The TraM protein of the conjugative plasmid F binds to the origin of transfer of the F and ColE1 plasmids

Laurenzio, Laura Di; Frost, Laura S.; Paranchych, W.

doi:10.1111/j.1365-2958.1992.tb01754.x

Cited by 60 publications

(76 citation statements)

References 31 publications

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“…If TraM signals the cell about the formation of stable mating pairs, it should at least temporarily contact the cell envelope. Previous studies of the localization of TraM support this idea, particularly since small amounts of the cytoplasmic protein could be found in the inner membrane (5,6,23).In this study, we identify by different experimental approaches the TraD protein as one F gene product which mediates contact between the TraM protein and the inner membrane. We furthermore identified a new host membrane protein with a mass of 35 kDa which also shows affinity to TraM.…”

supporting

confidence: 59%

supporting

confidence: 58%

“…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 for TraM (sbmA and sbmB) overlap with the two traM promoters, which means TraM autoregulates its own expression (21,24).…”

mentioning

confidence: 99%

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

Disqué-Kochem

Dreiseikelmann

1997

J Bacteriol

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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...

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confidence: 59%

supporting

confidence: 58%

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

Disqué-Kochem

Dreiseikelmann

1997

J Bacteriol

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“…4A, contains several inverted repeats, a direct repeat and two intrinsic bends, in addition to nic (Frost et al, 1994;Tsai et al, 1990). DNA footprinting analysis has shown specific binding sites for several proteins including TraI, IHF, TraY and TraM (the product of the F plasmid traM gene) Tsai et al, 1990;DiLaurenzio et al, 1992;Nelson et al, 1993). As noted above, there are two IHF binding sites.…”

Section: Relaxosome Assemblymentioning

confidence: 99%

Nicking by transesterification: the reaction catalysed by a relaxase

Byrd¹,

Matson

1997

Molecular Microbiology

108

117

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SummaryDNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site-and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3Ј-OH and the relaxase covalently bound to the 5Ј end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.

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“…Presence of the IHF in combination with TraY is known to stimulate the efficiency of the nicking reaction catalysed by TraI at the transfer origin (Nelson et al, 1995). Additionally, TraM protein binds to multiple extended sites within this region (Schwab et al, 1991;Abo et al, 1991;Di Laurenzio et al, 1992;. The binding sites for TraM partially overlap the traM promoters, such that binding inhibits expression of the traM gene (Schwab et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network

Pölzleitner

Zechner

Renner

et al. 1997

Molecular Microbiology

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SummarySite-directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a transferderepressed (finO ¹ ) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1-16 was 10 7 -fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.

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The TraM protein of the conjugative plasmid F binds to the origin of transfer of the F and ColE1 plasmids

Cited by 60 publications

References 31 publications

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

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

Nicking by transesterification: the reaction catalysed by a relaxase

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network

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