The F plasmid‐encoded TraM protein stimulates relaxosome‐mediated cleavage at <i>oriT</i> through an interaction with TraI

Ragonese, Heather M.; Haisch, Debi; Villareal, Ernesto; Choi, June‐Hyuk; Matson, Steven W.

doi:10.1111/j.1365-2958.2006.05576.x

Cited by 39 publications

(43 citation statements)

References 55 publications

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“…Neither the DNA-dependent ATPase activity nor oligonucleotide cleaving and recombination reactions of TraI were altered by the presence of the effector proteins. Recent evidence suggests that TraM and TraI of plasmid F interact physically via the TraI C terminus (59). Although a similar interaction between R1 components is likely and may be important to regulation of the transesterase, that contact did not alter other TraI catalytic activities under our test conditions.…”

Section: Discussionmentioning

confidence: 48%

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

et al. 2009

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Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membranespanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7,8,10,30,41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site-and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5Ј end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3Ј-OH generated at nic and the subsequent DNA unwind...

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Section: Discussionmentioning

confidence: 48%

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

et al. 2009

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“…To test this hypothesis, a kinetic assay using fluorophore-labeled oriT ssDNA for cleavage by the F TraI relaxase domain (TraI N300) was developed to complement existing radiolabel-based techniques (50,51). Imidobisphosphate (PNP), a simple and relatively stable bisphosphonate, was the first compound examined in this assay ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Lujan

Guogas²,

Ragonese³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Conjugative transfer of plasmid DNA via close cell-cell junctions is the main route by which antibiotic resistance genes spread between bacterial strains. Relaxases are essential for conjugative transfer and act by cleaving DNA strands and forming covalent phosphotyrosine linkages. Based on data indicating that multityrosine relaxase enzymes can accommodate two phosphotyrosine intermediates within their divalent metal-containing active sites, we hypothesized that bisphosphonates would inhibit relaxase activity and conjugative DNA transfer. We identified bisphosphonates that are nanomolar inhibitors of the F plasmid conjugative relaxase in vitro. Furthermore, we used cell-based assays to demonstrate that these compounds are highly effective at preventing DNA transfer and at selectively killing cells harboring conjugative plasmids. Two potent inhibitors, clodronate and etidronate, are already clinically approved to treat bone loss. Thus, the inhibition of conjugative relaxases is a potentially novel antimicrobial approach, one that selectively targets bacteria capable of transferring antibiotic resistance and generating multidrug resistant strains.antimicrobial ͉ bacterial conjugation ͉ bisphosphonates ͉ F plasmid TraI ͉ relaxase inhibition C onjugative elements are responsible for the majority of horizontal gene transfers within and between bacterial strains (reviewed in ref. 1), as first described for the Escherichia coli F plasmid by Lederberg and Tatum in 1946 (2). Conjugative DNA transfer is also the central mechanism by which antibiotic resistance and virulence factors are propagated in bacterial populations (reviewed in ref.3). Indeed, it is well established that antibiotic resistance can be rapidly acquired in clinical settings and that such acquisition is critically dependent on conjugative DNA transfer (reviewed in ref. 4). Small-molecule inhibition of conjugation could prove to be a powerful method for curbing the generation and spread of multidrug-resistant strains. Past studies suggested that various antibiotics, polycyclic chemicals, and crude extracts inhibit conjugation at concentrations less than the antibacterial minimum inhibitory concentration (5-11); however, most of these effects have been attributed to nonconjugation-specific inhibition of bacterial growth or DNA synthesis (12)(13)(14)(15). This study describes a bottom-up approach used to identify the first small molecule inhibitors of conjugative DNA transfer that target an enzyme of the conjugative system.The DNA relaxase is a central enzyme in each conjugative system (16-18) and thus is a prime target for inhibition. The conjugative relaxase initiates DNA transfer with a site-and strand-specific ssDNA nick in the transferred strand (T-strand) at the origin of transfer (oriT), forming a covalent 5Ј-phosphotyrosine intermediate (16,(19)(20)(21)(22)(23). The nicked T-strand moves from the donor cell (plasmid ϩ ) to the recipient cell (plasmid Ϫ ) via an intercellular junction mediated by a type IV secretion system (reviewed in refs. 19, 24,...

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“…8, steps I and II). Within the relaxosome, TraI transesterase in the step II binding modus is proficient for cleaving-joining activity at nic and is stimulated most substantially by IHF but also independently by the presence of TraY and TraM (10,27,33,41,44,48). The relaxosome is anchored to the T4CP TraD at the cytoplasmic membrane via interactions with TraM (2,15,39,40,49) and TraI (step III).…”

Section: Discussionmentioning

confidence: 99%

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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The F plasmid‐encoded TraM protein stimulates relaxosome‐mediated cleavage at oriT through an interaction with TraI

Cited by 39 publications

References 55 publications

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

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

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