A clinical isolate of transposon Tn5 expressing streptomycin resistance in Escherichia coli.

The central region of transposon Tn5 carries three antibiotic resistance markers: neo, ble, and str. The str gene codes for a phosphotransferase that inactivates streptomycin. This activity is phenotypically expressed in several gram-negative bacteria but not in Escherichia coli. We identified a Tn5 variant in E. coli clinical isolates that express streptomycin resistance. This transposon carries a 6-base-pair deletion within the str gene, near the 3' end. The same kind of mutation had been previously obtained experimentally from Tn5.     

Tn5 carries a streptomycin resistance determinant downstream from the kanamycin resistance gene

In Rhizobium meliloti, Tn5 conferred resistance not only to kanamycin but to streptomycin, as well, in Escherichia coli, however only to kanamycin. Using in vitro recombinant DNA techniques, it was shown that the streptomycin resistance determinant was located downstream from the kanamycin resistance gene in the unique central region of Tn5. Expression of various cloned fragments of Tn5 suggested that both kanamycin and streptomycin resistance genes were transcribed from the same promoter. E. coli mutants allowing the expression of streptomycin resistance from Tn5 were isolated. The differential expression of the streptomycin resistance gene provides a simple selection/counterselection criterion, using only streptomycin in transfer experiments of Tn5 between E. coli and R. meliloti.     

The transposon Tn5 carries a bleomycin-resistance determinant

Transposon Tn5 carries a determinant for resistance to bleomycin (Bm). Deletion mapping and cloning experiments have shown that this determinant, gene ble, is located between the determinant for kanamycin (Km) and neomycin resistance (gene neo) and the determinant for streptomycin resistance (gene str). Genes neo, ble, and str belong to an operon controlled by the common promoter. The Mr of the ble product, as determined by polyacrylamide gel electrophoresis, is 12000 to 13000.     

Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in E. coli. A Tn5* mutant able to express streptomycin resistance in E. coli was isolated. With this mutant, it was demonstrated that in E. coli the expression of the three resistance genes is coordinated in a single operon.     

Shuttle vectors conferring hygromycin B resistance to E. coli and to mammalian cells. Differential expression of carboxyterminal fusion proteins.

Shuttle vectors expressing resistance to hygromycin B in both E. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of the neo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive in E. coli.     

Identification of a Tn5 determinant conferring resistance to phleomycins, bleomycins, and tallysomycins

Tn5 conferred resistance to the related antibiotics, phleomycins, bleomycins, and tallysomycins in Escherichia coli and Salmonella typhimurium. For pure phleomycins the level of resistance was influenced by the structure of the terminal basic group. Deletion derivatives of a pBR322::Tn5 plasmid were used to show that the phleomycin resistance determinant is located between the previously identified neomycin and streptomycin resistance determinants. The pattern of expression of phleomycin and neomycin resistance in the deletion derivatives suggests that the phleomycin resistance gene is transcribed from the same promoter, PL, which is essential for expression of neomycin and streptomycin resistance. The location of the phleomycin resistance determinant correlates with the location of an open reading frame in the Tn5 sequence, which codes for a polypeptide of 126 amino acids.     

Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells.

A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli. This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter. It induced kanamycin resistance in E. coli, as well as resistance to the drug G418 in rat and mouse fibroblasts. Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction. After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium. In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E. coli. In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418. Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts. However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies.     

Plasmid pB8 is closely related to the prototype IncP-1beta plasmid R751 but transfers poorly to Escherichia coli and carries a new transposon encoding a small multidrug resistance efflux protein

The IncP-1beta plasmid pB8, which confers resistance to amoxicillin, spectinomycin, streptomycin, and sulfonamides, was previously isolated from a sewage treatment plant. It was found to possess abnormal conjugative transfer properties, i.e., transfer to Escherichia coli by conjugation or electroporation could not be detected. We showed in this study that plasmid pB8 is transferable to E. coli by conjugation, but only at low frequencies and under specific experimental conditions, a phenomenon that is very unusual for IncP-1 plasmids. Determination of the complete 57,198bp pB8 nucleotide sequence revealed that the backbone of the plasmid consists of a complete set of IncP-1beta-specific genes for replication initiation, conjugative plasmid transfer, stable inheritance, and plasmid control with an organisation identical to that of the prototype IncP-1beta plasmid R751. All of the minor differences in the pB8 backbone sequence compared to that of R751 were also found in other IncP-1beta plasmids known to transfer to and replicate in E. coli. Plasmids pB8 and R751 can be distinguished with respect to their accessory genetic elements. First, the pB8 region downstream of the replication initiation gene trfA contains two transposable elements one of which is similar to Tn5501. The latter transposon encodes a putative post-segregational-killing system and the small multidrug resistance (SMR) protein QacF, mediating quaternary ammonium compound resistance. The accessory genes in this region are not responsible for the poor plasmid transfer to E. coli since a pB8 deletion derivative devoid of all genes in that region showed the same conjugative transfer properties as pB8. A Tn5090/Tn402 derivative carrying a class 1 integron is located between the conjugative transfer modules. The Tn5090/Tn402 integration-sites are exactly identical on pB8 and R751 but in contrast to R751 the pB8 element carries the resistance gene cassettes oxa-2 for amoxicillin resistance and aadA4 for streptomycin/spectinomycin resistance, the integron-specific conserved segment consisting of the genes qacEDelta1, sul1, and orf5, and a truncated tni transposition module (tniAB). Although future work will have to determine the molecular basis for the poor transfer of pB8 to E. coli, our findings demonstrate that the host-range of typical IncP-1 plasmids may be less broad than expected.     

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.     

Mutations in multicopy Tn10 tet plasmids that confer resistance to inhibitory effects of inducers of tet gene expression.

Escherichia coli K-12 strains that carry the Tn10 tetracycline resistance determinant (tet) on multicopy plasmids are hypersensitive to 5a,6-anhydrotetracycline and heated chlortetracycline, two tetracycline derivatives that are relatively more effective as inducers of tet gene expression than as inhibitors of bacterial growth. Twenty spontaneous mutations that confer resistance to anhydrotetracycline (Atr) and resistance to heated chlortetracycline (Ctr) were isolated and characterized. All of these Atr mutations are located in the Tn10 tet region; the majority (18 of 20) have no effect on tetR repressor function. Atr mutations can increase, reduce, or eliminate the phenotypic expression of plasmid tetracycline resistance (Tcr). IS insertions that result in an Atr Tcs phenotype are clustered in a 150-base-pair promoter-proximal region of the tetA resistance gene. Some Atr mutations reduce expression of the tetA gene by altering either the tetR repressor or the tetA promoter. In addition, it appears that E. coli cannot tolerate constitutive expression of the wild-type tetA gene from a multicopy plasmid containing a tetR deletion. These observations support the proposal that high level expression of the 36-kilodalton tetA gene product inhibits the growth of E. coli. We speculate that this inhibition is related to the interaction of the tetA gene product with the cytoplasmic membrane.     

Analysis of the nourseothricin-resistance gene (nat) of Streptomyces noursei

A gene (nat) conferring resistance to the streptothricin antibiotic nourseothricin (Nc) was cloned from the producer Streptomyces noursei into Streptomyces lividans on the vector pIJ702 to form pNAT1. The nat gene was localized on a 1-kb SalI-MboI fragment, which also carries the nat promoter. Divergent promoter activity from the nat promoter region was identified on the cloned fragment using promoter probe plasmids pIJ486 and pIJ487. The nat gene is not expressed from its own promoter in Escherichia coli as shown by its failure to promote cat expression in promoter-less plasmid pBB100 and by the expression of NcR in only one orientation, when cloned in pUC19. In S. lividans 7A, harbouring plasmid pNAT1, an Nc-acetylating activity (NAT) was associated with the cloned resistance gene. The substrate specificity of NAT correlated well with the substrate range of the acetyltransferase in S. noursei and Tn1825-determined streptothricin resistance in Gram-negative bacteria. Moreover, an extract of S. lividans carrying pNAT1 showed specific serological cross-reactivity with an extract of E. coli carrying Tn1825.     

Inversion of the metE-oriC-rpsE chromosome segment in Escherichia coli K12

We have described recently a large inversion of the Escherichia coli chromosome (designated udpPf1), including region of the chromosomal replication region (oriC). The udpPf1 inversion was induced by Tn10 transposon (metE::Tn10). It results in increased expression of the uridine phosphorylase gene (udp) which is closely linked to the metE gene. The data of conjugational and transductional experiments presented in this report demonstrate that the udpPf1 inversion covers a chromosomal segment extending over 12 min of the E. coli genetic map and including the rpsE, crp and metE::Tn5 markers. The results are presented indicating that the increased uridine phosphorylase activity is due to fusion of the udp gene to a more strong promoter located, probably, in the operon for ribosomal proteins cluster, near 73 min on the E. coli chromosome.     

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.