The site of action of the F transfer inhibitor

Summary The mechanism of inhibition of F transfer from E. coli K12 cells containing the fin ⁺ R factor R100 was studied. For this, a series of Flac double mutants carrying both a traO ^-mutation, which prevents function of the transfer inhibitor, and a suppressible mutation in one of ten genes required for conjugational DNA transfer, were constructed. The levels of retransfer of these elements from cells carrying a wild-type Fhis element and R100 showed that of the ten transfer genes, only traJ was directly affected by the transfer inhibitor. Furthermore, in the case of R100 and therefore probably in the case of F itself, it was shown that the products of the other nine genes are absent from the cell during transfer inhibition, suggesting that the traJ product is required for their synthesis.D.F. acknowledges the support of a George Murray Scholarship from the University of Adelaide, Australia.     

Further characterization of the F fertility inhibition systems of "unusual" Fin+ plasmids.

Flac mutants insensitive to transfer inhibition by R factors. JR66a and R485 were isolated and characterized. Representative mutations were cis dominant and are therefore presumed to be at the sites of action, fisU and fisV, respectively, of the FinU and FinV transfer inhibition systems encoded by JR66a and R485. The mutants were used to confirm that the FinU and FinV fertility inhibition systems are different from each other and from the FinOP, FinQ, and FinW systems of R100, R62, and R455, respectively. Together with traO and fisQ mutants of Flac, the new mutants were also used to investigate the nature of the F fertility inhibition systems encoded by a further group of "unusual" Fin+ plasmids. Of these, two incompatibility group X plasmids were found to carry finO+ genes, and of five incompatibility group I plasmids, three encoded FinQ systems, one the FinU system, and one a new system (FinR). Transfer of a variety of derepressed F-like plasmids was inhibited by the FinQ, FinU, and FinV systems, but a quantitatively very different levels; this emphasizes the differences as well as the similarities between the conjugation systems of F-like plasmids.     

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.     

Two classes of Flac mutants insensitive to transfer inhibition by an F-like R factor

Summary Mutants of Flac episomes whose transfer is no longer inhibited by the fi ⁺ R factor R100 are shown to be of at least two types. One is recessive in transient heterozygotes containing Fhis and R100 in addition to the Flac mutant. The other is dominant. The occurrence of recessive mutants suggests that inhibition of F transfer by R100 requires an F-specified gene product in addition to that produced by the R factor. Synthesis of the F product or its interaction with the R100 product to give the true inhibitor seems to be a slow process. Since the inhibitor and mutations of the transfer gene traJ both affect a plasmid-specific transfer product, F-pilus formation, and surface exclusion, we propose that the inhibitor prevents the synthesis or function of the traJ product.Supported by a George Murray Scholarship from the University of Adelaide, Australia.     

Identification and characterization of four new tra cistrons on the E. coli K12 sex factor F.

Thirty-eight amber-suppressible transfer-deficient mutants of the Flac element F13-1 have been analyzed in conjugational complementation tests. Many of the mutations were in tra genes that have been identified previously (including traI), but several were in four new genes, traN, traU, traV, and traW. Results for representative mutations in the new genes were confirmed by complementation tests using λtra transducing phages and these also allowed the genes to be mapped quickly and simply. Approximate physical locations were found by complementation with a series of chimeric plasmids carrying various EcoRI fragments of the F transfer region. All four genes are located in the transfer operon, the order being traA … traBtraVtraWtraCtraUtraNtraF … traD. While traU, traV, and traW mutants were resistant to male-specific phages, traN mutants were sensitive.     

tra cistrons and proteins encoded by the Escherichia coli antibiotic resistance plasmid R6-5

Summary Hybrid plasmids obtained by cloning individual EcoRI and HindIII fragments of the conjugative plasmid, R6-5, were analyzed for their ability to complement transfer-deficient point mutations of Flac. As a result, the locations of 10 tra cistrons were defined on the physical map of R6-5. Two cistrons, traE and traG, are interrupted by EcoRI restriction sites and one cistron, traC, probably contains a HindIII restriction site. The origin of DNA transfer, oriT, was also localized. Surprisingly the hybrid plasmid carrying oriT is mobilized by the F factor as well as by R6-5. The surface exclusion cistrons, traS and traT, were mapped and their biological expression analyzed. A total of 18 proteins encoded by cistrons within the tra region were detected by SDS polyacrylamide gel electrophoresis of proteins synthesized in minicells; they represent about 53% of the coding capacity of the cloned DNA. R6-5 DNA fragments containing the cistrons traC, traE, and traT directed the synthesis of proteins which comigrated during SDS gel electrophoresis with the F-coded proteins previously characterized as TraCp, TraEp, and TraTp. A further two proteins encoded by R6-5 comigrated with F-encoded (but genetically unidentified) proteins whose cistrons map in the corresponding part of the tra region. In contrast, no R6-5 proteins corresponding to F proteins TraAp, TraDp, TraJp, TraMp, 6a or 6c were detected. These results are discussed in relation to known DNA sequence homologies between the F and R6-5 plasmids. A preliminary physical map of the tra region of R6-5 is presented and compared with that of F.     

The interaction of an I-like R factor and transfer-deficient mutants of Flac in E. coli K 12

Summary Strains carrying an I-like R factor, R64, or its derepressed derivative, R64-11, together with an Flac episome mutant in one of ten cistrons determining transfer-proficiency, transferred the Flac mutant at a frequency equivalent to about 1% of the level of R factor transfer. Similarly, R64, R64-11 and transfer-deficient mutants of R64-11, were transferred at increased frequencies in the presence of wild-type Flac. Experiments using RecA^– strains showed that mobilisation by recA ⁺-promoted recombination was not involved, and others using strains carrying transfer-deficient mutants of both R64-11 and Flac suggested that even inefficient complementation between R64-11 and Flac transfer mutants did not occur. The transfer systems of the two plasmids seemed, therefore, to be unrelated, and plasmid-specific, although at a low frequency the entire transfer system of one, not just the pilus, could transfer a transfer-deficient mutant of the other.     

A new activity in the Ftra operon which is required for F-pilin synthesis

Summary Membrane preparations from a series of Hfr mutant strains of Escherichia coli K12 deleted in the promoter distal end of the F transfer operon were analyzed. Deletions which extended into traG, as expected, had no discernible effect on synthesis of membrane F-pilin. A more extensive deletion in strain KI777 which eliminated traH activity similarly had no effect on F-pilin synthesis. Membranes from three other TraF⁺ TraH^- deletion strains, as well as membranes from all strains carrying deletions extending into traF or further, lacked F-pilin, however. Since traH amber mutations do not affect synthesis of membrane pilin (Moore et al. 1981 b) we conclude that a gene required for F-pilin biosynthesis is located between traF and traH. We have named this gene traQ.Further evidence for traQ and an assay for its activity was obtained by examining the products of a TraM⁺ TraJ⁺ TraA⁺ lambda transducing phage, KI13, in UV irradiated cells. Infection of F^- cells with KI13 does not result in F-pilin synthesis. Membrane pilin is synthesized as a product of the transducing phage if an Flac or Hfr irradiated host is used, however. Mutant analysis demonstrated that this synthesis is independent of host expression of traA, traL, traE, traK, traB, traV, traW, traC, traU, traF, or traH, but dependent on expression of the traF-traH region. We interpret our data to indicate that an activity encoded by traQ is required for the conversion of traA product to F-pilin.     

Growth and reactivation of single stranded DNA phage phiX174 in E. coli undergoing "thymineless death".

Summary Flac maintenance was aberrant at permissive temperature in a temperature-sensitive dnaC mutant of Salmonella typhimurium when the normally resident pLT2 plasmid was present. Flac was, however, efficiently transferred into the dnaC pLT2⁺ strain and the resulting Flac derivative was almost as efficient in transferring Flac as were dnaC ⁺ pLT2⁺ Flac strains indicating that aberrant Flac maintenance was not associated with appreciable inhibition of transfer replication. A range of F-like plasmids behaved like pLT2 in causing aberrant Flac maintenance when present in the dnaC pLT2^- strain. Flac was, however, stably maintained in the dnaC strain in the absence of other plasmids. Although the F-like plasmids destabilized Flac, each was stably maintained when introduced into strain 11G dnaC pLT2⁺ and pLT2 was also apparently stable under these conditions. The destabilizing effect of pLT2 and other fi ⁺ plasmids was not consequent upon their inhibiting the formation of a repressible F transfer component needed for Flac replication in the dnaC strain. Incompatibility between Flac and the other plasmids induced by the dnaC lesion also appeared unlikely to be a cause of the aberrant Flac maintenance. The possibility is discussed that the initiation of Flac replication differs from that of pLT2 and the F-like plasmids with F competing less effectively than the others for the DnaC gene product.     

Construction and characterization of multicopy plasmids containing the entire F transfer region

The restriction endonucleases HindIII and/or BamI have been used to clone the entire F transfer region into pBR322 to create a series of transfer-proficient multicopy plasmids. Despite the insertion of 40.7- to 55.9-kilobase F fragments, the plasmid copy numbers remained high, at 25–40 copies per cell. One of the chimeric plasmids contained the F replication and incompatibility region, and its high copy number confirmed that replication of the cointegrate was governed by pBR322. Despite the 30- to 40-fold increase in tra gene copy number compared to Flac, the transfer frequencies, number of pili per cell, and syntheses of the individual traT and traI proteins were increased only by about 5-fold. The level of tra mRNA in cells carrying the multicopy transfer-proficient plasmids was also increased only by about 5-fold, suggesting that its relative synthesis or stability was reduced in this situation. Nonetheless, the increased production of tra DNA, mRNA, and protein makes cells carrying the multicopy conjugative plasmids excellent sources of these products.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Early transfer of genes determining transfer functions by some Hfr strains in Escherichia coli K12

Summary Sixty-eight Hfr strains were examined for their ability to transfer early in conjugation the transfer genes carried by the integrated sex factor. This was measured by mating these strains with F^- phenocopied recipient cultures of strains carrying transfer-deficient Flac ⁺ factors, and then measuring the ability of the recipient strains to transfer lac ⁺ to a further recipient strain. Most Hfr strains did not complement the missing transfer functions, though in some strains complementation was observed. It is concluded that on the sex factors of different Hfr strains either the site at which integration occurs or the origin of transfer must vary.     

Incompatibility between Flac, R386, and F:pSC101 recombinant plasmids: the specificity of F incompatibility genes.

The specificity of F incompatibility genes (inc⁺) has been studied with the Flac and R386 plasmids, members of the IncFI incompatibility group. Recently, two inc⁺ regions, incA (46.4–49.3F) and incB (43.1–46.4F) were identified by cloning these F sequences onto pSC101 and subsequently demonstrating incompatibility of the recombinants with Flac. It is shown here that the FincA⁺ recombinant is incompatible with both Flac and R386 while the FincB⁺ recombinant is incompatible only with Flac. Also, a plasmid mutant is described that has reduced incompatibility against Flac and R386. The mutation is located on the BamHI restriction fragment that contains the FincA region. These genetic findings are consistent with the deduction of Palchaudhuri and Maas, based on heteroduplex analysis of IncFI plasmids, that placed the IncFI determinant in the 46.4–48.6F region. The findings also indicate that the FincB⁺ gene product, which has been implicated in negative control of F copy number, is specific for the F replicon.     

Effect of tra mutations on F factor-specified immunity to lethal zygosis

Summary Hfr, F⁺, and F-prime cells are, unlike F^– cells, insensitive to an excess of Hfr donor cells, indicating that there is an F factor mediated immunity to lethal zygosis (Ilz). Results with Flac episomes carrying traJ, traS or various polar mutations in the tra region indicate that this immunity is independent of surface exclusion, of traJ control, and of all known genes within the tra operon. However, analysis of a series of strains with deletions in the F factor, extending from the right into the tra region, suggests that a gene for immunity to lethal zygosis is located within the tra region. We therefore conclude that Ilz is genetically complex, and present a hypothesis to account for these results.     

The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids

The effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating-pair formation in liquid media by the transfer systems of the F-Iike plasmids pOX38 (F), ColB2 and R100-1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II-heptose III region of the LPS (rfaF) whereas R100-1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating-pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F-like plasmids (F, ColB2, R100-1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.     

Fertility inhibition in Rhizobium lupini by the resistance plasmid RP4

Summary R plasmid RP4 inhibits the fertility of R. lupini. An RP4 carrying R. lupini donor strain is no longer capable of transferring chromosomal genes. After loss of RP4 the R. lupini fertility reappears. Plasmid RP4 spontaneously mutates at high frequency in R. lupini. RP4 mutants which do not inhibit fertility were isolated. These mutants were always transfer-defective, too. It is postulated that the genetic information for fertility inhibition in R. lupini is a substantial part of the transfer unit of the RP4 plasmid.     

Relaxation of stable RNA synthesis by a plasmid-borne locus

Summary The presence of Clo DF13 copy mutants in Escherichia coli (Flac) cells results, in contrast to the presence of Clo DF13 wt plasmids, in a decreased transfer of Flac and a decrease in the efficiency of plating (EOP) of male specific RNA phages.The degree of reduction of these processes is correlated to the number of Clo DF13 copies per cell as was found by the use of copy mutants and a thermosensitive copy control mutant of Clo DF13. For instance, the presence of the Clo DF13 cop3 plasmid results in a hundredfold decrease in EOP of RNA phages and a tenfold decrease in transfer of the F plasmid. No interaction with the efficiency of plating of male specific RNA phages was measured when the wild type Clo DF13, ColE1, ColE2, ColE3 or ColK plasmid is present in the cell. Studies with both, insertion and deletion mutants of CLo DF13 cop3 showed that these effects are not due to a high number of plasmid DNA molecules itself but due to a high amount of plasmid gene products in the cell. Furthermore these studies enabled us to locate the genes involved in these interactions on the Clo DF13 physical map. It turned out that two Clo DF13 genes are involved in the observed phenomena: one gene, coding for polypeptide B (molecular weight 61,000 daltons) which is also involved in the mobilisation of Clo DF13, and one gene coding for polypeptide D (molecular weight 21,000 daltons). The possible role of these Clo DF13 gene products, involved in the decrease in transfer of Flac as well as the decrease in efficiency of plating of male specific RNA phages, is discussed.     

Early and late transfer of F genes by Hfr donors of E. coli K-12

Summary The results of short interrupted matings between an Hfr donor and a recipient strain carrying a temperature-sensitive replication mutant (frp ^–) of Flac demonstrate that the Hfr strain transfers this frp gene of F early in conjugation. This frp gene was also shown to function in the maintenance of mutant F plasmids which appear to be generated from the DNA transferred early in conjugation by Hfr donors. In the course of these experiments, it was further demonstrated that certain Hfr strains which had been described as transferring the tra genes early in fact transfer that region of F late in conjugation.     

The maintenance affinities of KLF-1 proximal F merogenotes inEscherichia coli

Summary In conjugation with donor strains carrying proximal F merogenotes of KLF-1 type about 100-fold lower frequency ofLeu ⁺ orLac ⁺ recombinants was found. The determination of the level of β-galactosidase synthesis during the initial period of mating indicated that the transfer process of plasmid DNA was not impaired. Among the recombinants selected a large fraction have not expressed the plasmic fertility functions. This phenomenon was found to be replicon specific and was observed only with proximal F merogenotes but not with classical F'lac and F'ORF-1 elements or RI-19 plasmid. The expression of KLF-1 plasmid functions in the cell seems to be affected by a chromosomal gene of the proximal F merogenote closely linked toleu marker.     

Investigations of the F conjugation gene traI:traI mutants and lambdatraI transducing phages.

Summary A series of traI point and deletion mutants of Flac, and a traM mutant, were characterised. Complementation tests with an amber Flac traI mutant confirmed their genotypes, and in addition all the traI mutants, but not the traM mutant, were complemented by pRS31 (pSC101 traDI) and ED109 (traI). Judging from the efficiencies of plating of F-specific phages, none of the mutations affected pilus formation. The traI products of F and of the F-like plasmid R1 were interchangeable with each other but not with that of R100, while the traM product of F could not be replaced by those of R1 or of R100. Neither traI nor traM were needed for conjugal transfer of ColE1.Three transducing phages carrying traI were isolated by in vivo or in vitro techniques, and characterised by genetic complementation tests, by analysis of the fragments produced by restriction endonucleases, and by measurement of heteroduplex molecules. The genetic structures together with the sizes and F coordinates, of the transfer regions carried by the phages were thereby determined.Comparison of the proteins synthesised in UV-irradiated cells by one of the raI phages with those made by a derivative carrying an amber traImutation, allowed the traI product to be identified as a protein of molecular weight 174,000. In addition, the molecular weights of the traD (84,000), traS (18,000), and traT (25,000) products made by the traSTDI phage ED107 were measured.The possible roles of the traI and traM products in conjugation are discussed.