TraM of plasmid R1 regulates its own expression

Schwab, Margit; Reisenzein, Helga; Högenauer, Gregor

doi:10.1111/j.1365-2958.1993.tb01170.x

Cited by 28 publications

(31 citation statements)

References 23 publications

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“…The TraI and MobA proteins of various plasmids are included in the same family and function in the initiation of plasmid transfer (10,17). On the other hand, TraM had characteristics similar to those of MbeD; both had low isoelectric points (pIs of MbeD and F TraM are 4.5 and 5.1, respectively), faster migration in SDS-polyacrylamide gels, an N-terminal amphipathic helix, and the ability to associate with the inner membrane (6,22). Moreover, F TraM binds to one site at oriT of the ColE1 plasmid (6).…”

Section: Discussionmentioning

confidence: 98%

A ColE1-encoded gene directs entry exclusion of the plasmid

Yamada

Nakazawa

1995

J Bacteriol

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To detect entry exclusion of the ColE1 plasmid, we established an assay system that was not influenced by incompatibility of extant plasmids in the recipient cells or by the viability of the cells due to the killing action of colicin E1 protein. The assay revealed that exc1 and exc2, assigned as genes directing entry exclusion, had no exclusion activity. Instead, mbeD, which had been characterized as a gene for plasmid mobilization, directed the exclusion activity. MbeD was overexpressed and identified as a 35 S-labeled protein, which was recovered in both the soluble and membrane fractions, particularly in the inner membrane fraction. An amphipathic helical structure was predicted in the N-terminal region of MbeD as well as in the corresponding homologous proteins of ColA and ColK. These proteins may bind to the inner membrane via the N-terminal amphipathic helix and function in entry exclusion.The ColE1 plasmid is transmissible from a donor to a recipient cell only in the presence of a conjugative plasmid such as the F plasmid within the donor cell. Entry (surface) exclusion can be defined as the ability of the recipient cells containing a plasmid to reduce the transmission frequency in conjugal matings from other cells containing a closely related or identical plasmid. Inselburg (11) first noted this activity of ColE1, which is unrelated to plasmid incompatibility.The F plasmid encodes two surface exclusion genes, traS and traT, which provide exclusion activity by two independent mechanisms (7, 13). The traS gene encodes a 18-kDa inner membrane protein. The protein expressed in the recipient cell prevents DNA transfer even when a stable mating aggregate is formed (8). The product of traT is a 25-kDa protein found in large amounts in the outer membrane of F-plasmid-containing cells (1). This protein in the recipient cell provides surface exclusion activity probably by reducing stable mating aggregate formation.The entry exclusion activity of small and non-self-transmissible plasmids such as ColE1 has not been analyzed in detail. Inselburg (11) roughly defined a ColE1 region that encodes exclusion activity. Chan et al. (5) predicted that the genes related to entry exclusion were exc1 and exc2, on the basis of comparison of the lengths of the structural genes with the lengths of traS and traT of the F plasmid. Upstream of exc1 and exc2, there are genes required for ColE1 mobilization. Boyd et al. (4) have shown that the region which includes mbeA, mbeB, mbeC, and mbeD (mob3, mob6, mob2, and mob7, respectively, in the terminology of Chan et al. [5]) is essential for ColE1 mobilization with the R64drd 11 plasmid.To detect the entry exclusion ability of ColE1, we established a system in which the entry exclusion assay is not influenced by incompatibility and colicin E1-killing activity. Using this system, we analyzed the gene and its product involved in entry exclusion. MATERIALS AND METHODS Materials.Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo (Kyoto, Japan). [ Bacterial strains and plas...

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Section: Discussionmentioning

confidence: 98%

A ColE1-encoded gene directs entry exclusion of the plasmid

Yamada

Nakazawa

1995

J Bacteriol

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“…1) decreases mating efficiency 100-fold, while the additional deletion of sbmC results in a further 100-fold decrease in the efficiency of mobilization of a plasmid containing a cloned version of oriT (22). TraM from the F-like plasmids R100-1 and R1 also autoregulate their transcription (2,46). The amino-terminal region of TraM is responsible for DNA binding in a plasmid-specific manner (31).…”

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confidence: 99%

Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity

Fekete

Frost

2000

J Bacteriol

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Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

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“…In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10,12,29,33,51,52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2,15,49).…”

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Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

et al. 2009

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Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genomemanipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the doublestranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1,9,17,31,45,66). The mechanisms supporting localized unwinding within the complex include initiatorinduced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10,12,29,33,51,52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2,15,49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36,50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV ...

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TraM of plasmid R1 regulates its own expression

Cited by 28 publications

References 23 publications

A ColE1-encoded gene directs entry exclusion of the plasmid

A ColE1-encoded gene directs entry exclusion of the plasmid

Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity

Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

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