Genetic regulation of the function of E. coli plasmid pAP42 transfer genes

Sensitivity of the genetic transfer system of F-like plasmid pAP42 marked with the transposons Tn1 and TN9 to fertility inhibitors of six reference Fin-groups was studied. It was shown that transfer function and donor-specific piliformation of the plasmid under study were inhibited by reference plasmids of FinU and FinV groups, surface exclusion by plasmids of FinU and FinQ groups. The different influence of the FinOP group plasmid on transfer functions of the marked plasmids pAP42::Tn1 and pAP42::Tn9 that is likely to be connected with the effect of incorporated transposons was determined.     

The use of Pseudomonas sp. M plasmid pM3 for the transposon mutagenesis of Erwinia

The transposon containing derivatives pMTF9 (Tn9), pMTF10 (Tn10) and pMTF59 (Tn5, Tn9) of the Pseudomonas sp. M conjugative plasmid pM3 demonstrating temperature-dependent instability in Erwinia cells incubated at 37 degrees C have been isolated. The obtained plasmids have been shown to be usable for transposon-mediated mutability in the bacterial cells of Erwinia generum incubated at 37 degrees C.     

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.     

Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates]

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.     

The systems of the genetic regulation of plasmid transfer fin K, fin L, fin M and fin N]

F-like plasmids pAP19-1::Tn9, pAP20::Tn9, pAP22-1::Tn1, pAP27 characterized by the presence of unique genetic plasmid transfer regulatory systems in their genomes have been found. These systems were named fin K, fin L, fin M, finN, consequently. They were characterized from the point of view of specificity of their action on F-factor and F-like conjugative function. Dependence of fin N-system expression on host-cell and on the order of plasmid entering into host-cell was shown.     

Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20-45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn4653 and the multiple candidates of IRs for Tn4676 and Tn4653-like element. We also found distinct regions with high and low G+C contents within Tn4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn4676 had been captured.     

Replication of staphylococcal multiresistance plasmids

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.     

Broad-host-range plasmids from agricultural soils have IncP-1 backbones with diverse accessory genes

Broad-host-range plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to antibiotics and heavy metals or degradation of pollutants. Although some broad-host-range plasmids have been extensively studied, their evolutionary history and genetic diversity remain largely unknown. The goal of this study was to analyze and compare the genomes of 12 broad-host-range plasmids that were previously isolated from Norwegian soils by exogenous plasmid isolation and that encode mercury resistance. Complete nucleotide sequencing followed by phylogenetic analyses based on the relaxase gene traI showed that all the plasmids belong to one of two subgroups (β and ε) of the well-studied incompatibility group IncP-1. A diverse array of accessory genes was found to be involved in resistance to antimicrobials (streptomycin, spectinomycin, and sulfonamides), degradation of herbicides (2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenoxypropionic acid), and a putative new catabolic pathway. Intramolecular transposition of insertion sequences followed by deletion was found to contribute to the diversity of some of these plasmids. The previous observation that the insertion sites of a Tn501-related element are identical in four IncP-1β plasmids (pJP4, pB10, R906, and R772) was further extended to three more IncP-1β plasmids (pAKD15, pAKD18, and pAKD29). We proposed a hypothesis for the evolution of these Tn501-bearing IncP-1β plasmids that predicts recent diversification followed by worldwide spread. Our study increases the available collection of complete IncP-1 plasmid genome sequences by 50% and will aid future studies to enhance our understanding of the evolution and function of this important plasmid family.     

Comparative study of non-conjugative R-plasmids from enterobacteria and Pseudomonas aeruginosa

Nonconjugative R-plasmids pBS76 and pBS94 (Sm Su), pBS95 and pBS96 (Sm Su Ap) isolated from clinical strains of Pseudomonas aeruginosa and plasmids pKMR281-pKMN284 (Sm Su), pKMR285-pKMR286 (Sm Su Tc) isolated from clinical strains of enterobacteria have been studied. Restriction maps of these plasmids are presented in the paper with some of plasmid genes for antibiotic resistance localized on them. The resistance determinants of plasmids pBS95 and pBS96 are shown to be included in transposon Tn3612 analogous to Tn3. Plasmids pBS76, pBS94-96 are of the wide host range and belong to incompatibility group P4 (IncQ). Plasmids pKMR281-pKMR286 are mutually incompatible and share the conspicuous DNA homology. They are inherited only by enterobacteria and are compatible with IncQ plasmids but in contrast to them are mobilized by RP4 plasmid with lower frequency.     

Region-specific insertion of transposons in combination with selection for high plasmid transferability and stability accounts for the structural similarity of IncP-1 plasmids

The overall architecture of IncP-1 plasmids is very conserved in that the accessory genes are typically located in one or two specific regions: between oriV and trfA and between the tra and trb operons. Various hypotheses have been formulated to explain this, but none have been tested experimentally. We investigated whether this structural similarity is due to region-specific transposition alone or also is reliant on selection for plasmids with insertions limited to these two regions. We first examined the transposition of Tn21Km into IncP-1beta plasmid pBP136 and found that most Tn21Km insertions (67%) were located around oriV. A similar experiment using the oriV region of IncP-1beta plasmid pUO1 confirmed these results. We then tested the transferability, stability, and fitness cost of different pBP136 derivatives to determine if impairment of these key plasmid characters explained the conserved plasmid architecture. Most of the pBP136 derivatives with insertions in transfer genes were no longer transferable. The plasmids with insertions in the oriV-trfA and tra-trb regions were more stable than other plasmid variants, and one of these also showed a significantly lower fitness cost. In addition, our detailed sequence analysis of IncP-1 plasmids showed that Tn402/5053-like transposons are situated predominantly between the tra and trb operons and close to the putative resolution site for the ParA resolvase, a potential hot spot for those transposons. Our study presents the first empirical evidence that region-specific insertion of transposons in combination with selection for transferable and stable plasmids explains the structural similarity of IncP-1 plasmids.     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.     

Conjugativity and incompatibility of derepressed pAP11-2 plasmid

It has been demonstrated during investigation of Colplasmid pAP11-2 and its varieties labeled with transpozone (Tn1 and Tn9) that this plasmid is a derepressed one in terms of transfer functions in E. coli strain K-12 cells as well as in some of serologically typed strains of this type. The plasmid under study is incompatible with reference plasmids belonging to two different groups (FI and FIV) and is marked by a number of the properties common to the system of genetic control over Tra-functions.     

Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system

The transposons Tn916 and Tn4001 and a series of integrating plasmids derived from their antibiotic resistance genes were used to examine polyethylene glycol-mediated transformation of Mycoplasma pulmonis. Under optimal conditions, Tn916 and Tn4001 could be introduced into M. pulmonis at frequencies of 1 x 10(-6) and 5 x 10(-5) per CFU, respectively. Integrating plasmids were constructed with the cloned antibiotic resistance determinants of Tn916 and Tn4001, a pMB1-derived plasmid replicon, and mycoplasmal chromosomal DNA and were used to examine recombinational events after transformation into M. pulmonis. Under optimal conditions, chromosomal integrations could be recovered at a frequency of 1 x 10(-4) to 1 x 10(-6) per CFU, depending on the size and nature of the chromosomal insert and the parental plasmid. Integrated plasmids were stable in the absence of selection and could be rescued in Escherichia coli along with adjacent mycoplasma DNA. These studies provide the first direct evidence of a recombination system in the Mollicutes and describe the first E. coli-M. pulmonis shuttle vectors.     

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.     

Fine Structure Genetic and Physical Map of the Phage P22 Tail Protein Gene

Bacteriophage P22 which are incapable of making functional tail protein can be propagated by the addition of purified mature tail protein trimers to either liquid or solidified medium. This unique in vitro complementation condition has allowed us to isolate 74 absolute lethal tail protein mutants of P22 after hydroxylamine mutagenesis. These phage mutants have an absolute requirement for purified P22 tail protein to be present in a soft agar overlay in order to form plaques and do not grow on any nonsense suppressing strains of Salmonella typhimurium. In order to genetically map and physically locate these mutations we have constructed two complementary sets of fine structure deletion mapping strains using a collection of Tn1 insertions in gene 9, the structural gene for the tail protein. Fourteen bacteriophage P22 strains carrying unique Tn1 transposon insertions (Ap phage) in gene 9 have been crossed with Ap phage carrying Tn1 insertions in gene 20. Phage carrying deletions that arose from homologous recombination between the Tn1 elements were isolated as P22 lysogens. The deletion prophage were shown to be missing all genetic information bracketed by the parental Tn1 elements and thus form a set of deletions into gene 9 from the 5' end of the gene. From the frequency of production of these deletion phage the orientation of the Tn1 insertions in gene 9 could be deduced. The genetic end points of the deletions in gene 9 and thus the order of Tn1 insertions were determined by marker rescue experiments using the original Ap phage. The genetic end points of the deletions in gene 20 were determined in similar experiments using nonsense mutations in gene 20. To locate the physical end points of these deletions in gene 9, DNA containing the Tn1 element has been cloned from each of the original Ap phage into plasmids. The precise point of insertion of Tn1 into gene 9 was determined by restriction enzyme mapping and DNA sequencing of the relevant portions of each of these plasmids. In vitro deletion of different 3' gene 9 sequences in the plasmid clones was accomplished through the use of unique restriction endonuclease sites in Tn1. The resulting plasmids form a set of deletions extending into the 3' end of the gene which are complementary compared to the deletion lysogens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.