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.     

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).     

Restriction map of the genetic transfer factor pAP42

Based on the calculated molecular weights of EcoR1, HindIII, and SalI fragments of the genetic transfer factor pAP42 the restriction map of this plasmid was designed. Sites recognizing restrictases are mostly located in the plasmid fragment with a molecular weight of 5.7 MD.     

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.     

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 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.     

Incompatibility and replication of plasmids]

The identified basic replicons rep1 and rep2 of plasmid pAP42 belong to different groups of incompatibility (inc FIX and inc FVIII). The replicons are partly incompatibile with other inc F-groups too. The results indicate connection between plasmid incompatibility and their replication.     

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.     

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.     

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.     

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.     

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+.     

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.     

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.     

Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids.

Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome.     

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.     

Effect of parB on plasmid stability and gene expression in Xanthomonas campestris

The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and introduced into Xanthomonas campestris pv campestris and X.c.pv manihotis. Analysis of the stability of plasmid pAP2 (parB-) and pAP23 (parB+) showed that the parB locus decreased significantly the plasmid loss rate mainly by X.c.pv campestris. The lower efficiency of stabilization in X.c.pv manihotis was probably due to the incompatibility system between the native plasmids and the newly introduced pAP23. Although parB had conferred higher stability, it determined a lower rate of alpha-amylase activity even by the strain Cm where its stabilization rate was higher.     

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 a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages

Phage-host interactions remain poorly understood in lactic acid bacteria and essentially in all Gram-positive bacteria. The aim of this study was to identify the phage genetic determinant (anti-receptor) involved in the recognition of Streptococcus thermophilus hosts. The complete genomic sequence of the lytic S. thermophilus phage DT1 was determined previously, and bioinformatic analysis indicated that orf18 might be the anti-receptor gene. The orf18 of six additional S. thermophilus phages was determined (DT2, DT4, MD1, MD2, MD4 and Q5) and compared with the orf18 of DT1. The deduced ORF18 was divided into three domains. The first domain, which contains the N-terminal part of the protein, was conserved in all seven phages. The second domain was detected in only two phages and flanked by a motif called collagen-like repeats. The second domain also contained a variable region (VR1). All seven phages had a third domain that consisted of the C-terminal section of the protein as well as another variable region (VR2). Chimeric DT1 phages were constructed by recombination; a portion of its orf18 was replaced by the corresponding section in orf18 of the phage MD4. All DT1 chimeric phages acquired the host range of phage MD4. Analysis of the orf18 in the chimeric phages revealed that host specificity in phages DT1 and MD4 resulted from VR2. This is the first report on the identification and characterization of a phage gene involved in the host recognition process of Gram-positive bacteria.