Characterization of the <i>oriT</i> region of the IncFV plasmid pED208

Laurenzio, Laura Di; Frost, Laura S.; Finlay, B. Brett; Paranchych, W.

doi:10.1111/j.1365-2958.1991.tb01927.x

Cited by 17 publications

(15 citation statements)

References 43 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.…”

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confidence: 60%

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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: 60%

supporting

confidence: 59%

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 precise nic site in F was identified by in vivo (Thompson et al, 1989) and in vitro assays (Matson & Morton, 1991) and attributed to TraI or DNA helicase I (DHI), as it was then known (Traxler & Minkley, 1988). Other F-like plasmid oriT regions have also been characterized in detail including R100 Inamoto et al, 1991), R1 (Schwab et al, 1991), pED208 (Di Laurenzio et al, 1991) and summarized in Frost et al (1994). Whereas the nic site is conserved, small sequence changes in the adjacent 9 bp and inverted repeat define the specificity of TraI recognition and cleavage (Harley & Schildbach, 2003).…”

Section: Organization Of Mob F Orit Regionsmentioning

confidence: 99%

Conjugative DNA metabolism in Gram-negative bacteria

et al. 2010

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Bacterial conjugation in Gram-negative bacteria is triggered by a signal that connects the relaxosome to the coupling protein (T4CP) and transferosome, a type IV secretion system. The relaxosome, a nucleoprotein complex formed at the origin of transfer (oriT), consists of a relaxase, directed to the nic site by auxiliary DNA-binding proteins. The nic site undergoes cleavage and religation during vegetative growth, but this is converted to a cleavage and unwinding reaction when a competent mating pair has formed. Here, we review the biochemistry of relaxosomes and ponder some of the remaining questions about the nature of the signal that begins the process.

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“…The TraM protein of pED208 binds to four sites in the oriT region, which contain 15 of the 16 short 5-bp repeats (GANTC) in phase along the DNA (Fig. 5) (48). Similarly, the group III oriT segment of the Rl plasmid, which includes two large binding sites for the Rl TraM protein, also contains Hinfl-like sequences, although they are not as frequent or equally spaced as in pED208 (Fig.…”

Section: Nicking and Initiation Of Transfer At Thementioning

confidence: 99%

“…Eveiy 10th position is marked by a dot below the sequence, and the number of residues in a sequence is noted as a running total in the right margin. The sequence in F(I) from nt 101 (Fig. 2) to the start of the traM gene (nt 465) (243, 244) is compared with equivalent sequences in ColB4-K98 (II) (61), R1-19 (III) (62), R100 (IV) (58, 61), and pED208 (V) (48). In addition, the sequences for the onT regions of pSU233 (222), pSU316 (152), and P307 (98) are presented.…”

Section: Tray Proteinmentioning

confidence: 99%

Analysis of the sequence and gene products of the transfer region of the F sex factor.

Frost

Ippen‐Ihler

Skurray

1994

Microbiological Reviews

288

274

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within this region are marked as a double-dashed line. Signal sequence cleavage sites are indicated by vertical arrows below the line. Features of interest, including homology to signature sequences mentioned in the legends to Fig. 3 and 4, are underlined and described below the line. The nick site (A, nt 140 to 141 according to reference 173), the site of the IS3 insertion, and the positions of frameshift mutations (V) are indicated below and above the line, respectively. The restriction sites for common restriction endonucleases are given in boldface type above the line, and the nucleotide on the 5' side of the cut site is also in boldface type. Nucleotides in parentheses above the line in the traX-finO region (nt 32466 to end) represent the sequence of the F plasmid as reported by Yoshioka et al. (274). MICROBIOL. REV.

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Characterization of the oriT region of the IncFV plasmid pED208

Cited by 17 publications

References 43 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

Conjugative DNA metabolism in Gram-negative bacteria

Analysis of the sequence and gene products of the transfer region of the F sex factor.

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