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.     

Five control systems preventing transfer of Escherichia coli K-12 sex factor F.

The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62. All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system. None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself. In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485. Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither. All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ. The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.     

Inhibition of Flac Transfer by the Fin+ I-Like Plasmid R62

Flac mutants have been isolated in Escherichia coli K-12 which carry dominant mutations resulting in insensitivity to transfer inhibition by the Fin(+) I-like plasmid R62. These mutants were still sensitive to transfer inhibition by the fin(+) F-like plasmid R100 and, conversely, FlactraO(-) and traP(-) mutants, which are insensitive to R100 inhibition, were still sensitive to R62. The sites of action of the two inhibition systems are therefore different. Furthermore, inhibition by R62, unlike R100, did not require an F-specified product. Like R100, R62 prevented transfer, pilus formation, and surface exclusion and, therefore, probably inhibits expression of the transfer operon traA through traI. However, R62 was different from R100 in inhibiting transfer of J-independent mutants, indicating that its effect on the transfer operon is probably direct rather than via traJ. This is consistent with the different sites of action of the two inhibition systems. None of the Flac mutants overproduced pili in the absence of R62, although one mutant differing from those described above showed increased levels of transfer and surface exclusion.     

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.     

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.     

Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1

The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific. These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.     

Plasmid Specificity of Two Proteins required for Conjugation in <Emphasis Type="Italic">E. coli</Emphasis> K12

IN recent years, many episomes other than the F factor of E. coli K12 have been discovered, such as the colicinogenic factors and the drug resistance transfer factors. Some of these seem to be related to F since they determine a pilus similar to the F pilus in serological properties and male specific phage sensitivity¹. These F-like plasmids frequently inhibit their own transfer and of F when present in the same cell, but mutants have been obtained which do not produce the inhibiting substance and which allow transfer at a high rate^1,2. One such mutant, R100-1, was used in the work to be described here.     

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.     

Analysis and characterization of the IncFV plasmid pED208 transfer region

pED208 is a transfer-derepressed mutant of the IncFV plasmid, F(0)lac, which has an IS2 element inserted in its traY gene, resulting in constitutive overexpression of its transfer (tra) region. The pED208 transfer region, which encodes proteins responsible for pilus synthesis and conjugative plasmid transfer, was sequenced and found to be very similar to the F tra region in terms of its organization although most pED208 tra proteins share only about 45% amino acid identity. All the essential genes for F transfer had homologs within the pED208 transfer region with the exception of traQ, which encodes the chaperone for stable F-pilin expression. F(0)lac appears to have a fertility inhibition system different than the FinOP system of other F-like plasmids, and its transfer efficiency was increased in the presence of F or R100, suggesting that it could be mobilized by these plasmids. The F-like transfer systems specified by F, R100, and F(0)lac were highly specific for their cognate origins of transfer (oriT) as measured by their abilities to mobilize chimeric oriT-containing plasmids.     

Deletion Map of the Chloramphenicol Resistance Region of R1 and R100-1

Recombination between single-site and multisite chloramphenicol-sensitive mutants of the F-like R factors R1 and R100-1 indicates that the chloramphenicol resistance region is a single structural gene coding for the 20,000-molecular weight subunit of chloramphenicol acetyltransferase.     

Further characterization of the recipient ability of Escherichia coli K-12 bacteriophage-resistant mutants.

We extended the study of Escherichia coli mutants defective in conjugation and showed that the mutants with altered lipopolysaccharide, which are defective as recipients with F-like donors, are also defective with the I-like plasmid R64-11. However, the extent of reduction in recipient ability for I-like donors does not correlate either with the effect on recipient ability for F-like donors or with the degree of alteration to the lipopolysaccharide.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: II. Structure of drug resistance (R) factors and f factors

The colicinogenic plasmid Col V-K94 and the class of transferable drug-resistance factors that inhibit fertility of the sex factor, F, are believed to be genetically similar to F. Heteroduplexes between these various F-like plasmids were studied by electron microscopy in order to identify and define the DNA segments that contain genes coding for fertility and other common functions. It was found that approximately 44% of the F factor is homologous to several different fi⁺ R factors and to Col V-K94, and that all of the homology between these plasmids and F is restricted to a region comprising only one-half of the F-factor molecule. These data, and previously reported electron microscope mapping of deletions of F factors, suggest that the genes active in the process of transfer are contained in one-half of the F factor, and therefore imply that the other half of F might not be required for expression of its fertility functions.DNA sequence homology among the R factors R6, R100 and R1 was also studied by heteroduplex formation. All of the DNA sequences contained in R100 were observed to be present in R6, and approximately 85% of the DNA sequences of R1 were included in R6. In the $\text{R1}\text{R6}$ heteroduplex, homology is distributed equally in the R-factor region homologous to the F factor, and the region containing antibiotic-resistance determinants (i.e. the segment deleted in the resistance transfer factor unit, RTF). The position of certain drugresistance determinants has been mapped by using various R-factor deletion mutants and correlating the pattern of drug resistance with the sequence homology observed among different R factors. Unlike the other resistance-determinant genes identified, the tetracycline marker has been mapped in the segment of R6 that is largely homologous to the F factor.Inverted repeats, which consist of DNA segments repeated in reverse sequence on the same single strand of DNA, were observed in R6, R100 and Col V-K94. One inverted repeat of R6 is located near the tetraeycline marker. Further duplication of one of the complementary sequences of the inverted repeat, and insertion of this duplication into the plasmid genome apparently inactivates the tetracycline-resistance determinant of R6.     

Genetic and physical characteristics of an enterotoxin plasmid.

We are engaged in the genetic and physical characterization of an enterotoxin (Ent) plasmid, Ent P307, which contains genes for the production of a hear-labile and a heat-stable enterotoxin. We are using an Escherichia coli K-12 strain, 711 (P307), constructed by S. Falkow, which contains no other plasmids besides Ent P307. Our genetic studies have shown that the plasmid is incompatible with the sex factor F, both in the integrated (Hfr) and the autonomous (F-prime) state. Ent P307 can thus be assigned to incompatibility group FI. An R factor, R386, which belongs to the same incompatibility group, was also found to be incompatibile with Ent P307, whereas five other R factors belonging to different incompatibility groups were compatible with Ent P307. In the presence of Ent P307, conjugal transfer and sensitivity to a male-specific phage of a derepressed F-like R factor, R1drd19, were repressed. Ent P307 is, thus, finO+. Presumably, it also causes repression of its own transfer genes since conjugal transfer of Ent P307 could not be demonstrated. Unlike F, it does not restrict the growth of female-specific phage phiII. From physical studies on extracted deoxyribonucleic acid, the molecular weight of Ent P307 was determined to be 54 X 10(6). By electron microscope heteroduplex analysis, the plasmid was found to be homologous with F in four regions, encompassing about half of its length. One long region and two short ones contain genes for conjugal transfer; the other short region carries genes for replication and incompatibility.     

Regulation of conjugal transfer by Lrp and Dam methylation in plasmid R100.

Conjugal transfer of the F-like plasmid R100 occurs at higher frequencies in the absence of DNA adenine methylation. Lower levels of R100-encoded FinP RNA were found in a Dam host, suggesting that Dam methylation regulates FinP RNA synthesis. Lack of the leucine-responsive regulatory protein (Lrp) causes a decrease in R100 plasmid transfer, indicating that Lrp is an activator of R100-mediated conjugation. Hence, host-encoded regulators previously described for the Salmonella virulence plasmid (pSLT) seem to play analogous roles in R100. Repression of conjugal transfer in rich medium is an additional trait shared by R100 and pSLT. DNA sequence comparisons in regulatory loci support the view that R100 and pSLT are closely related.     

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.     

F-Like Plasmid Sequences in Enteric Bacteria of Diverse Origin, with Implication of Horizontal Transfer and Plasmid Host Range

Seventy-eight bacterial isolates from human, animal, and plant hosts, representing eight species of the family Enterobacteriaceae, were screened for F-like plasmid sequences. Of the examined human Escherichia coli strains, 28% harbored one or two of the three F-like, RepFI replication regions, while 35% of the examined animal and all phytopathogenic strains harbored RepFIA-specific sequences. Comparative analysis of Salmonella, Shigella, Erwinia, and E. coli plasmid RepFI sequences showed 100% or very high homology, indicating frequent and recent interspecies gene transfer. The high incidence of RepFIA sequences in enteric bacterial species, including Klebsiella and Erwinia, showed that F-like plasmids are successful in avoiding natural barriers to establishment of horizontally transferred DNA and that in the natural environment conjugal transfer is efficient in diverse ecological niches. Received: 26 March 2001 / Accepted 12 July 2001     

Mechanisms of strand replacement synthesis for plasmid DNA transferred by conjugation

A single strand of plasmid DNA is transferred during conjugation. We examined the mechanism of complementary strand synthesis in recipient cells following conjugative mobilization of derivatives of the IncQ plasmid R1162. A system for electroporation of donor cells, followed by immediate mating, was used to eliminate plasmid-specific replicative functions. Under these conditions, Escherichia coli recipients provided a robust mechanism for initiation of complementary strand synthesis on transferred DNA. In contrast, plasmid functions were important for efficient strand replacement in recipient cells of Salmonella enterica serovar Typhimurium. The mobilizing vector for R1162 transfer, the IncP1 plasmid R751, encodes a DNA primase with low specificity for initiation. This protein increased the frequency of transfer of R751 into Salmonella, but despite its low specificity, it was inactive on the R1162 derivatives. The R751 primase was slightly inhibitory for the transfer of both R751 and R1162 into E. coli. The results show that there is a chromosomally encoded mechanism for complementary strand synthesis of incoming transferred DNA in E. coli, while plasmid-specific mechanisms for this synthesis are important in Salmonella.     

Physical Properties and Mechanism of Transfer of R Factors in Escherichia coli

The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.