Atypical incompatibility of the F-like factors of pA22-4, pAP39 and pA41 genetic transfer with the F-group incompatibility plasmids

A study was made of compatibility of three F-like factors of the genetic transfer (pAP22-4, pAP39, pAP41) identified in the cells of serologically typed E. coli strains with F-group incompatibility reference plasmids. The factors of pAP22-4 and pAP41 transfer are partly incompatible with groups FII, FIII, FIV, and FI, FIV, respectively, while the factors of pAP39 transfer are completely incompatible both with groups FI and FIV plasmids.     

Genetic transfer of pAP38, pAP39, and pAP41 plasmids into E. coli, Erwinia, and Hafnia strains

Genetic transfer factors pAP38, pAP39 and pAP41 can be transferred to E. coli, both typed and untypecd, as well as to Erwinia and Hafnia strains, with different frequency (10(-2) to 10(-8)). The transconjugates thus obtained possess donor activity and can transfer the factors they have received to recipient E. coli strains. After transfer these factors are stably maintained in a new host for at least 10 days. Under the action of ethidium bromide or elevated temperature the elimination of the transfer factors from host bacteria is observed. The studied transfer factors pAP38, pAP39 and pAP41 (F-like factors) are plasmids fi+.     

Systems regulating the tra genes of derepressed F-like plasmids

A study was made of the ability of reference plasmids of the 6 known Fin-groups to inhibit the functions of transfer genes (tra-genes) of the 4 derepressed F-like plasmids (pAP22-2, pAP38, pAP43, pAP53). It was shown that unlike the derepressed Flac plasmid, the conjugation transfer of pAP38 and pAP53 plasmids was inhibited only by, the FinV plasmid, whereas pAP22-2 plasmids by Fin V and Fin V plasmids. The formation of donor-specific pili in case of pAP38 plasmid was inhibited by Fin Q, Fin U and Fin V plasmids, in case of pAP43 plasmid by Fin U Fin V and Fin W plasmids.     

Identification of the number of mutant copies of the genetic transfer factor AP42

The copy number mutant of genetic transfer factor pAP42 (incFIX) was induced by the treatment of E. coli AP115 cells carrying the plasmid by N-methyl-N-nitro-N-nitrosoguanidine. The copy number mutant pAP42::Tn1cop1 is characterized by the increased copy number. The cells carrying the copy number mutant have a higher ampicillin resistance and higher beta-lactamase activity. Mutant plasmid pAP42: :Tn1cop1 is incompatible with plasmid pAP42 and compatible with plasmids of the other inc-groups of F-like plasmids.     

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.     

Range of the transmissivity of the genetic transfer factors pAP38, pAP39, pAP41 and pAP42

A study was made of the transmissivity range of the genetic transfer factors pAP38, pAP39, pAP41 and pAP42 identified in E. coli. It was demonstrated that these factors are not capable of transfer to the cells of P. putida, P. fluorescens, R. leguminosarum, A. lipoferum, A. tumefaciens. Factor pAP42 is mobilized to transfer to P. putida and R. leguminosarum with the aid of plasmid RP4. It is assumed that in the course of mobilization, the cointegrative structures are formed between plasmids pAP42 and RP4.     

Effect of transposons Tn1 and Tn9 on the genetic regulation system of transfer and incompatibility functions of Col-plasmid pAP19-1

F-like pAP19-1 Col-plasmid was labeled with transposons Tn1 and Tn9 and transfer functions of its derepressed mutants were investigated. The plasmid indicated was compatible with reference plasmids of 9 F-like incompatibility groups. Thus it belongs to the new incompatibility group FX. The ability of Tn9 to change the incompatibility of the plasmid investigated was discovered.     

Genetic characteristics of plasmid pAP53 derepressed in transfer functions detected in the cells of an opportunistic E. coli strain

A study was made of plasmid pAP53 derepressed as regards transfer functions (Tra-functions) detected in E. coli strain cells, serogroup 0128, after its labeling with transpozones Tn1 and Tn9. The compatibility tests demonstrated that the plasmid belongs to the incompatibility group FIII and is partially incompatible with the group FII reference-plasmid. Plasmid pAP53 is unable to inhibit Tra-functions of plasmid F'lac and is not inhibited by the fin type genetic regulation on the OP group plasmids under study. At the same time Tra-functions of plasmid pAP53 are inhibited in the presence of pAP41 plasmid, which indicates that this plasmid has a special type of genetic regulation.     

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.     

Characterisation of plasmids purified from Acetobacter pasteurianus 2374

Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.     

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.     

Mutations of the genetic region Fin of the F-like plasmid pAP18-1]

With help of nitrosoguanidine 60 mutants of F-like plasmids pAP18-1 drd::Tn 5 and pAP18-1::Tn 9 were induced which determined resistance of E. coli cells of specific phage MS2. Mutational changes in fin-locus of those plasmids were accompanied by phenotypic reversion Fin(-)-Fin+.     

Cloning of DNA fragments of the genetic transfer factor pAP39

Large HindIII digested fragments of the plasmid pAP39 have been cloned on the cosmid vector pHC79. The study of the structure of HindIII fragments of plasmid pAP39 in the recombinant plasmids has shown that these fragments are represented by f1 + f2 fragments from the plasmid pAP1055, by f1 + f6 fragments from the plasmid pAP1056, by f2 + f3 fragments from the plasmid pAP1057 and by two f3 fragment from the plasmid pAP1058. Physical maps of the recombinant plasmids have been constructed. The plasmid pAP39 is shown to contain two functionally active tra regions.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

Genetic properties of transposons Tn6-1, Tn6-2 and Tn19-1

The level and range transposition of the transposons Tn6-1, Tn6-2, Tn19-1, and their ability to influence plasmid transfer has been studied. The widest range of transposition was shown for transposon Tn6-2. Insertions of each of the studied transposons into different conjugative plasmids genomes resulted in change of frequencies of plasmids transfer and change of plasmids mobilization activity.     

Plasmid complex of E. coli B-13

The plasmid complex was identified in a wild type strain B-13 of Escherichia coli. The complex was found to contain four conjugative R-plasmids / pAP24 -1 fi+, pAP24 -2 fi-, pAP24 -3 fi-, pAP24 -4 fi-/, one conjugative Col-plasmid / pAP24 -5/ and one conjugative F-like plasmid Ent/ pAP10 -2 fi+/. The molecular weight of pAP24 -1 is 53.6 X 10(6), pAP24 -2 - 40.9 X 10(6), pAP24 -3 - 73.8 X 10(6), pAP24 -4 - 51,3 X 10(6). It is suggested that an autonomous transfer factor exists in the plasmid complex.     

Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens.

We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome. First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45. We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5. By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations. We also constructed isolates of A. tumefaciens containing an insertion of Tn5 in the chromosome. We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients. Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested. Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies. Donors were shown to transfer chromosomal markers in a polarized fashion. Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent. The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A. tumefaciens chromosomal functions.     

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.     

Prime plasmids derived from the IncP-10 plasmid R91-5 in Pseudomonas putida

Abstract Plasmid primes carrying various fragments of Pseudomonas putida chromosome have been derived from pMO22, a derivative of R91-5 loaded with Tn 501 . These prime plasmids transfer efficiently to P. aeruginosa where they effectively complement various auxotrophic markers. Proof of prime plasmid formation has been provided by the high-frequency transfer of plasmid and chromosomal markers, the unselected cotransfer of either plasmid or chromosomal markers into P. aeruginosa and by transformation of both plasmid and chromosomal markers using prime plasmid DNA. Such prime plasmids have been used to map the location of new markers on the P. putida chromosome.     

Conjugal retrotransfer of chromosomal markers inAzotobacter vinelandii

The IncP plasmids R68.45 and pJB3JI mediate retrotransfer (i.e., transfer of chromosomal markers from the recipient bacterium to the original donor) in homologous matings withAzotobacter vinelandii. Retrotransfer is not an early event inAzotobacter. On the contrary, it begins after genetic transfer in the usual direction, i.e., from the donor bacterium to the recipient, has been taking place for some time. Transfer of chromosomal markers mediated by the RP4/Tn5-Mob system does not undergo retrotransfer. The simplest hypothesis compatible with our results is that IncP plasmids that confer a high chromosome-mobilizing ability promote retrotransfer when transferred to the recipient bacteria.