F Plasmid Conjugative DNA Transfer

Matson, Steven W.; Sampson, Juliana K.; Byrd, Devon R.

doi:10.1074/jbc.m008728200

Cited by 50 publications

(43 citation statements)

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“…[1][2][3][4]. The TraI protein of IncF plasmids (first characterized as DNA helicase I of Escherichia coli (5)) is essential for the transmission of bacterial genes during conjugation (6,7). In that process a copy of a conjugative plasmid is transferred unidirectionally in single-stranded form from one bacterial cell to another (for reviews, see Refs.…”

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confidence: 99%

“…The DNA helicase activities are located in C-terminal domains of the bifunctional F TraI and R388 TrwC proteins (28 -33). For reasons that are poorly understood, efficient gene transfer requires physical linkage of the relaxase and helicase domains (7,34).This bifunctional arrangement of DNA transesterase and helicase activities is shared by adeno-associated virus replication initiation (Rep) 1 proteins (35). As revealed by very recent structural data, the endonuclease domain of adeno-associated virus-5 Rep proteins (36) bear the closest structural and functional relatedness to the N-terminal DNA transesterase domains of the F TraI and R388 TrwC proteins (37).…”

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Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Csitkovits

Zechner

2003

Journal of Biological Chemistry

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The IncF plasmid protein TraI functions during bacterial conjugation as a site-and strand-specific DNA transesterase and a highly processive 5 to 3 DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5 to 3 unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5 tail of singlestranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of singlestranded DNA of defined length was created and its structure verified. We found that substrates containing >27 nucleotides of single-stranded DNA 5 to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.Helicases are ubiquitous enzymes involved in nucleic acid metabolism (for review, see Refs. 1-4). The TraI protein of IncF plasmids (first characterized as DNA helicase I of Escherichia coli (5)) is essential for the transmission of bacterial genes during conjugation (6, 7). In that process a copy of a conjugative plasmid is transferred unidirectionally in single-stranded form from one bacterial cell to another (for reviews, see Refs. 8 and 9). The TraI protein of IncF plasmids contributes to conjugative transfer in several ways. It is a component of a nucleoprotein complex, the relaxosome, which assembles with site specificity at the plasmid origin of transfer (oriT). Relaxosomes initiate the series of DNA-processing reactions that prepare plasmid DNA for transfer to a recipient cell. They are common to all self-transmissible and mobilizable plasmids in different degrees of complexity (for reviews, see Refs. 8, 10, and 11). The simplest systems employ a plasmid-encoded DNA transesterase, or relaxase, protein that acts on a specific phosphodiester bond, nic, in the transfer origin. Cleavage at this site provides a point of origin for the directed 5Ј to 3Ј transmission of the plasmid genome to a recipient cell. Relaxase proteins are also active in relaxosomes containing auxiliary DNA-binding proteins of host or plasmid origin. IncF, IncW, IncP, and IncQ systems offer well studied examples (12-21). Among IncF plasmids factors known to stimulate nic cleavage include E. coli integration host factor and plasmid proteins . In the case of IncW plasmid R388, TrwA protein performs this role (13,14). The IncF system and the functionally analogous IncW-IncN family of DNA-mobilizing systems are (thus far) unique in that they additionally specify a DNA helicase activity esse...

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Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Csitkovits

Zechner

2003

Journal of Biological Chemistry

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“…The relaxase completes DNA transfer by reversing the covalent phosphotyrosine linkage and releasing the T-strand. In the F plasmid, this relaxase is located in the N-terminal domain of a large multifunctional protein, TraI (DNA helicase I) (22,23,(26)(27)(28). Some conjugative relaxases use one active-site tyrosine [e.g., IncQ RSF1010 MobA (29), IncP RP4 TraI (30, 31), IncI R64 NikA (32), Agrobacterium Ti VirD2 (33), and Tn5252 MocA/BmgA (34), where R indicates plasmids that propagate antibiotic resistance].…”

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“…The relaxase completes DNA transfer by reversing the covalent phosphotyrosine linkage and releasing the T-strand. In the F plasmid, this relaxase is located in the N-terminal domain of a large multifunctional protein, TraI (DNA helicase I) (22,23,(26)(27)(28) (38)(39)(40)(41)]. Optimal relaxase cleavage, ligation, and transfer of ssDNA require the metal ion and two catalytic tyrosines, one from each pair (42).…”

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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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Conjugation, Bacterial

2004

Encyclopedic Dictionary of Genetics, Genomics and Proteomics

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F Plasmid Conjugative DNA Transfer

Cited by 50 publications

References 44 publications

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Conjugation, Bacterial

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