Neil Willetts

Genetic structure, function and regulation of the transposable element IS21

SummaryThe IncP plasmid R68.45 and other plasmids carrying tandem repeats of the insertion sequence IS21 [=(IS21)2] produce replicon fusions via transposition at high frequencies in Escherichia coli and other gram-negative bacteria, whereas plasmids with a single IS21 copy, e.g. R68, give replicon fusions rarely. The 2131 bp nucleotide sequence of IS21 was determined; at the ends there were 11 bp inverted repeats with one mismatch. Two adjacent open reading frames, istA and istB, were located on one DNA strand of IS21. In E. coli maxicells, polypeptides of 46 kDa (the istA gene product) and 30 kDa (the istB gene product) were expressed by (IS21)2 plasmids, but not by IS21 plasmids. Genetic analysis of (IS21)2 plasmids indicates that the IS21-IS21 junctions form a promoter, which initiates transcription of the istAB operon in one of the two IS21 elements. A single IS21 element fused to an inducible external tac promoter expressed both proteins after induction, but did not promote effective replicon fusion, unless an IS21-IS21 junction (the preferred site for IS21 transposase action) was also present on the plasmid carrying the tac-IS21 construct. The sequences located between the IS21 elements in (IS21)2, 3 bp in R68.45 or 2 bp in pME28, were not recovered in the replicon fusion products. Homologous recombination between the directly oriented IS21 elements in the fusion products led to plasmids with a single IS21 insertion. Analysis of the latter showed that IS21 had a low, but not totally random specificity of insertion and created target duplications of 4 bp (occasionally 5 bp). These and previous results support the following model: plasmids like R68.45 are opened by IS21 transposase at the IS21-IS21 junction and then integrated into a target replicon by simple insertion, with loss of the 2–3 bp junction sequence.

The transcriptional control of fertility in F-like plasmids.

Two λtra transducing phages were characterized, one carrying the control gene traJ, and the other carrying traL traE traK, three genes of the transfer operon. DNA from these phages was used in filter hybridization assays which indicated firstly that during FinOP transfer inhibition of Flac or R100 the transcription of traJ is repressed, and secondly that in the absence of the traJ gene product, the transfer operon is not transcribed. This confirms that traJ is a positive control gene central to this complex mechanism of fertility control prevalent amongst F-like plasmids.

The requirements for conjugal DNA synthesis in the donor strain during flac transfer.

Abstract Although neither rifampicin nor spectinomycin had any effect on the frequency of F lac transfer by a sensitive donor, rifampicin but not spectinomycin prevented donor conjugal DNA synthesis as measured in matings between a dnaB donor and a tdk recipient. An untranslated RNA species is therefore probably required for this synthesis, although transfer took place even in its absence. Donor conjugal DNA synthesis was abolished in a dnaE donor, showing that DNA polymerase III is responsible for this process; again, plasmid DNA transfer was not affected. F lac mutants lacking the F pilus gave neither donor conjugal DNA synthesis nor plasmid DNA transfer, probably because they could not receive a “mating signal” to activate the transfer process. The products of traI and traM were also required both for donor conjugal DNA synthesis and for physical transfer of plasmid DNA, probably being involved in the conversion of covalently closed circular plasmid DNA into the open circular form that is the substrate for the independent although normally simultaneous synthesis and transfer steps. In contrast, donor conjugal DNA synthesis took place at a normal rate in both piliated traG and traN mutants, and at a reduced rate in traD mutants, although in no case was there physical transfer of plasmid DNA. These gene products are therefore required for DNA transfer to the recipient, and in addition, the absence of the traD product may hinder DNA synthesis. Based upon these results, a scheme for the processing of DNA during conjugation is presented.

Characterisation of an in vivo system for nicking at the origin of conjugal DNA transfer of the sex factor F.

Abstract Transfer of the F plasmid between conjugating Escherichia coli cells has been assumed to require endonucleolytic cleavage at a specific site ( oriT ) on a specific strand of the F molecule. Using a lambda transducing phage which contains oriT we have detected this nicking process in vivo . Nicking of DNA occurred in the strand that included the “transferred” F strand and at a location within the transducing segment consistent with all previous genetic and restriction enzyme cleavage data on the position of oriT in F. Genetic study of the nicking process using F lac tra − point and deletion mutants, and also λ tra phages which carried various parts of the transfer region, indicated that the products of two transfer operon genes, traY and the previously unidentified gene traZ , were directly involved in nicking at oriT . The product of traJ was also required for nicking, but the possibility that this was solely due to the regulatory function of the traJ product could not be excluded. The plasmid specificities of oriT, traY and traZ between F and the related F-like plasmids R1-19 and R100-1 were investigated using the λoriT nicking system, and shown to be consistent with those determined in genetic complementation tests. The differences in specificity observed imply that the oriT sequence of F differs from those of R1-19 and R100-1. The products of the traM and traI genes are known to be required for the initiation of DNA transfer; their possible roles in modulating the activity of the traY Z endonuclease are discussed.

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

SummaryMutants 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.

A physical and genetic map of the IncN plasmid R46

Abstract A combined physical and genetic map of the conjugative IncN plasmid R46 was obtained by restriction endonuclease cleavage analysis, followed by the construction and analysis of deletion and recombinant derivatives. The genetic determinants for the antibiotic resistance and uv-protection phenotypes were located, as well as the regions necessary for plasmid replication and for conjugal transfer. The end points of the deletion giving rise to the R46 derivative pKM101 were localized.

The site of action of the F transfer inhibitor.

SummaryThe 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.

Specificities of IncF plasmid conjugation genes

The conjugation regions of IncF plasmids are closely related in that they share extensive DNA homology, and that they specify related pili. Variations between individual conjugation gene products of different IncF plasmids have, however, been noted. We have extended these observations by carrying out a systematic survey of twelve such plasmids, to examine the numbers and the groupings of the plasmid-specific alleles of several genes required for conjugation and its control. Using vector plasmids carrying cloned origins of transfer (oriT), four different specificities were recognized, and these were correlated with the specificities of the genes with products that may act at this site (traM, traY and traZ). The traY gene is the first gene of the major transfer operon, and is therefore located close to the site at which the traJ protein acts to induce expression of the operon: correspondingly, correlation was observed between the oriT/traMYZ and traJ specificities in most of the plasmids. In turn, traJ is negatively regulated by the finO and finP products acting in concert: the finO product was relatively non-specific, but six finP alleles were identified, again with , specificities correlated with those of traJ. Our explanation for this unexpectedly large number of finP alleles derives from the concept that the finP product is an RNA molecule rather than a protein. Although the conjugative pili encoded by IncF plasmids are closely related, they confer different efficiencies of plating of the various F-specific bacteriophages. We distinguished four groups on this basis, presumably resulting from differences in the primary amino-acid sequences of the pilin proteins. These groups could be related to the surface exclusion system specificities, consistent with the hypothesis that surface exclusion acts at least in part by preventing interaction between the pilus and the recipient cell surface.

The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks.

The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT, has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT‐rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand‐specific nicks acquired by a lambda oriT genome upon propagation in Flac‐carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .

Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region.

The entire region required for mobilization of the non-conjugative plasmid RSF1010 has been cloned into a mobilization-deficient pBR322 derivative. The segment of DNA cloned was approximately 1.8 kb and included the origin of conjugal DNA transfer (oriT). The DNA sequence of the mobilization region has been determined, and revealed the presence of several overlapping reading frames. The isolation and mapping of both Tn1725 and BamH1-linker insertions and comparison with the DNA sequence data has allowed the identification of three genes required for mobilization. Two of these genes are overlapping and encode proteins of 16 kDa and greater than 65 kDa (although the truncated protein is functional, the gene extends outside the region cloned). The third gene is transcribed in the opposite direction. Promoters capable of transcribing these genes were located by S1 mapping in the inter-cistronic region between these divergently transcribed genes. The oriT site is located in this region, and the transcriptional patterns observed for mob+ and mob- plasmids implied that the promoters may be regulated by two of the mobilization proteins binding to the oriT site.