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.     

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.     

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 genome localization of the plasmid pAP27 fin N system]

Plasmid transfer genetic regulatory system fin N of F-like plasmid pAP27 have been localized on BamHI-restriction fragment f3 (length 8.7 kb). Plasmid pUC19 Fin-activity have been cleared up and characterized from the point of view of its specificity of action on some plasmid transfer functions.     

Compatibility between F-like genetic transfer factors

A study has been made of compatibility among four F-like factors of genetic transfer (pAP22-4, pAP38, pAP39 and pAP41) labeled separately by transpozones Tn1 and Tn9. It has been established that pAP38 transfer factor is compatible with plasmids pAP22-4, pAP39 and pAP412, while pAP41 transfer factor is compatible with plasmids pAP22-4 and pAP38 but is incompatible with plasmid pAP39.     

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+.     

Genetic organization of transfer region of F-like plasmid pAP18-1

The results of complementation analysis of nitrosoguanidine-induced mutants of F-like drd-plasmid pAP18-1 (Tc, ColV) testified to the existence of at least 3 tra regions (tra1, tra2, tra3) and regulating locus fin V in the genome of this plasmid. By means of molecular cloning of tra2 region and locus fin V of plasmid pAP18-1drd were located in Sall-fragment f5 (3.9 MD).     

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.     

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.     

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.     

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.     

Molecular genetic study of the changes in the incompatibility and regulation of the transfer of the F-like pAP18-1 plasmid induced by nitrosoguanidine and the Tn5 and Tn9 transposons

Relation between induced mutations of plasmid pAP18-1 (Tc, Col) and alterations in it's restriction map was studied. Nitrosoguanidine induced mutations of transfer regulation system and incompatibility of this plasmid related with alteration in the situation of recognition sites for restrictases EcoR1 and Sal1 in map positions 42.2-4.3 and 12.9-17.9 MD. Insertions of transposons Tn5 and Tn9 into the plasmid DNA resulted in a decrease of incompatibility level.     

Plasmid pAP20 controlling the hemolytic activity of E. coli

The authors investigated pAP20 plasmid identified in E. coli cells isolated from man. According to the evidence obtained pAP20 plasmid determines the synthesis of alpha-hemolysin, being an F-like plasmid of the drd type. Having medium molecular size, the plasmid belongs to the inc FIV group and is partly incompatible with pAP38 plasmid which is a reference plasmid of the inc FVII group.     

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.     

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+.     

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.     

Plasmid Specificity of The Origin of Transfer of Sex Factor F

The ability of F-like plasmids to promote transfer from the F origin of transfer was determined. Chromosome transfer was measured from plasmid derivatives of RecA(-) Hfr deletion strains which had lost all the F transfer genes but which in some cases retained, and in others had also lost, the origin sequence. ColV2 and ColVBtrp could initiate transfer from the F origin, but R100-1, R1-19, and R538-1 drd could not. These results can be correlated with the plasmid specificity of the traI components of the different plasmid transfer systems, supporting the hypothesis that the origin of transfer is the site of action of the traI product. Most F-like plasmids, including R1-19 and R538-1 drd, could transfer ColE1, consistent with previous findings that the (plasmid-specific) traI product is not necessary for ColE1 transfer by Flac; ColE1 transfer may be initiated by a ColE1-or host-determined product. R100-1 and R136fin(-) could not transfer ColE1 efficiently, apparently because of differences residing in their pilus-forming genes.     

The molecular cloning of a genetic region determining the surface exclusion system of the F-like plasmid pAP42]

Molecular cloning of genetic region of F-like plasmid pAP42, coding its surface exclusion system (system Six V) was performed. Restriction and genetic analysis of recombinant plasmids showed that six V locus is situated in Sal I-fragment f5 (4.2 MD) of this plasmid.     

Study of the role of the insA open reading frame in the IS1 insertion element structure during transposition and resolution of the IS1 mediated cointegrates

In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained. The latter are equal or multiple of 9 b.p. in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon. The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame. One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates. For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1. It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E. coli. However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon. Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained. It was shown that in the E. coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e. they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X. The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.