The cytoplasmic protein TraM is one of four essential gene products of the F factor which are involved in DNA transfer after mating pair formation. TraM binds to three specific sites within the oriT region. Besides regulation of its own synthesis, the precise function of TraM during conjugation is not yet known. In the present work, the affinity of TraM to TraD was studied in vitro by an overlay assay and by affinity chromatography. Whether the interaction between TraM and TraD causes a transient or permanent anchoring of the F factor to the site of transfer is discussed. A 35-kDa host membrane protein of yet unknown function also shows affinity to TraM and may be involved in this anchoring process as well.Bacterial conjugation is a process during which DNA is transferred from a donor to a recipient across the envelope of both cells. One of the best-studied conjugative plasmids is the F factor of Escherichia coli (for review, see references 7, 8, 11, and 27). After pilus synthesis and mating pair formation during the conjugation of the F factor, four additional gene products of the tra operon are directly involved in a successful DNA translocation across the envelope of the donor. These products are encoded by the genes traI, traY, traD, and traM. Of these four essential Tra proteins, TraI is the best characterized. The TraI protein, also called helicase I, catalyzes the strand-and site-specific nicking of the F factor at oriT (16,22). Furthermore, TraI unwinds duplex DNA in an ATP-hydrolysis-dependent reaction (1-3). The precise functions of TraY, TraD, and TraM are not yet known. It was recently shown in vivo that the nicking reaction of TraI is stimulated by TraY and the integration host factor (IHF) of E. coli (18). Both proteins bend DNA when bound to specific sites within the oriT region (14,15,17,19,26). Nelson and coworkers (18) propose that TraY and IHF form a nucleoprotein complex with oriT DNA which consequently can be recognized and nicked by TraI more efficiently.While TraI and TraM function in the processing of the F DNA, TraD seems to be a component of the DNA transfer apparatus. It is generally accepted that export of the singlestranded F DNA across the envelope of the donor cell proceeds through a complex pore formed by or with participation of various Tra proteins. One of these proteins is probably TraD. The TraD protein with a molecular mass of about 82 kDa is located in the inner membrane (12,20) and was shown to bind to DNA cellulose, indicating nonspecific binding to nucleic acids (20). The amino acid sequence deduced from the nucleotide sequence contains an ATP-binding motif (9) and preliminary data suggest a DNA-dependent ATPase activity of TraD (20).TraM is a cytoplasmic protein with a molecular mass of 14.5 kDa. In the past, investigations of the TraM protein have mainly been focused on its regulatory function. Maximal traM transcription requires TraY in addition to expression of the tra operon. TraM binds to three sites within the oriT region of F (6). Two sites with high affinity fo...
The transfer regions of different conjugative plasmids show significant similarities in the genetic organization and in the amino acid sequence of some gene products, especially of proteins from the traG or trbB family. These similarities are also evident on the level of the nucleotide sequences. On the basis of conserved DNA regions we designed degenerate PCR primer pairs to detect specifically tra regions within a collection of bacterial clones isolated from an agricultural soil. Most of the potential transfer-proficient indigenous bacterial isolates were able to mobilize a derivative of the nonconjugative IncQ plasmid RSF1010 into recipient strains. With the help of the primers it should be possible to evaluate the genetic potential for horizontal gene transfer carried out by conjugative plasmids.
The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoI-HpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 bp downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.
At the XhoI site (45.08F) of plasmid mini-F a deletion of 649 bp was generated employing exonuclease Bal31. By this deletion nucleotide sequences functioning as origin II and the four 19 bp direct repeats constituting the incB region in front of the E protein gene were removed from the plasmid. Analysis of proteins radioactively labelled in Escherichia coli mini-cells indicated that all mini-F encoded proteins are expressed. However, the plasmid carrying the deletion was not capable of replicating from the primary origin (origin I, 42.6F). Recently a smaller deletion at the XhoI site (45.08F) of about 300 bp, removing only the region functioning as origin II and replicating from origin I, was described by Tanimoto and Iino (1984, 1985). The data presented suggest that the incB repeats are essential for the initiation of replication from origin I, and possibly also from origin II, and seem not to be engaged in the autoregulation of E protein expression.
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