Conferring Tetracycline Resistance Research Articles

Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1

The streptococcal plasmid pMV158 and its derivative pLSl are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLSl were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLSl doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLSl revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 M r protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 M r protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLSl was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.

Isolation of the replication region of an indigenous plasmid ofRhodobacter sphaeroides

The isolation of the replication region of an indigenous plasmid of 42 kb of the phototrophic bacterium Rhodobacter sphaeroides is described. This plasmid was digested with the BglII restriction enzyme, ligated to the 2.7 BglII fragment of transposon Tn10, which contains the tet genes conferring tetracycline resistance, and the mixture was transformed into the Escherichia coli MC1061 strain. One of several chimeric plasmids harboring the replication region of the 42-kb plasmid obtained by this process was named pUA33 and further characterized. Plasmid pUA33 is approx. 8.3 kb. A partial restriction map has been constructed. Plasmid pUA33 is stable in E. coli cells growing under non-selective conditions and is non-self-transmissible. All these data suggest that the pUA33 plasmid may be a very useful tool for gene cloning in R. spheroides.

Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli.

Transfer factor pBFTM10, isolated from the obligate anaerobic bacterium Bacteroides fragilis, carries a clindamycin resistance determinant which we have suggested is part of a transposable element. DNA homologous to this determinant is found in many Clnr Bacteroides isolates, either in the chromosome or on plasmids. We have now established that Ccr resides on a transposon, Tn4400. In addition to the Ccr determinant that functions under anaerobic conditions in B. fragilis, Tn4400 also carries a determinant for tetracycline resistance (Tcr) which only functions in Escherichia coli under aerobic conditions. The presence of Tn4400 on pBFTM10 does not confer tetracycline resistance on B. fragilis cells containing it. DNA from pBFTM10 was cloned in E. coli, with pDG5 as the cloning vector, to form pGAT500. Using a mobilization assay involving pGAT500 and an F factor derivative, pOX38, we determined that a 5.6-kilobase region of pBFTM10 DNA was capable of mediating replicon fusion and transposition. Most of the mobilization products resulted from inverse transposition reactions, while some were the result of true cointegrate formation. Analysis of the cointegrate molecules showed that three were formed by the action of one of the ends of Tn4400 (IS4400), and one was formed by the action of the whole element (Tn4400). The cointegrate molecule carrying intact copies of Tn4400 at the junction of the two plasmids could resolve to yield an unaltered donor plasmid (pGAT500) and a conjugal plasmid containing a copy of Tn4400 or a copy of one insertion sequence element (pOX38::Tn4400 or pOX38::IS4400). Thus, Tn4400 is a compound transposon containing active insertion sequence elements as directly repeated sequences at its ends.

Comparison of the transposon-like structures encoding clindamycin resistance in Bacteroides R-plasmids

The R-plasmids pBF4, pBFTM10, and pBI136 encode transmissible clindamycin resistance (Cc r) in Bacteroides spp. These plasmids are distinct replicons but the regions implicated in Cc r share some homology and appear to have a transposon-like structure. To better understand the mechanism of dissemination and to locate the Cc r determinant(s), the genetic and structural properties of the Cc r regions of each plasmid were compared and contrasted. For this work a single EcoRI restriction fragment containing the Cc r region from each plasmid was cloned into pBR322 in Escherichia coli. Results of restriction mapping and heteroduplex experiments showed that the pBF4 EcoRI-D and pBFTM10 EcoRI-B fragments shared more than 90% base sequence homology but that the EcoRI-C fragment of pBI136 had diverged significantly. The pBI136 fragment also did not confer tetracycline resistance in E. coli as shown for the pBF4 EcoRI-D fragment (D. G. Guiney, P. Hasegawa, and C. E. Davis, 1984, Plasmid 11, 248–252). Heteroduplex experiments showed that the pBI136 EcoRI-C and pBF4 EcoRI-D fragments shared a 1.2-kb region of homology attributed to a directly repeated sequence which bounds the Cc r region. Southern hybridization studies indicated that an additional 0.85 kb of the pBI136 EcoRI-C fragment was homologous to the EcoRI-D fragment of pBF4. This region was characterized by its sequential restriction endonuclease sites for HindIII, AvaII, and DdeI, and it is proposed that the Cc r gene(s) resides in this area.

Transformation of Clostridium perfringens.

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.

Tetracycline resistance determined by pBR322 is mediated by one polypeptide

Only one polypeptide specified by plasmid pBR322 is necessary to determine tetracycline resistance. Small deletions in pBR322 constructed in vitro which result in the lack of ability to confer tetracycline resistance in vivo also result in the absence or alteration of this polypeptide in vivo. Other deletions define the extent of material necessary to encode this polypeptide. A correction to the DNA sequence of the tetracycline resistance cistron has been determined which confirms these observations.

Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322

The Tn10 tetracycline resistance gene, tetA, encodes a tetracycline-inducible protein with an apparent Mr of 36 × 103. We have determined the nucleotide sequence of the Tn10 tetA gene. The extent of the tetA gene was determined by analysis of amino-terminal and carboxy-terminal deletion mutants. We conclude that a single Tn10 gene, the tetA gene, is sufficient to confer tetracycline resistance, The predicted Mr of the tetA protein is 43.2 × 103. The sequence homology between the Tn10 tetA gene and pBR322 tetracycline resistance determinant (49% nucleotide homology, 44% amino acid homology) indicates that these phenotypically distinct tetracycline-resistance determinants must have evolved from a common ancestral sequence. The markedly hydrophobic character of the predicted amino acid sequences of the Tn10 tetA and pBR322 tet-coded proteins suggests that a substantial portion of these proteins may be embedded within the cytoplasmic membrane.

Convenient construction of recA deletion derivatives of Escherichia coli.

Two Escherichia coli K-12 Hfr strains have been constructed which transfer a recA deletion, which is highly linked to a Tn10 insertion conferring tetracycline resistance, early during conjugation. These strains transfer the recA deletion in opposite directions with different origins of transfer, allowing for preservation of desirable recipient strain markers either clockwise or counterclockwise of recA.

Streptococcus plasmid pAM alpha 1 is a composite of two separable replicons, one of which is closely related to Bacillus plasmid pBC16.

A tetracycline resistance plasmid of Streptococcus faecalis, pAM alpha 1, is shown to contain two independent sets of replication functions, separated from each other on either side by short (300- to 400-base-pair) sequences of homology. The homologous sequences are oriented as direct repeats and therefore permit the dissociation of pAM alpha 1 into its component replicons, referred to here as pAM alpha 1 delta 1 and pAM alpha 1 delta 2, as the reciprocal products of a simple intramolecular recombination. pAM alpha 1 delta 1 is a 4.6-kilobase plasmid which carries the tet gene, and pAM alpha 1 delta 2 is a 5.1-kilobase plasmid which carries no known selectable marker. pAM alpha 1 delta 1 is shown to replicate efficiently in Bacillus subtilis and to confer tetracycline resistance on Bacillus hosts. We demonstrate by restriction mapping analysis that pAM alpha 1 delta 1 is virtually identical to a 4.6-kilobase tetracycline resistance plasmid of Bacillus cereus, pBC16, which is known to show extensive homology to plasmid isolates from Staphylococcus species (such as pUB110), as well as from other Bacillus species. The pAM alpha 1 delta 1-pBC16-pUB110 replicon thus exists naturally in at least three different gram-positive genera, indicating that these plasmids have a high degree of interspecific functional adaptability and supporting the view that plasmid DNA is commonly exchanged among many species of gram-positive bacteria in their natural environments.

Plasmid-determined antibiotic resistance in Shigella flexneri isolated in England and Wales between 1974 and 1978.

The majority of Shigella flexneri strains isolated in England and Wales are from infections contracted abroad. Most of these strains are drug-resistant, over 75% being resistant to streptomycin and sulphonamides, sulphonamides alone or streptomycin, sulphonamides and tetracyclines. A selection of resistant strains was tested for resistance transfer and the plasmids identified were characterized by compatibility grouping. Streptomycin and sulphonamide resistance was usually determined by a non-autotransferring plasmid which may be mobilized by standard transfer factors, or by the plasmid which conferred tetracycline resistance where this was present. The remaining resistant strains were predominantly resistant to four or more drugs. These strains carried autotransferring plasmids of a variety of compatibility groups, of which groups B, I1 and FII were the most common.

Simian virus 40 promoters direct expression of the tetracycline gene in plasmid pACYC184.

Insertion of HindIII DNA fragments into the HindIII site of plasmid pACYC184 destroys the promoter of the plasmid tetracycline resistance gene and causes Escherichia coli cells harboring recombinant plasmids to be tetracycline sensitive and chloramphenicol resistant. The HindIII-C DNA fragment of simian virus 40 contains the two virus promoters and the virus origin of replication. We report the isolation of recombinant plasmids that contained the simian virus 40 HindIII-C DNA fragment at the HindIII site but were capable of conferring tetracycline resistance to E. coli cells. The viral promoter sequences contained in the HindIII-C fragment presumably replaced the inactivated tetracycline resistance gene promoter sequences and enabled transcription of the tetracycline resistance gene.

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK.290 (Ditta et al., 1980) by inserting a 1.6-kb BglII fragment containing λ cos into the unique BglII site in pRK.290. The new vector, pLAFRl, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFRl using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tc r) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFRl cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tc r transconjugants were prototrophic.

Cloning of chromosomal genes in Streptococcus pneumoniae.

A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the malM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme. A 10-kilobase restriction fragment containing the sul-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment of a recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtilis are considered and compared.

Citrate-dependent iron transport system in Escherichia coli K-12.

Induction of the citrate-dependent iron transport system of Escherichia coli K-12 required 0.1 mM citrate and 0.1 micrometer iron in the growth medium. Five--ten-times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate-dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (FecA), belonging to the transport system, was induced in fecB mutants which were devoid of citrate-dependent iron transport. The intracellular citrate and iron concentrations were 10--100-times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm. The Tn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate-dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with the fecA and fecB markers suggested the gene order fecA fecB Tn10.

Isolation and Partial Characterization of Four Plasmids from Antibiotic-resistant Thermophilic Bacilli

Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4.9 X 10(6) (7.0 kilobases) and pAB118B of molecular weight 3.0 X 10(6) (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 X 10(6) (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2.5 X 10(6) (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis (168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.

Cleavage maps of a tetracycline plasmid from Staphylococcus aureus.

The cleavage maps of a Staphylococcus aureus plasmid, pSN1 (2.75 megadaltons), conferring tetracycline resistance, were determined. Cleavage maps are given for HpaI and HindIII restriction endonucleases by using the single HpaII site as a reference point. Nucleases EcoRI, BamHI, SalI, and HaeIII have no sites on this plasmid.

Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition

A novel genetic element, which carries genes conferring tetracycline resistance (flanked by a 1400 base-pair inverted repetition), is capable of translocation as a unit from one DNA molecule to another. The tet R element, which is found in nature on a variety of R-factors, was acquired by bacteriophage P22 (producing P22Tc-10 and P22Tc-106) and has now been observed to insert into a large number of different sites on the Salmonella chromosome. Insertion of the tet R element is mutagenic when it occurs within a structural gene, and polar when it occurs within an operon. Insertion of the element is usually precise, occurring without loss of information on the recipient DNA molecule. Excision, on the other hand, is usually not precise, although excisions precise enough to restore a gene function can always be detected at low frequencies. Both insertion and excision processes are independent of the recA function.

Analysis of the resistance mediated by several R-factors to tetracycline and minocycline.

SUMMARYThe resistance levels conferred by the T-determinants in four R-factors to Tetracycline and Minocycline in cells ofEscherichia coliK 12, before and after induction of maximum resistance by treatment with sub-inhibitory concentrations of the drugs, are measured by simple growth-and-challenge tests. The effect of a plasmid TKwhich confers tetracycline resistance on its hostKlebsiella aerogenesis tested in the same way. The five T-determinants fall into a high-level and a low-level group for resistance, the former giving 3- to 4-fold higher resistance in both induced and uninduced cells than the latter. The T-determinants all confer much lower resistance to Minocycline (a tetracycline molecule modified at the C-6 and C-7 positions) than to Tetracycline. The main cause of this difference is that cells carrying a T-determinant exclude Minocycline much less efficiently than Tetracycline, but in addition Minocycline is less effective than Tetracycline in inducing increased resistance. These results are discussed in the light of a model put forward to explain the inducible nature of R-factor resistance to the tetracyclines.

THE EFFECT OF GROWTH AT ELEVATED TEMPERATURES ON SOME HERITABLE PROPERTIES OF STAPHYLOCOCCUS AUREUS.

SUMMARY: Populations of a predominantly tetracycline-resistant, penicillinase-positive strain of Staphylococcus aureus grown at 43.44° gave rise to progressively increasing proportions of tetracycline-sensitive and penicillinase-negative cocci. The losses did not appear until after the elapse of a number of generations at the elevated temperature, and then apparently proceeded independently, tetracycline resistance being lost more rapidly than the ability to produce penicillinase. Tetracycline-sensitive and penicillinase-negative variants were extremely stable and the growth rates at 44° of the parent strain and the tetracycline-sensitive variants were indistinguishable. Screening for numerous other ‘marker’ properties revealed no changes under the test conditions. The evidence suggests that tetracycline resistance and penicillinase-forming ability probably depend on the presence of two different plasmids in the cocci; that the replication-rates of the plasmids at elevated temperature are less than that of the cocci; and that the plasmids, once lost, are not spontaneously resynthesized nor, at least for that conferring tetracycline resistance, regained by infection. However, such a hypothesis raises the question of how equilibrium, particularly between the tetracycline-resistant and sensitive cocci, is maintained in a population growing at 37° and observed to change in one direction but never in the reverse direction.