Expression of the gene for tetracycline resistance of plasmids R6 and RP4 in bacteria of the family Enterobacteriaceae]

It was found that manifestation of the tetracycline resistance gene depended on the type of the plasmid containing the gene. The tetracycline resistance gene was subject to less repression in plasmid R6 than in plasmid RP4. Sensitivity of the initial plasmid-free bacteria varied within lower dose ranges than that of the plasmid-carrying strains. Regulation of the tetracycline resistance gene manifestation in the given plasmid may change in different bacterial hosts, i.e. in different cytoplasmic environment at different gene background.     

Host dependence of RP1-specified resistance to ampicillin: differential expression in Escherichia coli and Rhizobium leguminosarum.

Rhizobium leguminosarum L4 is able to serve as a host for the plasmid RP1. Properties of R. leguminosarum [RP1] plasmid carrier suggest that the expression of RP1-coded Apr gene(s) is inhibited in this host, although the determinants of transfer and resistance to kanamycin and tetracycline are expressed. This system exemplifies a differential expression of plasmid genes in a new host.     

Isolation of a nontransmissible antibiotic resistance plasmid by transductional shortening of R factor RP1.

A plasmid segregant carrying tetracycline and carbenicillin resistance markers has been isolated from R factor RP1 by transductional shortening with phage P22. The new plasmid RP1-S2, which has a molecular weight of 23 times 10-6, has lost the transfer, phage sensitivity, and neomycin resistance functions of RP1. It combines readily with a W group plasmid, R388, to form a transmissible carbenicillin and trimethoprim resistance plasmid, RWP1.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.     

Effect of plasmid RP1 on phase changes in inner and outer membranes and lipopolysaccharide from Acinetobacter calcoaceticus: a Fourier transform infrared study

The successful transfer of the resistance plasmid RP1 into the Gram-negative bacterium Acinetobacter calcoaceticus resulted in increased resistance of this microorganism to the antibiotics kanamycin and tetracycline. Microorganisms harboring the RP1 plasmid showed altered fatty acid composition in the lipopolysaccharide fraction and increased outer membrane permeability compared to organisms without the plasmid. Thermotropic gel to liquid crystal lipid phase changes were detected in both inner and outer membranes and purified lipopolysaccharide by Fourier transform infrared spectroscopy. The phase transition temperatures observed in the outer membranes and isolated lipopolysaccharide of the plasmid-containing cells were significantly higher than those of the plasmid-free organisms, while little difference was observed for the inner membranes. The plasmid-induced decrease in outer membrane fluidity may play a mediating role in the mechanisms of antibiotic resistance and susceptibility to host immune cells in Gram-negative microorganisms.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Formation of plasmid pBR322 oligomers as depending on a tetracycline concentration

Escherichia coli K12 strains containing the plasmid pBR322 often show varying contents of plasmid oligomers, in which the monomer units are arranged in tandem. When the concentration of the plasmid-selective antibiotic tetracycline in the medium becomes increased selection of cells containing largely higher oligomers occurs. The number of monomer units organized in the oligomers increases with tetracycline concentration. recA- mutants are unable to generate oligomers under the same conditions and show lower tetracycline resistance. This observations suggest a selective advantage of oligomer containing cells in the presence of tetracycline as a result of higher gene dosage. But E. coli cells transformed with monomers, dimers, trimers, as well as tetramers of pBR322 are characterized by roughly the same plasmid DNA content as well as plasmid coded beta-lactamase and resistance to tetracycline.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola.

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Comparison of kinetics of active tetracycline uptake and active tetracycline efflux in sensitive and plasmid RP4-containing Pseudomonas putida.

Membrane vesicles prepared from tetracycline-sensitive cells of Pseudomonas putida took up tetracycline by an active transport system with an apparent Km of 2.5 mM and a Vmax of 50 nmol min-1 mg protein-1. In contrast, resistance determinant RP4-containing P. putida had an active high-affinity efflux system for tetracycline with a Km of 2.0 to 3.54 microM and a Vmax of 0.15 nmol min-1 mg protein-1. Thus, the efflux system of tetracycline-resistant P. putida(RP4) had an average of 1,000-fold greater affinity for tetracycline than the influx system of tetracycline-sensitive cells. From these results, it is clear that a major mechanism of tetracycline resistance in RP4-containing P. putida is an active tetracycline efflux mechanism. There was also evidence for a second tetracycline efflux system with low affinity for tetracycline n P. putida(RP4). This efflux system had a Km of 0.25 mM and a Vmax of 1.45 nmol min-1 protein-1. Whether this low-affinity efflux system was also present in tetracycline-sensitive P. putida could not be discerned from these experiments.     

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

Tetracycline resistance genes from Bacillus plasmid pAB124 confer decreased accumulation of the antibiotic in Bacillus subtilis but not in Escherichia coli

Expression of tetracycline resistance by genes originating in the Bacillus plasmid pAB124 was examined in both Bacillus subtilis and Escherichia coli host cells. Expression of resistance in B. subtilis by genes from pAB124 was inducible and associated with decreased accumulation of the antibiotic. A fragment of pAB124 carrying the genes coding for tetracycline resistance was cloned into the E. coli plasmid RSF2124. The cloned fragment conferred a low level of resistance in E. coli, but this was not associated with decreased uptake of tetracycline and was not inducible.     

Adenine + thymine content of different genes located on the broad host range plasmid RP4

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.     

Characteristics of RecA-independent recombination of plasmids in E. coli cells producing restriction endonuclease EcoRI

The restriction endonuclease EcoRI dependent recombination of compatible plasmids has been studied in RecA cells of Escherichia coli. Plasmid RP4 and the isogenic ColE1 type plasmids pSA14 or pSA25, differing in restriction-modification RM EcoRI genes, have been used to study this type of recombination. EcoRI dependent recombination of plasmids is demonstrated in RecA cells and, thus, is independent of general system of homologous recombination. The classes of recombinant plasmids isolated from RecA cells differ from the classes isolated from wild type cells. Levels of tetracycline resistance conferred by plasmid RP4 are shown to be dependent on the alleles of RecA+ gene, being extremely low in RecA cells. This property is demonstrated to be useful for obtaining the multicopy recombinant plasmids resulting from EcoRI dependent recombination in RecA cells of Escherichia coli.     

Introduction of bacteriophage Mu into bacteria of various genera and intergeneric gene transfer by RP4::Mu.

The host range of coliphage Mu was greatly expanded to various genera of gram-negative bacteria by using the hybrid plasmic RP4::Mu cts, which is temperature sensitive and which confers resistance to ampicillin, kanamycin, and tetracycline. These drug resistance genes were transferred from Escherichia coli to members of the general Klebsiella, Enterobacter, Citrobacter, Salmonella, Proteus, Erwinia, Serratia, Alcaligenes, Agrobacterium, Rhizobium, Pseudomonas, Acetobacter, and Bacillus. Mu phage was produced by thermal induction from the lysogens of all these drug-resistant bacteria except Bacillus. Mu phage and RP4 or the RP4::Mu plasmid were used to create intergeneric recombinant strains by transfer of some genes, including the arylsulfatase gene, between Klebsiella aerogenes and E. coli. Thus, genetic analysis and intergeneric gene transfer are possible in these RP4::Mu-sensitive bacteria.     

SOS-induction of the RP4 plasmid tet-determinant

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.     

Heterogeneity of tetracycline resistance determinants

We have found that tetracycline resistance on different naturally occurring bacterial plasmids is encoded by more than one genetic determinant. Using restriction enzyme analyses and DNA-DNA hybridization to specific ³²P-labeled genetic probes, we can define at least four genetically distinct tetracycline resistance determiants: Class A (the determinant on prototype plasmid RP1), Class B (that on R222), and Class C (that on plasmid pSC101). At least one other determinant, encoded by plasmid RA1, belongs to none of these three groups and has been designated Class D. These genetic classes confirm phenotypic differences in expression of resistance to tetracycline and tetracycline analogs encoded by the different plasmids.     

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.     

On the nature of tetracycline resistance controlled by the plasmid pSC101.

In vitro enzymatic alteration of plasmid phenotype and in vitro construction of recombinant plasmids containing genetic information derived from the plasmid pSC101 have been used to investigate the mechanism of function of tetracycline resistance determined by the plasmid pSC101. The resistance has been shown to be inducible and involves the increased synthesis of membrane-associated polypeptides of 34,000, 26,000 and 14,000 daltons that are encoded for by the plasmid. The 34,000 dalton polypeptide along with another plasmid-encoded polypeptide of 18,000 daltons function in an ATP-independent manner to prevent the accumulation of tetracycline by the cell. These polypeptides are sufficient for resistance. A second component of plasmid-determined resistance involves the 14,000 dalton polypeptide and reduces the initial adsorption of tetracycline by sensitive cells, but is not alone sufficient for the generation of resistance. The role of the 26,000 dalton polypeptide in tetracycline resistance has not been identified.     

Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons.

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.     

Detection of plasmid RP4 transfer in soil and rhizosphere,and the occurrence of homology to RP4 in soil bacteria

Plasmid RP4 transfer between introduced pseudomonads was studied in non-rhizosphere and rhizosphere soil. The addition of nutrients to the non-rhizosphere soil stimulated plasmid transfers between introduced donor and recipient cells, and no transfer was detected in nonamended soil. Transfer was also detected in soil in a model rhizosphere, but not in corresponding non-rhizosphere soil. Colony hybridization with whole plasmid RP4 DNA as a probe was employed to detect transfers to indigenous organisms in soil. Although transfers to introduced recipient cells were easily detected in parallel controls, no indigenous organisms were identified that had received RP4. Background levels of soil organisms with the RP4 resistance pattern were considerable, and about 10% of these populations contained DNA sequences with homology to RP4. However, no plasmids could be detected in any of 20 isolates, nor was resistance transfer to aPseudomonas fluorescens recipient detected in filter matings.