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.     

Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.     

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 novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ～65 kb. Complete sequence analysis of the ～65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ～65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.     

Complete nucleotide sequence of a virulence plasmid of Salmonella enterica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhimurium

The complete nucleotide sequence of pOU1113 (pSDVu), one of the two types of virulence plasmids of Salmonella enterica serovar Dublin, was determined. It contained 80 156 bp with 53.8 mol% G+C content. Approximately 70 genes could be discerned. Compared with pSTV, the virulence plasmid of serovar Typhimurium, pOU1113 was shorter owing to a missing region amounting to c. 10 kb; furthermore, except for a unique 10 849-bp region, the nucleotide as well as deduced amino acid sequences of pOU1113 were nearly identical to the corresponding regions of three S. enterica virulence plasmids, namely pSCV (virulence plasmid of Choleraesuis), pSTV and pSEV (virulence plasmids of Enteritidis), confirming their close phylogenetic relationship. Comparative analysis indicated that these virulence plasmids appeared to have descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. From a biological and evolutionary point of view, if the decreasing sizes of pOU1113 and pSCV truly reflect a process in which the virulence plasmid has been shedding unnecessary genes during evolution, our data suggest that some genes in the missing region, such as the pef and tra operons, could have a minimal role in maintaining the survival of the bacteria in their environmental niche.     

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.     

Transfer RNA genes of Drosophila melanogaster.

Three recombinant plasmids containing randomly sheared genomic D. melanogaster tRNAs have been identified and characterized in detail. One of these, the plasmid 14C4, has a D. melanogaster (Dm) DNA segment of 18 kb, and has three tRNA2Arg and two tRNAAsN genes. The second plasmid, 38B10, has tRNAHis genes, while the third plasmid, 63H5, contains coding sequences for tRNA2Asp. The Dm DNA segments in each recombinant plasmid are derived from unique cytogenetic loci. 14C4 is from 84 F, 38B10 is from 48 F and 63H5 is from 70 A.     

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.     

Genomic map of Clostridium perfringens strain 13

A physical and genetic map of Clostridium perfringens strain 13 was constructed. C. perfringens strain 13 was found to have a 3.1-Mb chromosome and a large 50-kb plasmid, indicating that strain 13 has a relatively small genome among C. perfringens strains. A total of 313 genetic markers were mapped on the chromosome of strain 13. Compared with the physical and genetic map of C. perfringens CPN50, strain 13 had a quite similar genome organization, but with a large deletion (approximately 400 kb) in a particular segment of the chromosome. Among several toxin genes, a beta2 toxin gene that is a novel virulence gene in C. perfringens was found to be located on the 50-kb plasmid.     

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.     

Toxin Plasmids of Clostridium perfringens

SUMMARY

In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch''s postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract.     

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.     

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.     

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.     

Analysis of core housekeeping and virulence genes reveals cryptic lineages of Clostridium perfringens that are associated with distinct disease presentations

Clostridium perfringens is an important human and animal pathogen that causes a number of diseases that vary in their etiology and severity. Differences between strains regarding toxin gene composition and toxin production partly explain why some strains cause radically different diseases than others. However, they do not provide a complete explanation. The purpose of this study was to determine if there is a phylogenetic component that explains the variance in C. perfringens strain virulence by assessing patterns of genetic polymorphism in genes (colA gyrA, plc, pfoS, and rplL) that form part of the core genome in 248 type A strains. We found that purifying selection plays a central role in shaping the patterns of nucleotide substitution and polymorphism in both housekeeping and virulence genes. In contrast, recombination was found to be a significant factor only for the virulence genes plc and colA and the housekeeping gene gyrA. Finally, we found that the strains grouped into five distinct evolutionary lineages that show evidence of host adaptation and the early stages of speciation. The discovery of these previously unknown lineages and their association with distinct disease presentations carries important implications for human and veterinary clostridial disease epidemiology and provides important insights into the pathways through which virulence has evolved in C. perfringens.     

Plasmid transformation of Clostridium perfringens by electroporation methods

Two electroporation methods were compared and modified to improve the frequencies of transfer of plasmid DNA into Clostridium perfringens. A plasmid shuttle vector, pSB92A2, containing chloramphenicol and ampicillin resistance genes and a clostridial origin of replication isolated from a cryptic C. perfringens plasmid, was constructed and successfully introduced into C. perfringens by both electrotransformation methods. Modifications which improved frequencies by 15-28 fold are described and may improve frequencies sufficiently for some vector/host combinations to consider the future use of more direct cloning strategies for the clostridia.     

DNA Sequence Analysis of Plasmids from Multidrug Resistant Salmonella enterica Serotype Heidelberg Isolates

Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc) A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.     

Genetic and functional properties of the self-transmissible Yersinia enterocolitica plasmid pYE854, which mobilizes the virulence plasmid pYV

Yersinia strains frequently harbor plasmids, of which the virulence plasmid pYV, indigenous in pathogenic strains, has been thoroughly characterized during the last decades. Yet, it has been unknown whether the nonconjugative pYV can be transferred by helper plasmids naturally occurring in this genus. We have isolated the conjugative plasmids pYE854 (95.5 kb) and pYE966 (70 kb) from a nonpathogenic and a pathogenic Yersinia enterocolitica strain, respectively, and demonstrate that both plasmids are able to mobilize pYV. The complete sequence of pYE854 has been determined. The transfer proteins and oriT of the plasmid reveal similarities to the F factor. However, the pYE854 replicon does not belong to the IncF group and is more closely related to a plasmid of gram-positive bacteria. Plasmid pYE966 is very similar to pYE854 but lacks two DNA regions of the larger plasmid that are dispensable for conjugation.