Ampicillin Resistance Transposon Research Articles

Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli.

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.

Cloning of the genes that control formation of the fimbrial colonization factor antigen III (CFA/III) from an enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli strain 260-1 produces colonization factor antigen III and heat-labile enterotoxin. A 55-kb plasmid controlling the expression of the colonization factor antigen was isolated from this strain after it was labeled with ampicillin resistance transposon, Tn3. When this plasmid was introduced into E. coli K-12 strains, it induced the formation of pili that were morphologically and immunologically identical to those on the surface of 260-1 cells, as examined by electron microscopic observation and with the specific antiserum. The physical map of the plasmid was constructed, and the 17.4-kb region was found to be responsible for the expression of the pili.

Tn 3 transposition immunity is conferred by the transposase-binding domain in the terminal inverted-repeat sequence of Tn 3

A series of mutant terminal inverted repeats (IRs), having2 bp subtitutions at various sites within the 38-bp IR sequence of the ampicillin-resistance transposon Tn 3, were tested for transposition immunity to Tn 3. Mutations within region 1–10 in the IR did not affect transposition immunity, while mutations within region 13–38 inactivated the immunity function. These two regions corresponded to domain A which was not bound specifically by Tn 3 transposase and to domain B which was bound by the transposase, respectively. This indicates that specific binding of transposase to domain B within the IR sequence is responsible for transposition immunity.

Expression of glucose transporter messenger RNA in human fetal and adult brain

We have used the mutagenic potential of the ampicillin resistance transposon Tn3 and in vitro deletion techniques to study essential regions for maintenance of mini-F plasmids. Our parental mini-F plasmid contains the 40.3 to 40.8F and 43.1 to 49.3F sequences, a total of 6.7 kilobases (kb). From a spectrum of insertion and deletion mutants, we find only two insertion regions; they map near the 45.8- and 46.4-kb coordinates. In each region the orientation of Tn3 insertion is unique and different from that of the other region. Spontaneous deletions extend from either region in a common direction which is toward the 49.3-kb coordinate. One deletion plasmid, pBK138-2, which arose from a combination of in vitro and spontaneous deletion events, contains just the 44- to 45.8-kb sequences and the ampicillin resistance gene of Tn3. As shown by J. Wechsler and B. C. Kline (1980, Plasmid 4, 276–280), the 45.8- to 46.3-kb sequences specify F sensitivity to the plasmid curing agent, acridine orange. Since sensitivity to acridine orange is a property of normal F maintenance, the 45.8- to 46.35-kb sequences also likely are required for normal plasmid maintenance.

Mechanisms of transposition in bacteria.

The transposable elements of bacteria are diverse in size, functional arrangement, DNA sequence, and in their modes of transposition. We review here data suggesting that the kanamycin-resistance transposon Tn5 moves without replicating (conservative transposition), but the ampicillin-resistance transposon Tn3 is duplicated when it transposes, and that both the chloramphenicol-resistance transposon Tn9 and bacteriophage Mu are replicated in some events but not in others. A model is presented in which conservative and replicative transposition are alternative branches of a single pathway.

Expression of lipopolysaccharide O antigen in Escherichia coli K-12 hybrids containing plasmid and chromosomal genes from Shigella dysenteriae 1.

The requirement for both plasmid and chromosomal genes in the biosynthesis of Shigella dysenteriae 1 lipopolysaccharide O antigen was demonstrated in Escherichia coli-Shigella hybrids. A 6-megadalton S. dysenteriae 1 plasmid, designated pWR23, was phenotypically tagged with the Tn3 ampicillin-resistance transposon. The tagged plasmid, designated pWR24, was transferred by transformation or conjugal mobilization to a rough E. coli K-12 recipient. Although the resultant hybrids were agglutinated in S. dysenteriae 1 antiserum, they did not remove all of the anti-Shiga agglutinins in absorption experiments. Modified lipid A core structure was detected in these hybrids, but Shiga O antigen was not expressed. When the his+ locus of the S. dysenteriae 1 chromosome was transferred by transduction to E. coli K-12 containing pWR24, complete Shiga O antigen was expressed. Lipopolysaccharide extracted from these hybrids was indistinguishable chemically, electrophoretically, and serologically from native S. dysenteriae 1 lipopolysaccharide.

Identification and characterization of a second copy number control gene in mini-F plasmids.

We previously reported the existence of a series of chemically induced trans recessive copy-number mutations (cop) for mini-F plasmids and the existence of a similar series of cop mutations induced by insertion of the ampicillin resistance transposon Tn3. In this paper we describe the experiments showing that these two series of mutations are in different genes. Briefly, the experiments show that the one mutant series can complement the other, that the mutations map in distinct but adjacent regions, that the copy numbers of double mutants are the products of the copy numbers determined by the single mutations, and that Tn3 does not elevate copy number by a polar effect on the adjacent cop gene defined by chemical mutagenesis. We term the latter gene copA and the gene mutated by Tn3, copB. We also demonstrate here that copB mutations are recessive to the wild type allele. Further, we have characterized copB by deletion and recombinational analysis as the series of five 19- to 22-base-pair directly repeated sequences that had previously been designated incC-that is, one of the incompatibility genes. The evidence for this conclusion is that plasmids lacking two, three or five direct repeats have their copy number elevated proportionately. Possible mechanisms for copB control of replication are discussed.

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn 3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance ( amp r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.

Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids of Escherichia coli

Plasmid Rscl3, a small derivative of the plasmid R1, contains a region necessary for replication as well as a complete copy (4957 bp) of the ampicillin resistance transposon, Tn 3. We determined the nucleotide sequence of the replication region of Rscl3 to be 2937 bp and then compared this region (designated the 2.9-kb region) to the analogous region of pSM1, a small derivative of the plasmid R100 which has common ancestry with R1. Rsc13 and pSM1 were 96% homologous in this 2.9-kb region except for a discrete region of about 250 bp which showed only 44% homology. The sequence and distribution of nucleotide substitutions between Rsc13 and pSM1 supported a map of possible genes and sites which have previously been seen in the replication region of pSM1. Most of these genes and sites were shown to evolve by single base substitutions, but not by insertions or deletions. However, one of these genes, termed rep A2 (11000 dallons), was found in that region of Rsc13 and pSM1 which showed only 44% homology. Analysis of the amino acid sequence and predicted conformation of the two RepA2 polypeptides, however, suggested that they were very similar. We proposed that the repA2 region of R1 and R100 was replaced by a substitution of a short DNA segment from another plasmid which was evolutionarily related to R1 and R100 but had more divergence. This event may have been mediated by a mechanism similar to that of gene conversion as described in eukaryotic systems.

Mobilization of nonconjugative antibiotic resistance plasmids in Haemophilus ducreyi.

A clinical isolate of Haemophilus ducreyi was found to harbor three plasmids: a 23.5-megadalton (Mdal) phenotypically cryptic plasmid, a 7.0-Mdal ampicillin resistance plasmid, and a 4.0-Mdal sulfonamide resistance plasmid. The two smaller plasmids were transferable by conjugation to Haemophilus recipients, but only if the donor cell harbored the 23.5-Mdal plasmid as well, indicating that this large plasmid had mobilizing capabilities. Transfer was also possible to Escherichia coli recipients. Haemophilus influenzae transconjugants which had acquired both the 23.5-Mdal plasmid and one of the R-plasmids could subsequently retransfer the R-plasmid to other Haemophilus recipients at higher frequencies. A derivative of the 23.5 Mdal plasmid was isolated which was shown by restriction endonuclease analysis to contain an ampicillin resistance transposon and to have retained its conjugative ability.

Origin of Haemophilus influenzae R factors

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.

Molecular nature of a streptomycin and sulfonamide resistance plasmid (pBP1) prevalent in clinical Escherichia coli strains and integration of an ampicillin resistance transposon (TnA).

A small, nonconjugative plasmid, designated pBP1, was originally found in different fecal Escherichia coli serotypes isolated from a healthy proband. Of a total number of 130 hospital strains of E. coli subsequently studied, 8.5% yielded plasmid of the pBP1 type. This R plasmid specifies resistance to streptomycin (Sm) and sulfonamides (Su) and has a mass of 4.0 megadaltons. Inactivation of streptomycin is due to the aminoglycoside phosphotransferase APH-(3 "). A physical map was constructed by analysis with restriction endonucleases. Another small plasmid, pBP1-1, was isolated from one of the hospital strains and characterized as an enlarged pBP1 replicon containing an additional deoxyribonucleic acid sequence identified as a transposable element for ampicillin resistance (TnA). Plasmid pBP1-1 was cleaved by restriction enzymes for identification of the transposon sequence which codes for a TEM 1 beta-lactamase. The sequence organizations in the Sm Su plasmids RSF1010 and pBP1 were shown to be identical for regions specifying streptomycin and sulfonamide resistance, but different for the region containing the origin of replication and genes for replicative functions. Thus, RSF1010, which has been considered as the prototype of Sm Su plasmids, and pBP1, which is at least as frequent in clinical isolates as RSF1010, do not have a single common ancestor.

Genetic studies of F plasmid maintenance genes

We have used the mutagenic potential of the ampicillin resistance transposon Tn 3 and in vitro deletion techniques to study essential regions for maintenance of mini-F plasmids. Our parental mini-F plasmid contains the 40.3 to 40.8F and 43.1 to 49.3F sequences, a total of 6.7 kilobases (kb). From a spectrum of insertion and deletion mutants, we find only two insertion regions; they map near the 45.8- and 46.4-kb coordinates. In each region the orientation of Tn 3 insertion is unique and different from that of the other region. Spontaneous deletions extend from either region in a common direction which is toward the 49.3-kb coordinate. One deletion plasmid, pBK138-2, which arose from a combination of in vitro and spontaneous deletion events, contains just the 44- to 45.8-kb sequences and the ampicillin resistance gene of Tn 3. As shown by J. Wechsler and B. C. Kline (1980, Plasmid 4, 276–280), the 45.8- to 46.3-kb sequences specify F sensitivity to the plasmid curing agent, acridine orange. Since sensitivity to acridine orange is a property of normal F maintenance, the 45.8- to 46.35-kb sequences also likely are required for normal plasmid maintenance.

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.

Transposition of the Escherichia coli insertion element gamma generates a five-base-pair repeat.

We have determined DNA sequences surrounding the termini of the Escherichia coli insertion element gamma delta, both at its normal locus on the F (fertility) factor and at three different sites of insertion into the plasmid pBR322. After transposition, a five-base-pair pBR322 sequence is duplicated and appears in direct orientation adjacent to each end of the element. No such duplication flanks the ends of gamma delta in F, and there is no apparent homology between the sequences surrounding gamma delta in F and the five-base-pair duplications generated by insertion. These findings suggest that the duplications are not essential for transposition and that they do not act to direct gamma delta to a homologous site in the target chromosome. In addition, we find that the 35-base-pair inverted repeat that comprises the termini of gamma delta is strikingly similar in sequence to the ends of both the ampicillin-resistance transposon Tn3 and a 200-nucleotide-long sequence on the plasmid pSC101 which has been shown to mediate recombination with phage f1 replicative form. Within the terminal region, there is a specific heptanucleotide sequence common to each of the above elements and to bacteriophage Mu, all of which generate five-base-pair repeats upon insertion.

Location of promoter sites on plasmid NTP1 which contains the ampicillin resistance transposon Tn1701

We have modified the gene 32 protein electron microscopic method of Delius et al. (1973) so that promoter regions on covalently closed circular DNA templates may be mapped relative to specific restriction endonuclease cleavage sites. Using this method, four promoter regions have been located on the physical map of plasmid NTP1 at 0.01, 0.12, 0.33 and 0.45 NTP1 units. Transcription from all four promoters is to the right. Two of these promoters, at 0.01 and 0.12 NTP1 units, are located within the ampicillin resistance transposon recently designated Tn1701. The promoter located at 0.01 NTP1 units was found to be the strongest of the four promoters; it is proximal to the β-lactamase gene which is responsible for ampicillin resistance.

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.

Vibrio cholerae hybrid sex factor that contains ampicillin transposon Tn1

The ampicillin resistance transposon Tn1 was translocated from the R plasmid RP4 to the Vibrio cholerae conjugative plasmid, P. The hybrid sex factor P::Tn1 was highly transmissible and expressed the biological activities of the P factor. In addition, P::Tn1 facilitated transfer of RP4 to V. cholerae recipients. Physical studies of P::Tn1 indicated that the Tn1 transposon was added to the otherwise unaltered P plasmid.

Nonintegrated plasmid-folded chromosome complexes: Genetic studies on formation and possible relationship to plasmid replication

When folded chromosomes are purified from plasmid-containing bacteria, a reproducible fraction of the host's covalently closed, circular (CCC) plasmid DNA copurifies with the chromosomes. From this copurification, we infer the existence of nonintegrative plasmid-chromosome (NPC) complexes. Previously, we noted that plasmids dependent on DNA polymerase III and with stringent control of replication complex to a greater extent than plasmids dependent on DNA polymerase I and with relaxed control of replication. We have examined this subject in more depth and find that: (i) The composite plasmids formed by in vitro recombination of a “stringent” with a “relaxed” replicon complex to chromosomes at the frequency of the component replicon which directs replication; (ii) all of the detectable replicative intermediates, but only 25% of the CCC forms, of plasmid ColE1 complex to chromosome; and (iii) when a mini-F plasmid is deleted for the DNA sequences which include the primary origin of replication, the complexing frequency decreases 30 to 40%. We conclude from these findings that NPC complexes either indirectly or directly relate to plasmid replication. Further, we find that the EcoRI kan + fragments of pML31 and the ampicillin resistance transposon, Tn 3, promote complexing of both ColE1 and mini-F plasmids to host chromosomes. The biological significance of this latter complexing is unknown. However, we conclude from these studies and from point (iii) that complexing is determined in part by unique plasmid sequences.

Molecular Properties of Transmissible R Factors of Haemophilus influenzae Determining Tetracycline Resistance

The tetracycline-resistant Haemophilus influenzae strains LU121 and FR16017, recently isolated in West Germany, each harbour a plasmid; that of the former (pLU12U) has a mol. wt of 31.5 X 10(6) and that of the latter (pFR16017) has a mol. wt of 33 X 10(6). Conjugation and DNA-DNA hybridization studies have shown that both plasmids are self-transmissible and carry tetracycline-resistance genes. The purified plasmid DNA of H. influenzae strain LU121 transformed a sensitive Escherichia coli strain to tetracycline resistance. The two R factors are closely related to the H. influenzae plasmid specifying ampicillin resistance (pKRE5367). Electron microscope DNA heteroduplex analysis indicated that pLU121 and pFR15017 probably carry the tetracycline-resistance transposon TnD and that pKRE5367 probably carries the ampicillin-resistance transposon TnA. There is more than one integration site for the insertion which probably represents TnD in pFR15017. All three plasmids have a similar plasmid core and could have a common evolutionary origin.