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

Fekete, Richard A.; Frost, Laura S.

doi:10.1128/jb.182.14.4022-4027.2000

Cited by 35 publications

(34 citation statements)

References 52 publications

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“…In vivo plasmid nicking and transfer experiments have demonstrated that TraI proteins from F and R100 can distinguish the two-base difference between their cognate TraI binding site (Fig. 1) and the site from the other plasmid (20,24,26,28). To quantify the binding differences, an expression construct for the R100 TraI relaxase domain (TraI36) was generated, the protein was purified, and its ssDNA binding was compared with that of the previously generated F TraI36 (27).…”

Section: Resultsmentioning

confidence: 99%

“…The ssDNA cleavage specificity results for F and R100 TraI36 are consistent with the results of in vivo nicking and transfer experiments using the full-length proteins. R100 TraI nicks and efficiently transfers chimeric plasmids that include the F TraI binding site (28), whereas F TraI is highly specific in its ssDNA cleavage and plasmid mobilization activities, and interacts less effectively with the R100 sequence (24, 28).…”

Section: Resultsmentioning

confidence: 99%

“…F TraI36 binds a single-stranded F oriT sequence with a subnanomolar K D , and some single-base substitutions over a 10-base region can reduce affinity by between 10-and 10,000-fold (24). In vivo, this specificity is reflected in the poor efficiency with which F TraI mobilizes plasmids containing the TraI-binding site for the highly homologous R100 plasmid, despite a difference of only two bases (24,28). To better understand the basis of ssDNA recognition by relaxases, we set out to identify F TraI36 amino acids that contribute to ssDNA recognition.…”

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confidence: 99%

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Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

Harley

Schildbach

2003

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

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Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential for plasmid transfer. Relaxases bind and cleave one plasmid strand siteand sequence-specifically before transfer of the cleaved strand. TraI36, a domain of F plasmid TraI that contains relaxase activity, binds a plasmid sequence in single-stranded form with subnanomolar KD and high sequence specificity. Despite 91% amino acid sequence identity, TraI36 domains from plasmids F and R100 discriminate between binding sites. The binding sites differ by 2 of 11 bases, but both proteins bind their cognate site with three orders of magnitude higher affinity than the other site. To identify specificity determinants, we generated variants having R100 amino acids in the F TraI36 background. Although most retain F specificity, the Q193R͞R201Q variant binds the R100 site with 10-fold greater affinity than the F site. The reverse switch (R193Q͞Q201R) in R100 TraI36 confers a wild-type F specificity on the variant. Nonadditivity of individual amino acid and base contributions to recognition suggests that the specificity difference derives from multiple interactions. The F TraI36 crystal structure shows positions 193 and 201 form opposite sides of a pocket within the binding cleft, suggesting binding involves knob-into-hole interactions. Specificity is presumably modulated by altering the composition of the pocket. Our results demonstrate that F-like relaxases can switch between highly sequence-specific recognition of different sequences with minimal amino acid substitution.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

Harley

Schildbach

2003

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

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“…Biochemical analysis of the F-plasmid proteins (16,28) and recent genetic data (11) suggest that one function of TraY is to impart stability to the complex. Further work is necessary for a detailed understanding of the activity and regulation of IncF relaxosomes.…”

Section: Discussionmentioning

confidence: 99%

Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed oriT Cleavage In Vivo

2001

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The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (oriT) in vivo was investigated. As expected, the cleavage reaction was not detected in Escherichia coli cells expressing tral and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at oriT in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1 oriT constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations, traY was dispensable for mobilization in the presence of traM, but mobilization did not occur in the absence of both traM and traY. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA.The traM and traY genes of IncF conjugation systems are essential for transfer proficiency (19,24,25,35). Numerous studies have established a role for traM and traY in regulation of transfer gene expression (6,7,34,35,41). Thus the transferdeficient phenotype of traY and traM mutant derivatives is certain to reflect disruption of positive gene regulation as a minimum and may further reflect the loss of additional functions.The TraM and TraY proteins of different IncF plasmids exhibit sequence-specific DNA binding activity at oriT (1,8,18,21,23,30,38,39), and they have been implicated in the initiation stage of conjugative DNA transfer. During initiation of conjugative plasmid transfer an enzyme known as relaxase cleaves a defined strand of oriT DNA at a specific position (nic) in a transesterification reaction. Cleaving activity at oriT is exhibited as part of a nucleoprotein complex called the relaxosome. This complex includes the relaxase and auxiliary proteins, which can be host or plasmid encoded (12). These accessory factors impart specificity and stability to the complex and enhance the efficiency of the cleavage reaction. In many cases specific interactions between the auxiliary proteins and oriT DNA promote localized melting of the duplex and facilitate access of relaxase to its recognition site (31-33, 37, 40, 42, 46-49).For the IncF system, extensive biochemical analysis has been dedicated to characterizing the relaxase-catalyzed cleavage ...

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“…Using in vitro assays, TraM has been shown to interact with TraD, an inner membrane component of the transferosome encoded by the F-plasmid (1,9). TraM is essential neither for the nicking reaction that involves other components of the relaxosome nor for F-pilus assembly or mating-pair formation that involve most of the transferosome components (10,11,19,22,32). Therefore, TraM was proposed to transmit a signal between the cytoplasmic relaxosome and the transferosome during F conjugation (13,43).…”

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confidence: 99%

Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

Frost

2005

J Bacteriol

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Conjugation is a major mechanism for disseminating genetic information in bacterial populations, but the signal that triggers it is poorly understood in gram-negative bacteria. F-plasmid-mediated conjugation requires TraM, a homotetramer, which binds cooperatively to three binding sites within the origin of transfer. Using in vitro assays, TraM has previously been shown to interact with the coupling protein TraD. Here we present evidence that F conjugation also requires TraM-TraD interactions in vivo. A three-plasmid system was used to select mutations in TraM that are defective for F conjugation but competent for tetramerization and cooperative DNA binding to the traM promoter region. One mutation, K99E, was particularly defective in conjugation and was further characterized by affinity chromatography and coimmunoprecipitation assays that suggested it was defective in interacting with TraD. A C-terminal deletion (S79*, where the asterisk represents a stop codon) and a missense mutation (F121S), which affects tetramerization, also reduced the affinity of TraM for TraD. We propose that the C-terminal region of TraM interacts with TraD, whereas its N-terminal domain is involved in DNA binding. This arrangement of functional domains could in part allow TraM to receive the mating signal generated by donor-recipient contact and transfer it to the relaxosome, thereby triggering DNA transfer.

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

Cited by 35 publications

References 52 publications

Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed oriT Cleavage In Vivo

Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

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