Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome.

Wong, Edwin M.; Muesing, Mark A.; Polisky, Barry

doi:10.1073/pnas.79.11.3570

Cited by 63 publications

(39 citation statements)

References 26 publications

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“…Bacteria were grown in 1% Bacto Tryptone-0.5% Bacto Yeast Extract-0.5% NaCl, supplemented as necessary with ampicillin (100 g/ml), chloramphenicol, nalidixic acid, or streptomycin (each at 25 g/ml). For selection of ampicillin-resistant transconjugants, medium also contained methicillin (1 mg/ml) (28).…”

Section: Methodsmentioning

confidence: 99%

The primase of broad-host-range plasmid R1162 is active in conjugal transfer

Henderson

Meyer

1996

J Bacteriol

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The broad-host-range plasmid R1162 is conjugally mobilized at high frequency by the IncP-1 plasmid R751 but is poorly mobilized by pOX38, a derivative of the F factor. In both cases, the origin of transfer (oriT) and the Mob proteins of R1162 are required, indicating that these plasmids are mobilized by similar mechanisms. R1162 encodes a primase, essential for vegetative replication of the plasmid, that is made both as a separate protein and as the carboxy-terminal domain of MobA, one of the R1162 mobilization proteins (P. Scholz, V. Haring, B. Wittman-Liebold, K. Ashman, M. Bagdasarian, and E. Scherzinger, Gene 75:271-288, 1989). When R751 is the mobilizing vector, the primase is not required for mobilization of plasmids containing cloned mob-oriT R1162 DNA. However, detectable mobilization of such plasmids by pOX38 requires both the primase and its cognate initiation site, oriented for synthesis of the complement to the transferred strand. The long form of the primase is required for optimal transfer: R1162 replicons lacking this form also are not transferred detectably by pOX38 and are less well mobilized by R751. The distance between oriT and the primase initiation site affects the frequency of mobilization, and this effect is polar in the direction of transfer. Our results indicate that the R1162 primase is active in mobilization of R1162 and suggest that the MobA-linked form is an adaptation increasing its effectiveness during transfer.

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Section: Methodsmentioning

confidence: 99%

The primase of broad-host-range plasmid R1162 is active in conjugal transfer

Henderson

Meyer

1996

J Bacteriol

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“…The majority of these mutations map within the region essential for autonomous replication, particularly in the part encoding RNA I (12,17,18,24,33). However, in the case of ColEl mutations outside this region were found that influenced the plasmid copy number (29).…”

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confidence: 99%

“…Those mutants are thermosensitive for their control of plasmid replication (3,17,33). A conditional CloDF13 copy mutation, designated cop-J(Ts), is located within the region involved in the initiation of preprimer synthesis, a region that overlaps with the transcription terminator of the cloacin operon (3,24) (Fig.…”

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confidence: 99%

Methylation-dependent transcription controls plasmid replication of the CloDF13 cop-1(Ts) mutant

Putten¹,

Lang²,

Veltkamp³

et al. 1986

J Bacteriol

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The CloDF13 cop-l(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42°C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-l(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42°C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA H synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed.

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“…The cells were resuspended in 40 pl of broth, placed on broth agar medium, and incubated at 37°C for 90 min. The mating mixture was then resuspended in 1 ml of broth and 100 pl of various dilutions, mixed with 50 pl of 2% X-Gal, and plated on medium containing nalidixic acid and carbenicillin supplemented with methicillin (2 mg/ml) (21). Donor cells were enumerated by plating the mating mixture on medium containing carbenicillin.…”

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confidence: 99%

Role of the origin of transfer in termination of strand transfer during bacterial conjugation

1992

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Conjugal transfer of the broad-host-range plasmid R1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (oriT). Termination involves ligation of the transferred single strand by the plasmid-encoded MobA protein. Several different assays were used to identify the oriT DNA required for termination. For plasmids containing two oniTs, with transfer initiated at one and terminated at the other, the inverted repeat within oniT is-important for termination. Deletion of the outer arm reduces the termination frequency-, those terminations that do occur probably depend upon nicking at this oriT prior to transfer. The locations of second-site suppressor mutations indicate that base pairing between the arms of the inverted repeat is important for termination. In vitro, the inverted repeat is not required for specific cleavage of single-stranded DNA at nic, but competition experiments indicate that oriTs with the inverted repeat are preferentially cleaved.We propose that the function of the oriT inverted repeat is to trap the plasmid-encoded MobA protein at the end of a round of strand transfer, thus ensuring that the protein is available for the ligation step.The basic steps in DNA processing during conjugal transfer are probably similar for many different plasmids in gram-negative bacteria. One of the DNA strands is first nicked within the origin of transfer (oriT), which is required in cis. The nicked strand is then linearly transferred into a recipient cell in the 5'-to-3' direction and subsequently recircularized. In both donor and recipient cells, the complementary strand of DNA is resynthesized to restore the duplex plasmid molecule. Many different plasmids can be isolated in the form of a relaxosome, a complex of proteins and supercoiled plasmid DNA, which can be induced to undergo site-and strand-specific nicking within oriT (for example, see references 5, 6, 12, and 16). This nicking models the initial DNA-processing step in plasmid transfer.R1162 (nearly identical to RSF1010) is a broad-host-range, IncQ plasmid that is efficiently mobilized during the conjugal transfer of IncPl group plasmids (15,20). The R1162 oriT consists of no more than 38 bp of DNA (Fig.

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Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome.

Cited by 63 publications

References 26 publications

The primase of broad-host-range plasmid R1162 is active in conjugal transfer

The primase of broad-host-range plasmid R1162 is active in conjugal transfer

Methylation-dependent transcription controls plasmid replication of the CloDF13 cop-1(Ts) mutant

Role of the origin of transfer in termination of strand transfer during bacterial conjugation

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