Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.     

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.     

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.     

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.     

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.     

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.     

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.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Characterization of sulfonamide resistance determinants and relatedness of Bordetella avium R plasmids.

Five plasmids, varying in size from 16 to 51.5 kb, were isolated from virulent strains of Bordetella avium and compared by restriction endonuclease digestion and DNA-DNA hybridization. These plasmids confer resistance to streptomycin and sulfonamides, and three of the five also confer resistance to tetracycline, but they are not closely related. Four of the plasmids, pRL100, p4093, pCW, and pWAM, carried determinants related to the heat-labile type I plasmid-mediated dihydropteroate synthase of the plasmid R388, while one plasmid, p4168, carried a determinant related to the heat-stable type II dihydropteroate synthase of pGS05.     

Identification of a transferable tetracycline resistance plasmid (pCW3) from Clostridium perfringens

A tetracycline- and chloramphenicol-resistant strain of Clostridium perfringens, CW92, was shown to carry two plasmids, pCW2 and pCW3. Twenty-four independently derived tetracycline-sensitive mutants were isolated using a variety of curing agents. All were missing pCW3 but still carried pCW2. Tetracycline resistance could be transferred to a sensitive recipient strain by what appears to be a conjugation-like process. The efficiency of transfer was 2.8 × 10⁻⁵ transcipients per viable donor cell after a 20-h mating. The transcipients transferred tetracycline resistance at a similar frequency. Ten independently derived tetracycline-resistant transcipients all carried pCW3 as shown by agarose gel electrophoresis. The identity of this plasmid in one of these strains was confirmed by electron microscopy and restriction endonuclease analysis. Therefore, pCW3 (30.6 megadaltons) is a transferable tetracycline-resistance plasmid. No chloramphenicol-sensitive mutants or chloramphenicol-resistant transcipients were isolated. Therefore, pCW2 (36.4 megadaltons) remains cryptic.     

Shuttle plasmids for Escherichia coli and Clostridium perfringens.

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens.

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

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.     

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.     

Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates

Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.     

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.     

Distribution and homology of plasmids in several strains of Cyanothece

The plasmid distribution of several clonal isolates of the unicellular, diazotrophic, cyanobacterium Cyanothece sp. has been analyzed. The Cyanothece isolates contain three to four plasmids ranging in size from 4.8 kb to 40 kb. The plasmid profiles of three Cyanothece strains (BH63, BH68, BH93) indicated that strains BH68 and BH93 were closely related and that strain BH63 may be more distantly related. A small 4.8-kb plasmid (pSE480), from the clonal isolate Cyanothece sp. strain BH68F, has been subcloned and restriction mapped. Ten restriction sites have been mapped, five of which are unique and suitable for further subcloning. Southern hybridization revealed that this plasmid was present in two out of five clonal isolates of strain BH68 and in one isolate of strain BH93. A 10-kb plasmid from strain BH68F (pSE1000) was found in all of the BH68 isolates and was absent in the BH93 isolate, Cyanothece sp. strain BH93A. No notable physiological changes were observed in the absence of either the 4.8-kb or 10-kb plasmids. Therefore, these plasmids remain cryptic. Further analysis of these plasmids may provide insight into the function of these plasmids and will allow the construction of shuttle vectors for gene transfer experiments.     

Analysis of two chloramphenicol resistance plasmids from Staphylococcus aureus: Insertional inactivation of Cm resistance, mapping of restriction sites, and construction of cloning vehicles

Chloramphenicol (Cm) resistance plasmids pCW7 and pC221 of Staphylococcus aureus have been characterized by the construction of detailed restriction maps and by the identification of restriction sites on both plasmids which map within either the structural gene encoding CAT or its controlling elements. The number and order of recognition sites for endonucleases AluI, HinfI, and TaqI on pCW7 and pC221 were determined from multiple enzyme digestion results and by the 5′-terminal labeling procedure of Smith and Birnstiel (1976). Endonuclease sites mapping within the Cm-resistance determinant were identified by the construction of recombinant plasmids in vitro from pC221 or pCW7 and the tetracycline-resistance plasmid pCW3. Site-specific mutations were then introduced by filling in the complementary ends of selected restriction sites present on pCW7 or pC221 with E. coli polymerase I followed by recircularization of the recombinant plasmid by blunt-end ligation. Pol I treatment of the BstEII site of pC221 and the BstEII or BglII site present on pCW7 resulted not only in the loss of those recognition sites but also in the loss of Cm resistance encoded by the plasmid. Circularization of the largest MboI fragment (1.8kb) from pC221 formed a stable replicon which encoded an inducible CAT but did not exhibit the relaxation phenomenon associated with pC221. The possible roles of several of the recombinant plasmids as cloning vehicles within S. aureus and Bacillus subtilis are discussed.     

Conjugal transfer of antibiotic resistances and plasmids from Campylobacter jejuni clinical isolates

Six Campylobacter jejuni clinical isolates were examined for the occurrence of plasmids in association with antibiotic resistances as well as conjugal transfer. All the isolates were found to carry three similar plasmids of 78 kb, 12.6 kb and 3.3 kb in size. Multiple resistance to at least three of the antibiotics tested was observed with resistance to tetracycline most common. En bloc transfer of donor resistances at frequencies ranging from 10(-8) to 10(-4) were seen in all but one of the isolates during conjugation. The conjugal transfer of erythromycin, neomycin and streptomycin were observed to occur at frequencies similar to that of chloramphenicol, kanamycin and tetracycline. In isolate ABA94, three different antibiotic resistance phenotypes of the transconjugants were seen. In addition to en bloc transfer of the donor resistances, in approximately 10% of the transconjugants the streptomycin resistance was lost although these transconjugants carried the donor complement of three plasmids. In a further 1% of the transconjugants, resistance to kanamycin only was detected and these transconjugants did not carry any plasmids.