Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer.

Transfer genes of the IncP plasmid RP4 are grouped in two separate regions, designated Tra1 and Tra2. Tra2 gene products are proposed to be mainly responsible for the formation of mating pairs in conjugating cells. To provide information relevant to understanding the function of Tra2 gene products, the nucleotide sequence of the entire RP4 Tra2 region is presented here. Twelve open reading frames were identified in the Tra2 core region, being essential for intraspecific Escherichia coli matings. Predicted sizes of 11 of the 12 Tra2 polypeptides could be verified by expression in E. coli. Based on hydropathy plot analysis, most of the Tra2 open reading frames encode proteins that may interact with membranes. Interestingly, six of the predicted Tra2 gene products exhibited significant sequence similarities to gene products encoded by the VirB operon of the Agrobacterium Ti plasmid. VirB proteins are thought to function in the formation of a transmembrane structure that mediates the passage of T-DNA molecules from bacteria into plant cells. Because of this analogy and the hydropathy of Tra2 gene products, we assume that the DNA transfer machineries acting in bacterial conjugation and T-DNA transfer are structurally and functionally similar. Therefore, the data presented here, support the hypothesis that Ti vir and IncP tra genes evolved from a common ancestor. This suggestion is favored by previous findings of sequence similarities between the IncP and Ti DNA transfer system.     

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.     

A new type IV secretion system promotes conjugal transfer in Agrobacterium tumefaciens

Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.     

The replicator of the nopaline-type Ti plasmid pTiC58 is a member of the repABC family and is influenced by the TraR-dependent quorum-sensing regulatory system

The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034-1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, and repC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids from Rhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region between traI and repA contained two copies of the tra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from the tra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal rep plasmid increased five- to sevenfold in strains expressing traR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.     

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.     

Assembly of a functional phage PRD1 receptor depends on 11 genes of the IncP plasmid mating pair formation complex.

PRD1, a lipid-containing double-stranded DNA bacteriophage, uses the mating pair formation (Mpf) complex encoded by conjugative IncP plasmids as a receptor. Functions responsible for conjugative transfer of IncP plasmids are encoded by two distinct regions, Tra1 and Tra2. Ten Tra2 region gene products (TrbB to TrbL) and one from the Tra1 region (TraF) form the Mpf complex. We carried out a mutational analysis of the PRD1 receptor complex proteins by isolating spontaneous PRD1-resistant mutants. The mutations were distributed among the trb genes in the Tra2 region and accumulated predominantly in three genes, trbC, trbE, and trbL. Three of 307 phage-resistant mutants were weakly transfer proficient. Mutations causing a phage adsorption-deficient, transfer-positive phenotype were analyzed by sequencing.     

The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F.

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.     

Essential Components of the Ti Plasmid trb System, a Type IV Macromolecular Transporter

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.     

Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex.

DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.     

vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells.     

Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4.

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.     

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.     

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.     

A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains.

The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown. The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids. Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine. Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine. Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli. Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags. Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes. Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes. ags is preceded by a promoter that functions only in the plant. Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium. Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence. Antibodies raised against the agcA product cross-reacted with the anabolic enzyme. These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium.     

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.     

Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes.

The conjugation system of the IncP alpha plasmid RK2/RP4 is encoded by transfer regions designated Tra1, Tra2, and Tra3. The Tra1 core region, cloned on plasmid pDG4 delta 22, consists of the origin of transfer (oriT) and 2.6 kilobases of flanking DNA providing IncP alpha plasmid-specific functions that allow pDG4 delta 22 to be mobilized by the heterologous IncP beta plasmid R751. Tn5 insertions in pDG4 delta 22 define a minimal 2.2-kilobase region required for plasmid-specific transfer of oriT. The Tra1 core contains the traJ and traK genes as well as an 18-kilodalton open reading frame downstream of traJ. The traJ and traK genes were shown to be required for transfer by complementation of inserts within these genes. Genetic evidence for the role of the 18-kilodalton open reading frame in transfer was obtained, although this protein has not been detected in cell lysates. These studies indicate that at least three transfer proteins are involved in plasmid-specific interactions at oriT.     

The octopine-type Ti plasmid pTiA6 of Agrobacterium tumefaciens contains a gene homologous to the chromosomal virulence gene acvB.

Although the majority of genes required for the transfer of T-DNA from Agrobacterium tumefaciens to plant nuclei are located on the Ti plasmid, some chromosomal genes, including the recently described acvB gene, are also required. We show that AcvB shows 50% identity with the product of an open reading frame, designated virJ, that is found between the virA and virB genes in the octopine-type Ti plasmid pTiA6. This reading frame is not found in the nopaline-type Ti plasmid pTiC58. acvB is required for tumorigenesis by a strain carrying a nopaline-type Ti plasmid, and virJ complements this nontumorigenic phenotype, indicating that the products of these genes have similar functions. A virJ-phoA fusion expressed enzymatically active alkaline phosphatase, indicating that VirJ is at least partially exported. virJ is induced in a VirA/VirG-dependent fashion by the vir gene inducer acetosyringone. Primer extension analysis and subcloning of the virJ-phoA fusion indicate that the acetosyringone-inducible promoter lies directly upstream of the virJ structural gene. Although the roles of the two homologous genes in tumorigenesis remain to be elucidated, strains lacking acvB and virJ (i) are proficient for induction of the vir regulon, (ii) are able to transfer their Ti plasmids by conjugation, and (iii) are resistant to plant wound extracts. Finally, mutations in these genes cannot be complemented extracellularly.     

Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa.

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.