In vitro construction of deletion mutants of the bacteriocinogenic plasmid Clo DF13

Stuitje, Antoine R.; Veltkamp, E.; Elzen, Peter J. M. van den; Nijkamp, H. J. J.

doi:10.1093/nar/5.6.1801

Cited by 22 publications

(20 citation statements)

References 21 publications

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“…In this latter host the plasmid is present to the extent of about 10 copies per cell (27). A small DNA region located between 1.8 and 11.5% of the CloDF13 genome is essential for autonomous replication (25,31). This region contains the origin of replication (oriV) and the information for two transcripts involved in plasmid replication, namely, a preprimer RNA of about 580 nucleotides (RNA II) and a small 108-nucleotide repressor RNA (RNA I) (30).…”

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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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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 DNA restriction fragments comprising the region from 0 to 15% and from 39 to 100% were prepared as follows. DNA of miniplasmid pEV35, a plasmid that contains a deletion from 15 to 39% (27), was cleaved with the enzymes HpaI and BamHI. The two resulting 0 to 15% and 39 to 100% DNA fragments were separated on a horizontal 1% agarose gel, and the fragments were recovered from the gel by use of hydroxylapatite, as described in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

“…In a first attempt to construct a transcription map of plasmid CloDF13, we focused our attention on the CloDF13 DNA region from 0 to 40%. Previous studies have shown that this region contains the genes coding from the bacteriocin cloacin DF13 (4), the immunity protein (38), and the origin of DNA replication (28), as well as the genetic information involved in plasmid replication control (27).…”

Section: Discussionmentioning

confidence: 99%

“…As revealed from experiments with E. coli minicells, CloDF13 encodes for the synthesis of at least eight proteins (4). The isolation of mutants by insertion of transposons (32) or deletion of particular DNA fragments (27) enabled us to construct a rather detailed genetic map (4,38) and to get more insight into functions specified by the plasmid. For instance, it was possible to identify the genes and gene products involved in cloacinogenity (4), immunity (38), mobilization (31), and the interaction with the propagation of RNA and DNA phages (32, 32a) ( Fig.…”

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Transcription of bacteriocinogenic plasmid CloDF13 in vivo and in vitro: structure of the cloacin immunity operon

Elzen¹,

Konings²,

Veltkamp³

et al. 1980

J Bacteriol

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Escherichia coli minicells harboring plasmid CloDF13 synthesized at least 25 messenger ribonucleic acid (RNA) species; three of these RNAs, a 2,400-, a 2,200-, and a 100-nucleotide RNA, were synthesized in relatively large amounts. Using insertion and deletion mutants of CloDF13 as well as an RNA blotting technique, we could demonstrate that these three RNAs are transcripts from the CloDF13 DNA region from 0 to 40%. This region contains the cloacin and immunity genes and the genetic information involved in plasmid DNA replication. A transcription map of this region is presented and discussed. The data indicate that the cloacin and immunity genes were coordinately transcribed into messenger RNAs of about 2,400 and 2,200 nucleotides, which differ in length at their 3' terminus. RNA polymerase binding studies and in vitro transcription assays indicated that transcription of these genes initiates at a promoter located around 32% on the CloDF13 map. Furthermore, it is shown that a 100-nucleotide RNA is encoded by the CloDF13 DNA region between 7.7 and 8.8% on the plasmid genome; the synthesis of this RNA proceeds in a direction opposite to the transcription of the cloacin and immunity genes.tively labeled DNA. Our results indicate that 579 on August 5, 2020 by guest

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“…Previous studies have shown that the bacteriocinogenic plasmid CloDF13 replicates unindirectionally from a unique origin, which is located at 2.8% on the CloDF13 physical map (1). In our study on the control of CloDF13 replication, we used both conditional (2,3) and nonconditional (4) plasmid copy number (cop) mutants. It was shown that the region between 1.8% and 11.5% of the plasmid genome contains sufficient information for autonomous plasmid DNA replication.…”

Section: Introductionmentioning

confidence: 99%

The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacterio-cinogenic plasmid Clo DF13

Stuitje¹,

Veltkamp²,

Maat

et al. 1980

Nucl Acids Res

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The nucleotide sequence from about 100 base-pairs downstream to about 600 base pairs upstream the CloDF13 replication origin has been determined. A comparison of this sequence with the corresponding ColE1 origin sequence reveals that: The sequence at the origin of replication is conserved. There are large differences in the nucleotide sequence downstream the replication origin, whereas there is a large homology in the region of about 410 base-pairs upstream the replication origin. This conserved region might code for a largely homologous basic, arginine rich polypeptide of about 45 amino-acids, for both ColE1 and CloDF13. Although there are large differences in the primary structure of the region coding for the 100 nucleotide RNA, the secondary structure of this region seems to be conserved.

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In vitro construction of deletion mutants of the bacteriocinogenic plasmid Clo DF13

Cited by 22 publications

References 21 publications

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

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

Transcription of bacteriocinogenic plasmid CloDF13 in vivo and in vitro: structure of the cloacin immunity operon

The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacterio-cinogenic plasmid Clo DF13

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