A restriction map of IncFIV plasmid R124

A physical and genetic map of the 125.7-kb IncFIV plasmid R124 was constructed using the restriction enzymes Sal1 and EcoR1. Two discrete regions involved in plasmid replication were identified on the plasmid genome. One region was located on a 4.66-kb segment of an EcoR1 fragment at map coordinates 73.87 to 78.53 kb. Another was located within an 8.05-kb segment of an EcoR1 fragment at map coordinates 113.40 to 121.45. This region was very unstable but, when ligated to the 3.21-kb EcoR1 fragment E13 located at map coordinates 18.83 to 22.06 kb, replication was stable. Thus, at least three regions of R124 widely separated around the genome are associated with plasmid replication and stable maintenance. Each of these three regions expressed incompatibility with R124. The Tc resistance gene of R124 was located on the contiguous EcoR1 fragments E8 and E12 located at map coordinates 100.49 to 113.40.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Tn10 mediated integration of the plasmid R100.1 into the bacterial chromosome: Inverse transposition

Summary Upon integration into the bacterial chromosome the drug resistance plasmid R100.1 often loses its tetracycline resistance character. We have analyzed an Hfr strain formed by such an integration and an R-prime plasmid derived from it. We find that integration took place within the Tn10 transposon, that the two IS10 sequences were retained, but that at least 80% of the transposon segment located between them, and carrying the tetracycline resistance genes, had been lost. We suggest that integration of R100.1 was mediated by an inverse transposition using the IS10 sequences.     

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.     

Molecular cloning and characterization of form I antigen genes of Shigella sonnei.

Sau3AI-generated DNA fragments of the Shigella sonnei large plasmid encoding the form I antigen were cloned into Escherichia coli with cosmid vector pHSG262. One resulting plasmid, designated pJK1137, was studied further. Restriction endonuclease mapping and analysis of transposon Tn3 insertion mutants demonstrated that the form I antigen genes were located within a region of about 12.6 kb consisting of the two contiguous HindIII fragments of 1.26 kb and 12.4 kb. The results of complementation studies between Tn3 insertion mutants of pJK1137 and recombinant plasmids carrying different parts of the form I antigen genes indicated that the 12.6 kb DNA sequence contained at least four gene clusters, regions A, B, C and D. Analysis of radioactively labelled proteins in minicells demonstrated that the DNA sequence of about 12.6 kb coded for at least four specific proteins of 42, 23, 48 and 39 kDa. The former two were coded by region A, the latter two by region D.     

The complete DNA sequence and analysis of R27, a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer

Salmonella typhi, the causative agent of typhoid fever, annually infects 16 million people and kills 600 000 world wide. Plasmid-encoded multiple drug resistance in S.typhi is always encoded by plasmids of incompatibility group H (IncH). The complete DNA sequence of the large temperature-sensitive conjugative plasmid R27, the prototype for the IncHI1 family of plasmids, has been compiled and analyzed. This 180 kb plasmid contains 210 open reading frames (ORFs), of which 14 have been previously identified and 56 exhibit similarity to other plasmid and prokaryotic ORFs. A number of insertion elements were found, including the full Tn10 transposon, which carries tetracycline resistance genes. Two transfer regions, Tra1 and Tra2, are present, which are separated by a minimum of 64 kb. Homologs of the DNA-binding proteins TlpA and H-NS that act as temperature-regulated repressors in other systems have been located in R27. Sequence analysis of transfer and replication regions supports a mosaic-like structure for R27. The genes responsible for conjugation and plasmid maintenance have been identified and mechanisms responsible for thermosensitive transfer are discussed.     

The complete nucleotide sequence of the resistance plasmid R478: defining the backbone components of incompatibility group H conjugative plasmids through comparative genomics

Horizontal transfer of resistance determinants amongst bacteria can be achieved by conjugative plasmid DNA elements. We have determined the complete 274,762 bp sequence of the incompatibility group H (IncH) plasmid R478, originally isolated from the Gram negative opportunistic pathogen Serratia marcescens. This self-transferable extrachromosomal genetic element contains 295 predicted genes, of which 144 are highly similar to coding sequences of IncH plasmids R27 and pHCM1. The regions of similarity among these three IncH plasmids principally encode core plasmid determinants (i.e., replication, partitioning and stability, and conjugative transfer) and we conducted a comparative analysis to define the minimal IncHI plasmid backbone determinants. No resistance determinants are included in the backbone and most of the sequences unique to R478 were contained in a large contiguous region between the two transfer regions. These findings indicate that plasmid evolution occurs through gene acquisition/loss predominantly in regions outside of the core determinants. Furthermore, a modular evolution for R478 was signified by the presence of gene neighbors or operons that were highly related to sequences from a wide range of chromosomal, transposon, and plasmid elements. The conjugative transfer regions are most similar to sequences encoded on SXT, Rts1, pCAR1, R391, and pRS241d. The dual partitioning modules encoded on R478 resemble numerous sequences; including pMT1, pCTX-M3, pCP301, P1, P7, and pB171. R478 also codes for resistance to tetracycline (Tn10), chloramphenicol (cat), kanamycin (aphA), mercury (similar to Tn21), silver (similar to pMG101), copper (similar to pRJ1004), arsenic (similar to pYV), and tellurite (two separate regions similar to IncHI2 ter determinants and IncP kla determinants). Other R478-encoded sequences are related to Tn7, IS26, tus, mucAB, and hok, where the latter is surrounded by insLKJ, and could potentially be involved in post-segregation killing. The similarity to a diverse set of bacterial sequences highlights the ability of horizontally transferable DNA elements to acquire and disseminate genetic traits through the bacterial gene pool.     

Isolation of the replication region of an indigenous plasmid of Rhodobacter sphaeroides

Abstract 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 Bgl II restriction enzyme, ligated to the 2.7 Bgl II fragment of transposon Tn 10 , 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 .     

Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux

The large conjugative multidrug resistance (MDR) plasmid pOLA52 was sequenced and annotated. The plasmid encodes two phenotypes normally associated with the chromosomes of opportunistic pathogens, namely MDR via a resistance-nodulation-division (RND)-type efflux-pump (oqxAB), and the formation of type 3 fimbriae (mrkABCDF). The plasmid was found to be 51,602 bp long with 68 putative genes. About half of the plasmid constituted a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin (TA) plasmid addiction system. The plasmid was also classified as IncX1 with incompatibility testing. The conjugal transfer and plasmid maintenance regions of pOLA52 therefore seem to represent IncX1 orthologues of the well-characterized IncX2 plasmid R6K. Sequence homology searches in GenBank also suggested a considerably higher prevalence of IncX1 group plasmids than IncX2. The 21 kb 'genetic load' region of pOLA52 was shown to consist of a mosaic, among other things a fragmented Tn3 transposon encoding ampicillin resistance. Most notably the oqxAB and mrkABCDF cassettes were contained within two composite transposons (Tn6010 and Tn6011) that seemed to originate from Klebsiella pneumoniae, thus demonstrating the capability of IncX1 plasmids of facilitating lateral transfer of gene cassettes between different Enterobacteriaceae.     

Characterization of maxi- and mini-derivatives of the IncHI1 plasmid R27

R27, an IncHI1 plasmid of 182 kilobases (kb), which was originally isolated fromSalmonella typhimurium, was found to contain two copies of IS1 in direct orientation. Deletion derivatives constructed in vitro were of two types: a maxi-derivative of 110 kb (pDT1047) with a single complete copy of IS1, and mini-derivatives of 5–6 kb which contained less than a complete copy of IS1. The IncHI1 miniplasmids also contained a portion of the tetracycline resistance determinant from R27 and a replication region related to the RepFIA replicon. Electron microscopic homoduplex studies demonstrated the presence of two other inverted repeat sequences within the miniplasmid that were unrelated to IS1.     

Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7

Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27. The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc-. Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C. These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible. Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible. Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour). Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids. The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa). A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site. Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites. Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27. Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27.     

Expression in Escherichia coli of cryptic tetracycline resistance genes from Bacteroides R plasmids

The putative clindamycin resistance region of the Bacteroides fragilis R plasmid pBF4 was cloned in the vector R300B in Escherichia coli. This 3.8-kb EcoRI D fragment from pBF4 expressed noninducible tetracycline resistance in E. coli under aerobic but not anaerobic growth conditions. The fragment does not express tetracycline resistance in Bacteroides, a strict anaerobe. The separate tetracycline resistance transfer system in the Bacteroides host strain V479-1 has no homology to the cryptic determinant on pBF4. In addition, this aerobic tetracycline resistance determinant is not homologous to the three major plasmid mediated tetracycline resistance regions found in facultative gram-negative bacteria, represented by R100, RK2, and pBR322. A similar cryptic tetracycline resistance fragment was cloned from pCP1, a separate clindamycin resistance plasmid from Bacteroides that shares homology with the EcoRI D fragment of pBF4. This study identifies cryptic drug resistance determinants in Bacteroides that are expressed when inserted into an aerobically growing organism.     

Constitutive expression of tetracycline resistance mediated by a Tn10-like element in Haemophilus parainfluenzae results from a mutation in the repressor gene.

The Tn10-like constitutively expressed tetracycline resistance determinant from a Haemophilus parainfluenzae strain was cloned in Escherichia coli. Toxicity resulting from expression on multicopy plasmids necessitated its being cloned on a low-copy plasmid vector or in cells containing the Tn10-encoded repressor. Constitutive expression of tetracycline resistance was found to result from the synthesis of a truncated inactive repressor molecule. Instead of the 23-kilodalton repressor found in other Tn10-containing strains, this determinant encoded a 14.5-kilodalton molecule. The DNA sequence of the 700-base-pair region spanning the repressor gene and promoter-operator regions of the Haemophilus determinant was identical to that of the same region of Tn10, except for the absence of a single T X A base pair in the repressor gene. This deletion leads to premature termination of the protein. Antisera to the repressor suggested that the repressor was also absent in a second independently isolated H. parainfluenzae strain bearing a Tn10-like constitutive tetracycline resistance determinant.     

Physical and genetic organization of the plasmid R15

The conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb) is cleaved by the hexanucleotide-specific endonucleases BglII, HindIII, EcoRI, BamHI, SmaI, SalI, PstI and XhoI into 9, 9, 6, 5, 4, 4, 4 and 2 fragments, respectively. The restriction sites were located on the physical map of the R15 genome. Distribution of the cleavage sites is strongly asymmetric. 28 of 32 sites for BamHI, EcoRI, HindIII, SalI, SmaI and PstI were located close to or within the sequences of transposable elements Tn2353 and Tn2354. According to the results of analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring fragments of R15, the genetic determinants for resistance to Sm, Su and Hg were mapped, as well as the regions necessary for EcoRII restriction--modification and for plasmid replication and conjugation. The features of physical and genetic structures of R15 and other IncN plasmids are discussed.     

Structure of a naturally occurring plasmid with genes for enterotoxin production and drug resistance.

A physical map of the 117-kilobase conjugative plasmid pCG86 was constructed using electron microscope heteroduplex analysis. This plasmid carries the genes elt, for heat-labile enterotoxin, and estA, for heat-stable enterotoxin, as well as the genes for resistance to tetracycline, streptomycin, sulfonamides, and mercury. These genes were mapped using deletions and Tn5 insertions as physical markers. Analysis of a heteroduplex between pCG86 and a previously described enterotoxin plasmid (EntP307) showed a 48-kilobase region of complete homology which included the genes elt and estA. An 8.8-kilobase BamHI fragment of EntP307 carrying elt, cloned by others, was also shown to be completely homologous with pCG86. The position of elt on the fragment was verified, and it was shown to carry estA as well. A 44-kilobase region of pCG86 showed partial homology with the region of EntP307 previously shown to contain conjugal transfer genes. The gene for tetracycline resistance is carried on a stem-loop structure with the dimensions of Tn10, and the genes for the other drug resistance markers are carried on a 14.6-kilobase segment that forms an insertion loop in heteroduplexes with EntP307. These studies suggest that pCG86 arose either by recombination between an enterotoxin plasmid of incompatibility group FI, like EntP307, and a multiple resistance factor of incompatibility group FII, or by transposition into EntP307 of two transposons.     

Physical and genetic structure of the IncN plasmid R15

Restriction sites for seven hexanucleotide-specific endonucleases were located on the map of the conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty-four sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI were located close to or within the sequences of an IS5-like element and the transposons Tn2353 and Tn2354. By analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring R15 fragments, the genetic determinants for the streptomycin, sulfonamide, and mercury resistances were mapped, as well as the regions necessary for EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed.     

The multiple antibiotic resistance IncP-1 plasmid pKJK5 isolated from a soil environment is phylogenetically divergent from members of the previously established alpha, beta and delta sub-groups

The 54,383bp plasmid pKJK5 was recovered from a soil environment by exogenous plasmid isolation and conveys resistance towards tetracycline and trimethoprim. Sequencing and annotation revealed a high level of structural similarity of the backbone genes to other IncP-1 plasmids containing a Tra1 and Tra2 region, a central control module and a replication initiation module. A considerable degree of divergence was associated with the backbone genes of pKJK5 as compared to homologous genes in the alpha, beta and delta subgroups, which indicates that pKJK5 may belong to a novel subgroup of IncP-1 plasmids, which may also accommodate the partially sequenced non-subgroup classified plasmid pEMT3. Individual backbone genes in pKJK5 have a GC-content, which is consistently lower (average 6.3%) than the homologous genes from the archetype IncP-1beta plasmid R751 indicating homogenous amelioration of IncP-1 plasmid backbone genes. Two discrete accessory elements of 2145bp (load 1) and 11678bp (load 2) respectively are situated between the Tra1 and Tra2 regions of pKJK5, both bounded by inverted repeats and direct flanking repeats indicative of transposon-mediated insertion. Load 1 consists of an insertion sequence ISPa17 and load 2 is a Tn402-derivative containing a class 1 integron, IS1326 and a fragment identical to a region of plasmid pTB11 harboring a tetracycline resistance determinant and part of an IncP-1alphaoriV region.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.     

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.