The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids

Paterson, E. Suzanne; Iyer, V. N.

doi:10.1128/jb.174.2.499-507.1992

Cited by 8 publications

(15 citation statements)

References 26 publications

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“…3; however, the sequence of these iterons does not have any identity between the two plasmids (18). The lack of iterons to the left of the pMUR274 oriT supports an earlier observation that the iterons do not play a significant role in the transfer of pCU1 (32).…”

Section: Resultssupporting

confidence: 72%

“…For sequencing double-stranded DNA, 7 l of Wizard miniprep DNA and 20 pmol of primer were boiled for 5 min and quick chilled. Sequenase buffer, dithiothreitol, label mix, and [␣- 32 P]dATP were added as described in the manufacturer's instructions. After the addition of 1.5 U of Sequenase, the reactions were mixed, quick spun, and immediately added to the termination mixtures to incubate for 5 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…While production of such a hybrid oriT would require sufficient sequence identity between the two parental regions to allow interaction of the Mob proteins with both oriTs, some sequence variation is also necessary to allow identification of the junction in the hybrid. The oriT of pCU1 lies on a 325-bp sequence (32) which has 30% sequence identity to the oriT region of IncW plasmid R388 (18). Unsuccessful attempts to detect site-specific recombination between the oriTs of pCU1 and R388 (19,31) suggest that these two plasmids do not have sufficient sequence identity to allow such an event to occur.…”

mentioning

confidence: 99%

“…Its ability to mobilize a plasmid carrying the mob region of pCU1 but not the oriT region alone indicates that its oriT sequence is not identical to that of pCU1 but that it is probably more similar than the sequence of the R388 oriT (32). In this paper, we describe the formation of a hybrid oriT by in vivo site-specific recombination between the oriTs of pCU1 and pMUR274 in an effort to localize the nic site of pCU1.…”

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confidence: 99%

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Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT

Paterson

Iyer

1997

J Bacteriol

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The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.During conjugal DNA transfer in gram-negative eubacteria, plasmid-encoded Mob proteins interact with a small cis-acting region called the origin of transfer (oriT) to form a DNAprotein complex called the relaxosome (40). Transfer is initiated by the binding of one or more small Mob proteins to the oriT region, following which a site-specific, single-stranded nick is made in the oriT by a second larger Mob protein which remains covalently bound to the 5Ј end of the nicked strand. The nicked strand is unwound in the 5Ј-3Ј direction, and complementary strand synthesis is initiated from the 3Ј end of the nicked strand. Once a complete strand has been unwound, a second nick is made in the reconstituted oriT and the 5Ј and 3Ј ends of the unwound strand are sealed. Both nicking and sealing are carried out by the same enzyme (3, 28). The nucleotide sequences of oriTs of different plasmids have only limited identity, even among plasmids encoding similar transfer systems. This lack of sequence identity is reflected in the plasmid specificity exhibited by the Mob proteins (8, 41).Brasch and Meyer (4) demonstrated that when two identical oriT sequences are placed on the same plasmid, transfer can be initiated at one oriT and terminated at the other, resulting in an intramolecular site-specific recombination event in which the DNA between the second and first oriT is deleted. This recombination event can be detected by the loss of a selectable marker on the deleted DNA. By placing mutations in eith...

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Section: Resultssupporting

confidence: 72%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT

Paterson

Iyer

1997

J Bacteriol

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“…The relaxase-helicase enzyme TraI, encoded by MOB F conjugative resistance plasmid pCU1, is responsible for DNA plasmid processing leading to cell-to-cell transfer of the pCU1 plasmid (50)(51)(52). The structure and function of the pCU1 TraI N-terminal relaxase domain have been well studied (53,54), with only limited data available for its C-terminal helicase.…”

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confidence: 99%

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

2014

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The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.H elicases are molecular motors that drive the separation of duplex nucleic acid strands through the coupling of ATP hydrolysis to unwinding (1-3). As a result, helicases are key players in a wide variety of DNA and RNA metabolic processes, such as recombination, chromatin remodeling, and DNA transport (4-6). Most recently, helicases have emerged as potential therapeutic targets for diseases ranging from cancer to Gram-positive bacterial infections (7-18). Classified into six superfamilies, superfamily 1 and 2 (SF1 and SF2) helicases are the most similar, sharing up to seven conserved sequence motifs and core RecA-like folds (1-3). Furthermore, SF1 helicases comprise the largest class and can be further subdivided into SF1A and SF1B, based on the direction of helicase translocation along the strand; SF1A helicases progress 3= to 5= and SF1B 5= to 3= (3). Despite their importance, SF1B helicases are still poorly characterized in comparison to SF1A and SF2 helicases, though some recent studies have expanded our knowledge (19-21). SF1B helicases are critical to a diverse range of processes, including DNA repair by human DNA helicase B (22), telomere regulation by Pif1 (23), and conjugative plasmid transfer by F TraI (24).Conjugative plasmid transfer (CPT) mediates the propagation of antibiotic resistance genes and virulence factors within commensal and pathogenic bacteria (25-27) and relies on the helicase domain of a bifunctional protein to facilitate plasmid DNA transfer (24,(28)(29)(30). Early work characteri...

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DNA Processing and Replication during Plasmid Transfer between Gram-Negative Bacteria

Wilkins

Lanka

1993

Bacterial Conjugation

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The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids

Cited by 8 publications

References 26 publications

Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT

Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

DNA Processing and Replication during Plasmid Transfer between Gram-Negative Bacteria

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