Physical and genetic analyses of IncI2 plasmid R721: evidence for the presence of shufflon

A physical map of the 75.1-kb IncI2 plasmid R721 was constructed by using 15 restriction enzymes, and the regions of several genetic determinants including the origins of replication and of conjugal DNA transfer were located on the physical map. It was found that R721 bears a DNA region which undergoes DNA rearrangement similar to the shufflon of R64.     

Distribution of shufflon among IncI plasmids.

A shufflon or clustered inversion is a novel type of DNA rearrangement originally discovered in the IncI1 plasmid R64 (T. Komano, A. Kubo, and T. Nisioka, Nucleic Acids Res. 15:1165-1172, 1987). In a 1.95-kilobase region of R64 DNA, four DNA segments inverted independently or in groups, resulting in a complex DNA rearrangement. We found similar types of shufflon in other IncI1 plasmids, including delta, pIP111, pIP565, pIP112, pIP186, R144, R163, R483, and R621a. A variant type of shufflon occurs in the IncI1 plasmid ColIb.     

Purification and characterization of the R64 shufflon-specific recombinase

The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.     

Mating variation by DNA inversions of shufflon in plasmid R64

Gene organization of the 54-kb transfer region of IncI1 plasmid R64 was deduced from the DNA sequence. Forty-eight ORFs were found in this region. A unique DNA rearrangement designated shufflon is located at the downstream region of an operon responsible for synthesis of thin pilus. The shufflon of R64 consists of four DNA segments, designated as A, B, C, and D, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination mediated by the product of the rci gene between any two inverted repeats results in a complex DNA rearrangement. An analysis of open reading frames revealed that the shufflon is a biological switch to select one of seven C-terminal segments of the pilV genes. The products of pilV genes were shown to be components of thin pilus which was required for liquid mating.Seven R64 derivatives where the pilV genes were fixed in the seven C-terminal segments were constructed and their transfer frequencies in liquid mating were measured using various bacterial strains as recipients. Transfer frequencies of R64 in liquid mating strongly depended on the combination of C-terminal segments of the pilV genes in donor cells and bacterial strains of recipient cells, suggesting that the shufflon determines the recipient specificity in liquid mating of plasmid R64.     

Cloning and nucleotide sequence of the ColIb shufflon

The R64 shufflon is a novel type of DNA rearrangement in which four DNA segments invert independently or in groups. The related plasmid ColIb carries a variant shufflon. The present sequence analysis shows that the ColIb shufflon consists of three DNA segments that are highly homologous to the A, B, and C segments of the R64 shufflon. The 329-bp D segment of R64 is not present in the ColIb shufflon. As in the case of R64, the ColIb shufflon may act as a biological switch to select one of the six open reading frames in which the N-terminal region is constant while the C-terminal region is variable.     

Twelve pil genes are required for biogenesis of the R64 thin pilus

The IncI1 plasmid R64 produces two kinds of sex pili: a thin pilus and a thick pilus. The thin pilus, which belongs to the type IV family, is required only for liquid matings. Fourteen genes, pilI to -V, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R. Kim and T. Komano, J. Bacteriol. 179:3594-3603, 1997). In this study, we introduced frameshift mutations into each of the 14 pil genes to test their requirement for R64 thin pilus biogenesis. From the analyses of extracellular secretion of thin pili and transfer frequency in liquid matings, we found that 12 genes, pilK to -V, are required for the formation of the thin pilus. Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes. Two genes, traBC, were previously shown to be required for the expression of the pil genes. In addition, the rci gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon. Altogether, 15 genes, traBC, pilK through pilV, and rci, are essential for R64 thin pilus formation and function.     

Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences

The shufflon of plasmid R64 consists of four DNA segments separated and flanked by seven sfx recombination sites. Rci-mediated recombination between any inverted sfx sequences causes inversion of the DNA segments independently or in groups. The R64 shufflon selects one of seven pilV genes encoding type IV pilus adhesins, in which the N-terminal region is constant, while the C-terminal regions are variable. The R64 sfx sequences are asymmetric. The sfx central region and right arm sequences are conserved, but left arm sequences are not. Here we constructed a symmetric sfx sequence, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence and made artificial shufflon segments carrying symmetric sfx sequences in inverted or direct orientations. The symmetric sfx sequence exhibited the highest inversion frequency in a shufflon segment flanked by two inverted sfx sequences. Rci-dependent deletion of a shufflon segment flanked by two direct symmetric sfx sequences was observed, suggesting that asymmetry of R64 sfx sequences inhibits recombination between direct sfx sequences. In addition, intermolecular recombination between symmetric sfx sequences was also observed. The extra C-terminal domain of Rci was shown to be essential for inversion of the R64 shufflon using asymmetric sfx sequences but not essential for recombination using symmetric sfx sequences, suggesting that the Rci C-terminal segment helps the binding of Rci to asymmetric sfx sequences. Rci protein lacking the C-terminal domain bound to both arms of symmetric sfx sequence but only to the right arm of asymmetric sfx sequence.     

Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.     

Highly mobile DNA segment of IncI alpha plasmid R64: a clustered inversion region.

When R64 DNA was digested with EcoRI, two DNA fragments not equimolar to the plasmid DNA were produced. A DNA region including these fragments was cloned (pKK009), and the pKK009 DNA sample was found to be a mixture of six or more DNA species with EcoRI, PstI, and AvaI cleavage sites at different positions, suggesting a complex rearrangement of DNA. When a part of the pKK009 DNA was removed by HindIII digestion, 33 different types of plasmids (pKK010-series plasmids) were obtained out of 58 clones tested, but no DNA rearrangement could be observed. On the basis of a comparison of the detailed restriction maps of these pKK010-series plasmids, we propose a model in which four DNA segments invert independently or in groups within the 1.95-kilobase region of R64, so that the arrangements of these four segments change randomly. The fixed pKK010-series plasmid DNA was again rearranged in the presence of R64, indicating that trans-acting gene function may be present to mediate the DNA rearrangement. The gene (tentatively designated as rci) was located on a 4.5-kilobase E9' fragment of R64.     

The transfer region of IncI1 plasmid R64: similarities between R64 tra and legionella icm/dot genes

The entire nucleotide sequence of the transfer region of IncI1 plasmid R64 was determined together with previously reported sequences. Twenty-two transfer genes, traE-Y and nuc, were newly identified in the present study. The protein products of 17 genes were detected by maxicell experiments or by the T7 RNA polymerase expression system. Mutagenesis experiments indicated that 16 genes were indispensable for R64 transfer both in liquid and on surfaces. In summary, the R64 transfer region located within an approximately 54 kb DNA segment was shown to encode the most complex transfer system so far studied. It contains at least 49 genes and may produce 58 different proteins as a result of shufflon DNA rearrangement and overlapping genes. Among the 49 genes, 23 tra, trb and nik genes have been shown to be indispensable for R64 conjugal transfer in liquid and on surfaces. Twelve additional pil genes are required only for liquid matings. The amino acid sequences of 10 R64 tra/trb products share similarity with those of the icm/dot products of Legionella pneumophila that are responsible for its virulence, suggesting that the R64 transfer and L. pneumophila icm/dot systems have evolved from a common ancestral genetic system.     

Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames.

The IncI alpha plasmid R64 was found to bear a highly mobile DNA segment which was designated as a clustered inversion region (J. Bacteriol. 165, 94-100, 1986). The clustered inversion region consists of four DNA segments designated respectively as A, B, C and D which differ in molecular size and restriction sites. The four DNA segments invert independently or in groups resulting in a complex DNA rearrangement. We now show the nucleotide sequence of the clustered inversion region of R64. The present results suggest that the clustered inversion region is a biological switch to select one of seven open reading frames whose primary structures at the region proximal to N-termini are constant while those at the C-terminal region are variable. A name, "Shufflon" was proposed to call this kind of the clustered inversion region.     

Antirestriction activity of the IncI1 transmissive plasmid R64 ArdA protein

The transmissive plasmid IncI1 R64 contains the ardA gene encoding the ArdA antirestriction protein. The R64 ardA gene locating in the leading region of plasmid R64 has been cloned and their sequence has been determined. Antirestriction proteins belonging to the Ard family are specific inhibitors of type I restriction-modification enzymes. The IncI1 ColIb-P9 and R64 are closely related plasmids, and the latter specifies an ArdA homologue that is predicted to be 97.6% (162 residues from 166) identical at the amino acid sequence level with the ColIb = P9 equivalent. However, the R64 ArdA selectively inhibits the restriction activity of EcoKi enzyme leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The ColIb-P9 ArdA inhibits restriction endonuclease and methyltransferase activities simultaneously. It is hypothesized that the ArdA protein forms two complexes with the type I restriction-modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonuclease. The association of the ColIb-P9 ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of the R64 ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.     

Thin pilus PilV adhesins of plasmid R64 recognize specific structures of the lipopolysaccharide molecules of recipient cells

IncI1 plasmid R64 encodes a type IV pilus called a thin pilus, which includes PilV adhesins. Seven different sequences for the C-terminal segments of PilV adhesins can be produced by shufflon DNA rearrangement. The expression of the seven PilV adhesins determines the recipient specificity in liquid matings of plasmid R64. Salmonella enterica serovar Typhimurium LT2 was recognized by the PilVA' and PilVB' adhesins, while Escherichia coli K-12 was recognized by the PilVA', PilVC, and PilVC' adhesins. Lipopolysaccharide (LPS) on the surfaces of recipient cells was previously shown to be the specific receptor for the seven PilV adhesins. To identify the specific receptor structures of LPS for various PilV adhesins, R64 liquid matings were carried out with recipient cells consisting of various S. enterica serovar Typhimurium LT2 and E. coli K-12 waa mutants and their derivatives carrying various waa genes of different origins. From the mating experiments, including inhibition experiments, we propose that the GlcNAc(alpha1-2)Glc and Glc(alpha1-2)Gal structures of the LPS core of S. enterica serovar Typhimurium LT2 function as receptors for the PilVB' and PilVC' adhesins, respectively, while the PilVC' receptor in the wild-type LT2 LPS core may be masked. We further propose that the GlcNAc(beta1-7)Hep and Glc(alpha1-2)Glc structures of the LPS core of E. coli K-12 function as receptors for the PilVC and PilVC' adhesins, respectively.     

Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection.

F-like plasmids require a number of genes for conjugation, including tra operon genes and genes traM and traJ, which lie outside the tra operon. We now establish that a gene in the "leading region," gene 19, provides an important function during conjugation and RNA phage infection. Mutational inactivation of gene 19 on plasmid R1-16 by introduction of two nonpolar stop codons results in a 10-fold decrease in the conjugation frequency. Furthermore, infection studies with the male-specific bacteriophage R17 revealed that the phage is not able to form clear plaques in Escherichia coli cells carrying an R1-16 plasmid with the defective copy of gene 19. The total number of cells infected by phage R17 is reduced by a factor of 10. Both the conjugation- and infection-attenuated phenotypes caused by the defective gene 19 can be complemented in trans by introducing gene 19 alleles encoding the wild-type protein. Restoration of the normal phenotypes is also possible by introduction of the pilT gene encoded by the unrelated IncI plasmid R64. Our functional studies and similarities of protein 19 to proteins encoded by other DNA transfer systems, as well as the presence of a conserved motif in all of these proteins (indicative for a putative muramidase activity) suggest that protein 19 of plasmid R1 facilitates the passage of DNA during conjugation and entry of RNA during phage infection.     

Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64.

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.     

Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.     

The genome sequence of the incompatibility group Iγ plasmid R621a: Evolution of IncI plasmids

We present the complete genome sequence of the tetracycline resistance plasmid R621a isolated from Salmonella typhimurium, which belongs to the incompatibility group Iγ. In the 93,185 bp circular double-stranded R621a genome, 96 complete ORFs are predicted. In addition, one and six different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of four regions: replication, leading, transfer, and miscellaneous regions. The R621a genome is similar to those of IncI1 plasmids such as R64 and ColIb-P9 and particularly to those of pEK204 and pEC_Bactec. Three major differences including inc, parAB, and excA regions were noted between R621a and prototype IncI1 plasmids. Seven nucleotide replacements and one nucleotide deletion in the putative Inc RNA sequence are found between R621a and IncI1 plasmids irrespective of close similarity in the other parts of the rep system. The sequences of R621a parAB and excA genes are significantly different from those of R64 and ColIb-P9, while those of R621a parAB and excA genes exhibit close similarity to those of pEK204 and pEC_Bactec, respectively. The R621a genome is suggested to be formed by acquiring parAB and excA genes from pEK204 and pEC_Bactec genomes, respectively, and then novel inc function by the mutations. The insertions in the R621a, pEK204, and pEC_Bactec genomes are flanked by direct repeats, suggesting that insertions accompanied by long target duplications have also played an important role in the evolution of IncI plasmids.     

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64.

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.     

Distribution of plasmid maintenance regions among IncFIV plasmids

The distribution of the IncFI basic replicons among IncFIV plasmids was assessed by DNA hybridization. In addition these and 20 other plasmids from 16 incompatibility groups were screened for the presence of IncIV, an incompatibility determinant recently found on the IncFIV plasmid R124. The IncIV determinant was found commonly but not universally among the IncFIV plasmids. It was also detected on the IncFI reference plasmid R386 and plasmids from IncB, IncI alpha and IncI gamma. The frequency and distribution of IncFI replicons among the IncFIV plasmids is similar to that observed in other F groups. The similarity of the IncFIV plasmids to plasmids of the other IncF groups and the failure to find replicons unique to IncFIV plasmids indicates that their division into a separate incompatibility group is not justified.