The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro

Abstract

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...

Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes

“…Whereas in plasmid RP4 the CP TraG interacts directly with the relaxase (5,6), in plasmid F the CP TraD interacts in vitro with the accessory nicking protein TraM (7). On the other hand, no interaction has ever been shown between a CP and a T4SS component, an analysis probably hampered by the fact that these are membrane proteins and thus most interactions probably take place within the membrane or at the periplasm.…”

Coupling Factors in Macromolecular Type-IV Secretion Machineries

“…This protein enhances relaxase activity and it is capable of binding DNA [141]. TraM specifically interacts with the TrwB orthologue in IncF plasmids, TraD, suggesting that TraM links the relaxosome with the DNA transfer apparatus [98]. Plasmid R388 lacks such a TraM orthologue.…”

“…TraG from RP4 interacts with the relaxosome relaxase [92,97]. In plasmid F, TraD contacts an accessory nicking protein, TraM [98], and in plasmid R27, TraG interacts with the T4SS protein TrhB [99].…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…The TraD F T4CP possesses a C-terminal extension that is required for efficient F transfer but is inhibitory for RSF1010 transfer (28,82,180). This region of TraD binds TraM, an accessory factor with an RHH DNA binding domain that functions as a tetramer.…”