitro into IncP-1 plasmid RP4 that fails to mobilize the P. mirabilis chromosome. RP4::HindIII alpha plasmids did not mobilize the latter chromosome, but rendered the Proteus host lac+. RP4::HindIII beta plasmids pMC1 and pMC17, containing the fragment in opposite orientations, mobilized the P. mirabilis chromsome. For pMC17, mobilization was indistinguishable from that of plasmid D, i.e. having the same orientation and the same single origin. However, mobilization promoted by pMC1 was from two distinctly different origins, different from that of pMC17. This apparently deviates from known examples where inversion of homologous DNA inserted into plasmids leads to mobilization from the same origin but in reverse direction.

The instability of plasmid pON5300 after its transformation into Escherichia coli K 12.

The plasmid R1drd-19 Km- and its derivative pON5300 were split by the PstI restriction enzyme; they differed in a 2.4 Mg/mol fragment which was present only in the pON5300. Both plasmids were present in 1-2 copies per chromosomal equivalent in E. coli JC5455 cells. The pON5300 is stable in its original mutagenized host but it segregates the enlarged region of the plasmid molecule after transformation into non-mutagenized E. coli JC5455. The possible explanation of this instability is discussed.

Experimental and mathematical models of Escherichia coli plasmid transfer in vitro and in vivo.

Little is known about the factors that govern plasmid transfers in natural ecosystems such as the gut. The consistent finding by earlier workers that plasmid transfer in the normal gut can be detected only at very low rates, if at all, has given rise to numerous speculations concerning the presence in vivo of various inhibitors of plasmid transfer. Plasmids R1, R1drd-19, and pBR322 were studied in Escherichia coli K-12 and wild-type E. coli hosts in two experimental systems: (i) gnotobiotic mice carrying a synthetic indigenous microflora (F-strains) which resemble in their function the normal indigenous microflora of the mouse large intestine, and (ii) anaerobic continuous-flow cultures of indigenous large intestinal microflora of the mouse, which can simulate bacterial interactions observed in the mouse gut. Mathematical models were developed to estimate plasmid transfer rates as a measure of the "fertility," i.e., of the intrinsic ability to transfer the plasmid under the environmental conditions of the gut. The models also evaluate the effects of plasmid segregation, reduction of the growth rates of plasmid-bearing bacterial hosts, repression of transfer functions, competition for nutrients, and bacterial attachment to the wall of the gut or culture vessel. Some confidence in the validity of these mathematical models was gained because they were able to reproduce a number of known phenomena such as the repression of fertility of the R1 plasmid, as well as known differences in the transmission and mobilization of the plasmids studied. Interpretation of the data obtained permitted a number of conclusions, some of which were rather unexpected. (i) Fertility of plasmid-bearing E. coli in the normal intestine was not impaired. The observed low rates of plasmid transfer in the normal gut can be explained on quantitative grounds alone and do not require hypothetical inhibitory mechanisms. (ii) Conditions for long-term spread and maintenance throughout human or animal populations of a diversity of conjugative and nonconjugative plasmids may be optimal among E. coli strains of low fertility, as are found among wild-type strains. (iii) E. coli strains carrying plasmid pBR322 plus R1drd-19 were impaired in their ability to transfer R1drd-19, but strains carrying pBR322 were significantly better recipients of R1drd-19 than a plasmid-free recipient E. coli. (iv) Long-term coexistence of plasmid-bearing and plasmid-free E. coli, in spite of undiminished fertility, appeared to be due to a detrimental effect of the plasmid on the growth rate of its host bacterium, rather than due to high rates of plasmid segregation. (v) Mathematical analysis of experimental data published by earlier investigators is consistent with the conclusion that plasmid transfer occurs consistently in the human gut, but that the resulting transconjugant E. coli populations are too small to be detected regularly with the culture methods used by earlier investigators. It is concluded that the long-term interactions observed were often the consequences of minor differences in parameters such as growth rates, fertility, rates of segregation, etc., which were too small to be detected except by precise mathematical analysis of long-term experiments, but which were nevertheless decisive determinants of the ultimate fates of the plasmids and their hosts.

Isolation of the kanamycin resistance region (Tn2350) of plasmid R1drd-19 as an autonomous replicon.

We have isolated a circular form of Tn2350, an IS1-flanked kanamycin resistance transposon forming part of the plasmid R1drd-19. This circle (pTn2350::9.6 kilobases) contains a single IS1 element and probably arises by recombination between the two directly repeated Is1 sequences of Tn2350. It can be used to transform Escherichia coli to kanamycin resistance. It is capable of autonomous replication but is not maintained stably in dividing cells and segregates under nonselective conditions. Cloning of a segment of pTn2350 on a conditional plasmid vector allowed us to assign the replication functions of this plasmid to a 1.6-kilobase restriction fragment. The plasmid R1drd-19 can thus be considered as a cointegrate between two replicons separated by IS1 sequences.

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1.

Specialized transducing lambda phages, lambda oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of lambda oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA+, but sensitive to the concerted action of PstI fragments F1 and F2. This mutant was previously assumed to be of dominant type. Physical mapping of the lambda oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%-2% per cell generation, which is consistent with their lack of the R1par region.

Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1.95 +/- 0.05 x 10(6) for plasmids pBY1 and pBY2, respectively. Plasmid DNA purified by cesium chloride-ethidium bromide gradient centrifugation was used to transform a restriction- and modification-negative strain of Escherichia coli. Penicillin G- and tetracycline-resistant transformants were screened for the presence of plasmid DNA. A plasmid band corresponding to a molecular weight of 1.95 x 10(6) was present in all transformants tested. Curing experiments demonstrated that the plasmid, referred to as pBY22 when present in transformants, was responsible for penicillin G and tetracycline resistance. Plasmid pBY22 was mobilized and transferred to other E. coli strains by plasmid R1drd-19. Stability of pBY22 was examined in different E. coli strains and was shown to be stably maintained in both restriction-negative and restriction-positive strains. Unexpectedly, pBY2 and pBY22 were resistant to digestion by 12 different restriction endonucleases.

The structure of R1drd19: a revised physical map of the plasmid.

We have analyzed derivatives of the plasmid R1drd19 carrying the transposon Tn10 by electron microscopy following denaturation and renaturation of the molecules, and by digestion with various restriction enzymes, gel electrophoresis and Southern blotting. We show: 1) that the published restriction map of R1drd19 is inconsistent with our results. We present a modified map which is consistent with our data. 2) that R1drd19 carries a single resident copy of the element IS10 which is normally associated with Tn10 as an inverted repeat, and 3) that R1drd19 carries three copies of the insertion element IS1 in the resistance determinant region.

The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19.

The region of plasmid R 1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wild-type R 1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.

Partitioning of plasmid R1 in Escherichia coli: II. Incompatibility properties of the partitioning system

A theoretical as well as an experimental study of the effect of the partitioning system on plasmid R1 drd-19 incompatibility was performed. The theoretical numerical analysis (by computer) was based upon the following assumptions: (i) The partitioning ( par) mechanism is independent of the replication ( rep) and replication control ( cop) mechanism. (ii) A par mutation causes random (binomial) distribution of plasmid copies between the daughter cells at cell division. (iii) In the par + case, the plasmid copies are equipartitioned and selected randomly for partitioning. (iv) Selection of plasmid copies for replication is random. (v) Two different replication control systems were considered: Model 1 assumes that the plasmid copy number is set to exactly 2n before cell division, whereas in Model 2 exactly n copies are synthesized per cell per cell cycle. Numerical analysis was performed for the n values 2–8. The result was that in all cases ( par +/ par +, par +/ par, par/par), steady states with respect to copy number distribution within the heteroplasmid population were rapidly (within five or six generations) established, giving constant loss rates. The rate of loss was slightly higher in the par/par case than in the other two. The two replication control models gave almost identical loss rates. In the par +/ par case, the par + plasmid had an advantage over the par plasmid. The experimental approach was to create heteroplasmid populations of Escherichia coli by introducing two genetically marked derivatives of plasmid R1 drd-19 and then follow the reduction in the relative size of this population during exponential growth in LB medium. The loss rate was essentially the same in the par +/ par + and par +/ par combination and slightly higher in the par/par case, suggesting that plasmid incompatibility mainly is caused by the replication and copy number control system. In the par +/ par situation, the par + plasmid had a pronounced advantage over the par plasmid. The par region of plasmid R1 (without the basic replicon) was cloned onto the vector pSF2124. A par (deletion) mutation was not complemented by par +. Plasmid pSF2124, which does not seem to carry a par system of its own, could use the R1 par system, adding to the conclusion that par is independent of rep and cop. Plasmids pSF2124 and R1 drd-19 are completely compatible, whereas plasmid pSF2124 carrying the R1 par system and plasmid R1 drd-19 showed a weak incompatibility although the copy numbers of the two plasmids were not affected in the heteroplasmid cells. Hence, the partitioning system causes incompatibility, but the effect is weak compared to that of the cop system. The result is consistent with some sort of assortment of plasmids before partitioning.

Isolation of an IS1 flanked kanamycin resistance transposon from R1drd19.

We have isolated and identified an IS1-flanked transposon from the plasmid R1drd19. This transposon specifies resistance to kanamycin and is 10.4 kg long. It exhibits a frequency of transposition two orders of magnitude lower than that of the smaller, IS1-flanked transposon Tn9. We have named it Tn2350.

Partitioning of plasmid R1 in Escherichia coli, : I. Kinetics of loss of plasmid derivatives deleted of the par region

The stability of inheritance of plasmid R1 drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage λ was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1 drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1 drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1 drd-19 carries genetic information for partitioning ( par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control ( cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene ( Yoshikawa, 1974, J. Bacteriol.,, 118, 1123–1131).

Plasmid R1 in Salmonella typhimurium: Molecular instability and gene dosage effects

Plasmid R1 drd-19 and two of its copy mutants (pKN102 and pKN103) were transferred from Escherichia coli to Salmonella typhimurium, where the expression of the copy mutations was studied further. The copy number (ratio of plasmid DNA to chromosomal DNA) was the same in S. typhimurium and in E. coli. The activities of the plasmid-coded antibiotic-metabolizing enzymes β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylyltransferase as well as the resistances to ampicillin and streptomycin were proportional to the gene dosage up to at least a threefold increase in the steady state plasmid copy number, whereas resistance to chloramphenicol showed no increase with increased number of plasmid copies per chromosome equivalent. Also the resistance to rifampicin was affected since S. typhimurium cells became more sensitive the higher the copy number of the resident plasmid. Furthermore, plasmid R1 showed molecular instability in S. typhimurium cells since there was a tendency to dissociate into resistance transfer factors and resistance determinants and also to form miniplasmids. This tendency to instability was more pronounced the higher the plasmid copy number.

Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19

Plasmid R1drd-19 is present in a small number of copies per cell of Escherichia coli. The plasmid was reduced in size by in vivo as well as in vitro (cloning) techniques, resulting in a series of plasmid derivatives of different molecular weight. All plasmids isolated contain a small region (about 2 x 10(6) daltons of deoxyribonucleic acid) of the resistance transfer factor part of the plasmid located close to one of the IS1 sequences that separates the resistance transfer factor part from the resistance determinant. All these derivatives were present at the same copy number, retained the incompatibility properties of plasmid R1drd-19, and were stably maintained during cell division. Genes mutated to yield copy mutations also were found to be located in the same region.

Cell-free synthesis of proteins coding for mobilisation functions of ColE1 and transposition functions of Tn3

A hybrid plasmid, pJC74, carrying the large (3.35 Md) EcoRI- PstI fragment of ColE1, the 1.3 and 1.8 Md PstI fragments of plasmid R1 drd19 (containing a part of Tn3 and known to specify transposition functions), and a part of λ bacteriophage DNA carrying the fused cohesive ends of λ ( cos) was constructed. Supercoiled DNA of pJC74 and a series of deletions (series pJC75) were used in a cell-free coupled transcription-translation system. Analysis of the proteins produced allowed the identification of a 28 to 30 kd protein and/or a 3 kd protein responsible for mobilisation of the plasmids in sex-factor-promoted conjugation. Furthermore, three protein bands of 12, 12.5 and 13 kd were correlated with the presence of a portion of the Tn 3 transposon previously. shown to code for transposition functions which can complement in trans. These latter and the 30 kd β-lactamase were the only proteins identified as Tn 3-specific by comparison of ColE1 with RSF2124.