Procedures are described for the detection and isolation of plasmids of various sizes (2.6 to 350 megadaltons) that are harbored in species of Agrobacterium, Rhizobium, Escherichia, Salmonella, Erwinia, Pseudomonas, and Xanthomonas. The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures. Proteins and cell debris were removed by extraction with phenol-chloroform. Under these conditions chromosomal DNA concentrations were reduced or eliminated. The clarified extract was used directly for electrophoretic analysis. These procedures also permitted the selective isolation of plasmid DNA that can be used directly in nick translation, restriction endonuclease analysis, transfornation, and DNA cloning experiments. was isolated from Cotoneaster sp. in this laboratory. Providencia stuartii 164 was sent by F. Bolivar. Serratia marcescens 5B was obtained from C. Mulder.

No abstract

TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.Horizontal gene transfer mediated by plasmid-borne conjugation from donor to recipient cells is initiated by a cell-to-cell bridging arrangement. These cellular interactions, whose complex is named mating pair formation (Mpf), 1 are initiated by conjugative pili encoded by broad-host range plasmids in Gram-negative bacteria. In the case of Agrobacterium-mediated T-DNA transfer to plant cells, the VirB complex is the Mpf structure (1, 2).Pili are usually long thin filaments extending from the surface of donor cells and upon close examination appear as tubelike structures with an outer diameter of about 8 -10 nm and a central, hydrophilic lumen of 2 nm (3, 4). With both the broadhost range plasmids of the IncP group and the Ti plasmids of the IncRh1 group, promiscuous pilus production requires genes of the Mpf system (5) and the virB operon (6, 7), respectively.Like the sex pili of F (8), pili of the above plasmids are essential for conjugative DNA transfer. Functional dissection of the Mpf apparatus and the virB genes has shown that DNA transfer is abolished by nonpolar inactivation of the pilin precursor genes or any gene of the pilus assembly machinery (5, 9). Besides DNA transfer, the transport of pilin subunits via the virB-encoded apparatus has been proposed to erect the T pilus (10). It remains unknown, however, if the transmembrane structures of the Mpf system and VirB complex are used as a channel to transfer DNA. Certainly, there exists a close evolutionary relationship between the IncP conjugative apparatus and the T-DNA transfer system (11,12). In this regard, the bacterial reliance of the pilus for DNA transfer raises a key question on whether or not the conjugative pilus is directly involved not only in bridging the donor to the recipient cell but also serving as a molecular conduit in DNA transmission. This resurrects an earlier key question raised on the possibility that the sex pilus, which appears to be tubular in structure, is used by the donor cell to transfer F-DNA rather than by direct cell-to-cell contact (3, 13). In this regard, the analyses of the structure as w...

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of BaciUus subtilis for use in the positive selection and isolation of insertion sequence (IS)Y'elements in gram-negative bacteria. Cells containing pUCD80 do not grow on medium containing 5% sucrose tinless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NTlRE by selection for growth on sucrose medium. This putative IS element'appears to be unique to Agrobacterium strains.

The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells. Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each virB gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined. In the present study, each virB gene was examined and was found to be unessential for T-pilin biosynthesis, except virB2, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation. We also find that the genes of the virD operon are neither involved in T-pilin export nor T-pilus formation. Critical analysis of three different virD4 mutants also showed that they are not involved in T-pilus biogenesis irrespective of the A. tumefaciens strains used. With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the A. tumefaciens rod-shaped cell in a polar manner. We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation. These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid vir genes. A search of the vir genes involved in controlling this biphasic reaction in induced A. tumefaciens cells revealed that virA on the Ti plasmid is involved and that neither virB nor virD genes are needed for this reaction. The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified vir gene in A. tumefaciens.Agrobacterium tumefaciens is uniquely adapted for the horizontal transmission of DNA. The DNA transfer system employed by this rhizoplane inhabitant is encoded by vir genes located on a resident Ti plasmid. The DNA transfer system is highly promiscuous, as evidenced by its transfer activity of the T-DNA into plants, fungi (8, 9, 14), and actinomycetes (23).The products of the vir genes have been extensively studied in order to gain insights into the DNA transfer mechanism. Comparative studies of the nucleotide sequences of the vir genes with those of broad-host-range plasmids have revealed significant homologies between genes involved in DNA processing as well as plasmid transfer via bacterial conjugation, suggesting that the mechanism for T-DNA transfer could be a conjugation process (24,29,30,35,41,43,48). Using the F plasmid as a classic example, bacterial conjugation requires a conjugative pilus, whose propilin subunit is encoded by traA (17). The TraA propilin is processed from a 12.7-kDa propilin into a 7.2-kDa pilin with an acetylated N terminus (17). VirB2, encoded by the virB operon of the Ti plasmid, is a homolog of TraA and is processed like TraA by generating a 7.2-kDa protein from a 12.3-kDa full-length holoprotein (2...