Cloning and analysis of the Clostridium perfringens tetracycline resistance plasmid, pCW3

Clostridium perfringens strain CW92 carries pCW3, a conjugative 47-kb plasmid that confers inducible resistance to tetracycline. The plasmid was examined by restriction endonuclease analysis and by cloning each of the five ClaI fragments of pCW3 in Escherichia coli, using pBR322. Analysis of the recombinant plasmids allowed the deduction of a detailed restriction map of pCW3. The tetracycline resistance determinant of pCW3 was mapped by examining the phenotype of recombinant E. coli clones derived from the cloning, into pUC vector plasmids, of EcoRI fragments from pCW3. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed EcoRI fragments which together encompass a 4-kb region of pCW3. Deletion experiments showed that the tetracycline resistance gene, and/or its control regions, contained internal EcoRI and SphI sites. E. coli strains that carried recombinant plasmids with only the 4-kb region were found to express tetracycline resistance constitutively. In contrast, recombinant plasmids harboring a 10.5-kb ClaI fragment of pCW3, that included the 4-kb region, coded for an inducible tetracycline resistance phenotype. The existence of a negatively regulated resistance gene, similar to that proposed for several other bacteria is postulated.     

Molecular analysis of transferable tetracycline resistance plasmids from Clostridium perfringens.

Conjugative tetracycline resistance plasmids from 15 Clostridium perfringens isolates from piggeries were analyzed by restriction endonuclease digestion and agarose gel electrophoresis. Seven isolates from one farm were found to carry a 47-kilobase pair (kb) plasmid, pJIR5, which had EcoRI, XbaI, and ClaI profiles that were identical to those of a previously characterized plasmid, pCW3. An isolate from a second farm was found to carry a plasmid, pJIR6, which also was indistinguishable from pCW3. Five additional isolates from a third farm carried a 67-kb plasmid, pJIR2, which had at least 29 kb of DNA in common with pCW3. Finally, two isolates from a fourth farm were found to carry a 50-kb plasmid pJIR4, which appeared to consist of an entire pCW3 molecule with a 3-kb insertion. Comparative restriction maps of pCW3, pJIR2, and pJIR4 that identified the regions of homology among these plasmids were constructed. We suggest that many conjugative tetracycline resistance plasmids in C. perfringens may contain a pCW3-like core.     

Hybridization analysis of the class P tetracycline resistance determinant from the Clostridium perfringens R-plasmid, pCW3

The tetracycline resistance determinant from pCW3, a conjugative plasmid from Clostridium perfringens, has been identified and the structural gene localized to within a 1.4-kb region. Hybridization analysis, which utilized an internal 0.8-kb specific gene probe, showed that eight nonconjugative tetracycline resistant C. perfringens strains all carried homologous resistance determinants. No homology was detected in DNA prepared from tetracycline resistant isolates of Clostridium difficile or Clostridium sporogenes. However, the one strain of Clostridium paraputrificum that was tested did contain an homologous determinant. No homology was found to any of the recognized classes of tetracycline resistance determinants. The C. perfringens tetracycline resistance determinant represents a new hybridization group, Class P.     

Identification of Tn4451 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens.

The recombinant plasmids pJIR45 and pJIR97 contain the chloramphenicol resistance determinants derived from the Clostridium perfringens R plasmids pIP401 and pJIR27, respectively. Escherichia coli cultures which harbored these recombinant plasmids rapidly became chloramphenicol sensitive when grown in the absence of chloramphenicol. The loss of resistance was associated with the loss of 6.2-kilobase (kb) segments from both plasmids. Detailed restriction analysis of E. coli- and C. perfringens-derived deletion plasmids indicated that deletion of these segments was essentially precise. Transposition of the 6.2-kb segments was demonstrated by cloning the determinants into a temperature-sensitive plasmid, curing the recombinant plasmids, and selecting chloramphenicol-resistant, plasmid-free clones. Southern hybridization analysis of chromosomal DNA isolated from these recA E. coli clones indicated that the 6.2-kb segments had transposed to different sites on the chromosome. Heteroduplex analysis and restriction mapping indicated that the transposons, Tn4451 (pIP401) and Tn4452 (pJIR27), were closely related and did not contain large inverted or directly repeated sequences. These transposons represent the first transposable elements from the clostridia to be identified and characterized.     

TcpA, an FtsK/SpoIIIE homolog, is essential for transfer of the conjugative plasmid pCW3 in Clostridium perfringens

The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3DeltatcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.     

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.     

The resolvase/invertase domain of the site-specific recombinase TnpX is functional and recognizes a target sequence that resembles the junction of the circular form of the Clostridium perfringens transposon Tn4451.

Tn4451 is a 6.3-kb chloramphenicol resistance transposon from Clostridium perfringens and is found on the conjugative plasmid pIP401. The element undergoes spontaneous excision from multicopy plasmids in Escherichia coli and C. perfringens and conjugative excision from pIP401 in C. perfringens. Tn4451 is excised as a circular molecule which is probably the transposition intermediate. Excision of Tn4451 is dependent upon the site-specific recombinase TnpX, which contains potential motifs associated with both the resolvase/invertase and integrase families of recombinases. Site-directed mutagenesis of conserved amino acid residues within these domains was used to show that the resolvase/invertase domain was essential for TnpX-mediated excision of Tn4451 from multicopy plasmids in E. coli. An analysis of Tn4451 target sites revealed that the transposition process showed target site specificity. The Tn4451 target sequence resembled the junction of the circular form, and insertion occurred at a GA dinucleotide. Tn4451 insertions were flanked by directly repeated GA dinucleotides, and there was also a GA at the junction of the circular form, where the left and right termini of Tn4451 were fused. We propose a model for Tn4451 excision and insertion in which the resolvase/invertase domain of TnpX introduces 2-bp staggered cuts at these GA dinucleotides. Analysis of Tn4451 derivatives with altered GA dinucleotides provided experimental evidence to support the model.     

Induction of pCW3-Encoded Tetracycline Resistance in Clostridium perfringens Involves a Host-Encoded Factor

The tetracycline resistance determinant Tet P, which is encoded by the conjugative plasmid pCW3 from Clostridium perfringens, is induced by subinhibitory concentrations of tetracycline. In this study we have shown that the inducible phenotype is strain dependent. When pCW3 is present in derivatives of the wild-type strains CW234 and CW362 resistance is inducible. However, transfer to derivatives of strain 13 leads to a constitutive phenotype that is only observed in this strain background. Based on these results it is proposed that induction of the pCW3-encoded tet(P) genes in C. perfringens requires a host-encoded factor that is either absent or nonfunctional in strain 13 derivatives.     

Conjugative R plasmids in group C and G streptococci.

Two streptococcal isolates of groups C and G harbored conjugative R plasmids with molecular weights of 17 X 10(6) (pIP646) and 20 X 10(6) (pIP920). These plasmids carried genetic markers for resistance to macrolides and related drugs, as well as to chloramphenicol (pIP920), and have very similar HindIII restriction enzyme patterns.     

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.     

Cloning of tetracycline-resistance genes from various strains of Clostridium perfringens and expression in Escherichia coli.

One hundred strains of Clostridium perfringens and 52 strains of other clostridia of human and animal origins were screened for tetracycline resistance. Fifty-six strains were resistant to tetracycline in the C. perfringens group. Ten strains were selected for their high level of resistance. In all of them, the tetracycline-resistance genes were found to be residing in large plasmids of about 50 kb, all showing homologies. Several tetracycline-resistance genes from plasmids of various strains of C. perfringens were cloned in plasmid pUC19 and the resistance was expressed in Escherichia coli. Hybridization analysis showed these genes to be homologous among themselves and also to tetP gene from the PCW3-type plasmid.     

Two Novel Membrane Proteins,TcpD and TcpE,Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.     

Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.     

Conjugative plasmids in Bordetella avium.

Three of five antibiotic-resistant plasmids isolated from virulent strains of Bordetella avium were found to be conjugative. A physical and genetic map of one of these plasmids, the 51.5-kb plasmid p4093, revealed that the area of p4093 responsible for streptomycin and tetracycline resistance was located in a region consisting of a cluster of restriction enzyme recognition sites, whereas the remainder of p4093 contained relatively few restriction sites. Additionally, the genes involved in the conjugative ability of p4093 were clustered in at least two widely separated regions of the plasmid.     

Characterization and construction of molecular cloning vehicles within Staphylococcus aureus.

Four chloramphenicol resistance (Cm) and four tetracycline resistance (Tc) plasmids from Staphylococcus aureus were characterized by restriction endonuclease mapping. All four Tc plasmids had molecular masses of 2.9 megadaltons (Mdaltons) and indistinguishable responses to seven different restriction endonucleases. The four Cm plasmids (pCW6, pCW7, pCW8, and pC221) had molecular masses of 2.6, 2.8, 1.9, and 2.9 Mdaltons, respectively. The four Cm plasmids also differed both in the level of resistance to Cm and in susceptibility to retriction endonucleases. Single restriction endonuclease sites contained within each plasmid included the following: in pCW6 for HindIII, XbaI, HpaII, and BstEII; in pCW7 for HindIII, BstEII, BglII, HaeIII, and HpaII; in pCW8 for HindIII, HaeIII, and HpaII; in pC221 for HindIII, BstEII, and EcoRI. The molecular cloning capabilities of pCW8 and pC221 were determined. Cm and erythromycin resistance (Em) recombinant plasmids pCW12, PCW13, and pCW14 were constructed and used to transform S. aureus 8325-4. A 2.8-Mdalton HindIII fragment from plasmid pI258 was found to encode Em resistance and contain single sites for the retriction endonucleases BglII, PstI, HaeIII, and HpaII. The largest EcoRI fragment (8 Mdaltons) from pI258 contained the HindIII fragment encoding Em resistance intact. Cloning of DNA into the BglII site of pCW14 did not alter Em resistance. Cloning of DNA into the HindIII site of pCW8 and the HindIII and EcoRI sites of pC221 did not disrupt either plasmid replication of Cm resistance.     

Antibiotic resistance in clinical strains of Pseudomonas aeruginosa isolated from 1979-1984

The levels and spectra of drug resistance were determined in 530 strains of P. aeruginosa isolated in hospitals of three cities of the USSR within 1979-1984. Their conjugative R plasmids were searched for and distribution of various type resistance determinants in the composition of these plasmids was investigated. The results were compared with the findings of analogous studies on clinical strains of P. aeruginosa isolated within 1976-1979. It was shown that there were a rise in the relative number of the strains resistant to kanamycin and a decrease in the occurrence of the P. aeruginosa strains resistant to streptomycin, tetracycline and sulfanilamides. The frequency of the kanamycin, carbenicillin and gentamicin resistance genes in the composition of the detected conjugative R plasmids increased. Hybridization of 32P-labeled probes containing various type antibiotic resistance determinants with strains of P. aeruginosa ML (PAO) containing conjugative R plasmids was indicative of wide spread of genes determining APH(3')II and APH(3") and determinants of classes A and C in the composition of the studied plasmids.     

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4-6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.     

Conjugative plasmids inBordetella avium

Three of five antibiotic-resistant plasmids isolated from virulent strains ofBordetella avium were found to be conjugative. A physical and genetic map of one of these plasmids, the 51.5-kb plasmid p4093, revealed that the area of p4093 responsible for streptomycin and tetracycline resistance was located in a region consisting of a cluster of restriction enzyme recognition sites, whereas the remainder of p4093 contained relatively few restriction sites. Additionally, the genes involved in the conjugative ability of p4093 were clustered in at least two widely separated regions of the plasmid.     

Conjugative transfer of multiple-antibiotic resistance markers in beta-hemolytic group A, B, F, and G streptococci in the absence of extrachromosomal deoxyribonucleic acid

Nine clinical isolates of group A, B, F, and G streptococci resistant to tetracycline, macrolides, lincosamides, and streptogramin B (MLS resistance) and to chloramphenicol were investigated for the conjugative transfer of the antibiotic-resistance markers into streptococcal recipients (groups B and D). The wild donors transferred the resistance markers en bloc, at a low frequency (10^?6 to 10^?8) and only into one of the two recipients tested. In addition, one of the strains transferred only the MLS resistance at a high frequency (10^?3). All attempts to detect extrachromosomal DNA in wild donors or in transconjugants were unsuccessful, except in one transconjugant. This plasmid DNA, designated pIP659, had a molecular weight of 17.5 × 10⁶ and a restriction fingerprint similar to other plasmids determining MLS resistance.     

Transfer of plasmid-borne resistance from a multiply-resistant Staphylococcus aureus isolate,WBG1022

Staphylococcus aureus isolate, WBG1022, was resistant to penicillin, kanamycin, neomycin, streptomycin, chloramphenicol, trimethoprim, cadmium, and ethidium bromide and harbored plasmids of 34.5, 24.5, 4.4, 3.2, and 2.6 kilobases. The plasmids were transferred in mixed-culture transfer and conjugation experiments. No resistance phenotype was associated with the 2.6-kb plasmid. The 3.2-kb and 4.4-kb plasmids encoded chloramphenicol and streptomycin resistance respectively. The 24.5-kb plasmid, pWBG626, encoded joint resistance to penicillin, kanamycin, neomycin, and ethidium bromide. Resistance to trimethoprim and cadmium were chromosomal. The 34.5-kb plasmid, pWBG661, had no resistance phenotype but was found to be conjugative. It also mobilized the 4.4-kb and 24.5-kb plasmids in WBG1022. Restriction endonuclease analysis of pWBG661 with EcoRI, ClaI, PvuII, and BglII restriction enzymes demonstrated that pWBG661 was identical to two previously isolated S. aureus conjugative plasmids, p WBG620 and pWBG637, that also lack resistance phenotypes.