Plasmid Origin Of Transfer Research Articles

Sequence comparisons of plasmids pBJS-O of Spiroplasma citri and pSKU146 of S. kunkelii: implications for plasmid evolution

BackgroundSpiroplasma citri BR3-3X and S. kunkelii CR2-3X cause serious diseases worldwide on citrus and maize species, respectively. S. citri BR3-3X harbors a plasmid, pBJS-Original (pBJS-O), that encodes the spiroplasma adhesion related protein 1 (SARP1), a protein implicated in binding of the pathogen to cells of its leafhopper vector, Circulifer tenellus. The S. kunkelii CR2-3X plasmid, pSKU146, encodes a homolog of SARP1, Sk-ARP1. Due to the close phylogenetic relationship of the two pathogens, we hypothesized that the two plasmids are closely related as well.ResultsThe nucleotide sequence of pBJS-O was determined and compared to the sequences of a plasmid from BR3-T (pBJS-T), which is a multiply passaged leafhopper transmissible derivative of BR3-3X, and to known plasmid sequences including that of pSKU146. In addition to arp1, the 13,374 bp pBJS-O sequence putatively contains nine genes, recognized as open reading frames (ORFs). Several pBJS-O ORFs have homologs on pSKU146. However, the sequences flanking soj-like genes on both plasmids were found to be more distant from one another than sequences in any other region. Further, unlike pSKU146, pBJS-O lacks the conserved oriT region characteristic of the IncP group of bacterial plasmids. We were unable to identify a region in pBJS-O resembling a known plasmid origin of transfer. In regions where sequence was available for the plasmid from both BR3-3X and BR3-T, the pBJS-T sequence had a 0.4 kb deletion relative to its progenitor, pBJS-O. Southern blot hybridization of extrachromosomal DNA from various S. citri strains and spiroplasma species to an arp-specific probe and a probe made from the entire plasmid DNA of BR3-3X revealed limited conservation of both sequences in the genus Spiroplasma. Finally, we also report the presence on the BR3-3X chromosome of arp2, an S. citri homolog of arp1 that encodes the predicted protein SARP2. The C-terminal domain of SARP2 is homologous to that of SARP1, but its N-terminal domain is distinct.ConclusionOur data suggest that pBJS is a novel S. citri plasmid that does not belong to any known plasmid incompatibility group. The differences between pBJS-O and pSKU146 suggest that one or more events of recombination have contributed to the divergence of the plasmids of the two sister Spiroplasma species; the plasmid from S. citri itself has diverged slightly during the derivation of S. citri BR3-T from BR3-3X. Our data also show that pBJS-O encodes the putative adhesin SARP1. The presence of traE and mob on pBJS-O suggests a role for the plasmid in spiroplasmal conjugation.

DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA.

The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer.

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region.

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro.

The traY Gene Product and Integration Host Factor Stimulate Escherichia coli DNA Helicase I-catalyzed Nicking at the F Plasmid oriT

F plasmid conjugative transfer is initiated by the introduction of a site- and strand-specific nick within the plasmid origin of transfer (oriT). Genetic studies have shown nick formation to be dependent on both the traI and traY genes. However, highly purified TraIp, the traI gene product, nicks oriT in a site- and strand-specific manner in the absence of the traY gene product (TraYp) in vitro (Matson, S.W., and Morton, B.S. (1991) J. Biol. Chem. 266, 16232-16237). Analysis of the oriT region has revealed binding sites for TraYp and the host protein integration host factor (IHF). To explore possible interactions occurring at oriT, highly purified TraIp, TraYp, and IHF were incubated with a supercoiled oriT-containing DNA substrate. A marked enhancement of the nicking reaction catalyzed by TraIp was observed in a reaction that required both TraYp and IHF. In addition, TraIp was able to nick a linear oriT-containing double-stranded DNA substrate when IHF and TraYp were present in the reaction; such a substrate is not nicked by TraIp alone. Individual protein concentration requirements for the supercoiled and linear nicking reactions were similar, and the reactions occurred at equal velocity, suggesting that they are biochemically identical. Concentrations of TraYp and IHF that yield half-maximal activity in the nicking assays compare well with the reported KD values for the IHF and TraYp binding sites in oriT. These data, coupled with data presented in the accompanying report, suggest that TraYp and IHF bind independent of one another, forming a nucleo-protein complex with oriT that can be recognized and nicked by TraIp.

Stepwise Assembly of a Relaxosome at the F Plasmid Origin of Transfer

A central step in the transfer of genetic information during bacterial conjugation of the Escherichia coli F plasmid involves the formation of a protein-DNA complex, called the relaxosome, at the origin of transfer. During conjugation, the relaxosome introduces a site- and strand-specific nick from which the physical transfer of a single strand of DNA is initiated. At least two F-encoded proteins, TraIp (traI gene product) and TraYp (traY gene product), and one host-encoded protein, integration host factor, are involved in this process. In this report, we use DNase I protection and electron microscopic techniques to investigate the mechanism of relaxosome formation. Our results show that TraYp and integration host factor form a protein-DNA complex that facilitates the binding of TraIp to assemble a relaxosome capable of introducing a site- and strand-specific nick at the origin of transfer. This nick is identical to that observed during conjugation.

Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer

Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I.

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.

In vitrocleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.

Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT

A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 205-209). We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene. The implications of this result are discussed. The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer. Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction.

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 .