Localization and nucleotide sequence of the bom region of Clo DF13

Snijders, Ambrosius P.; Putten, Arnold J. van; Veltkamp, E.; Nijkamp, H. J. J.

doi:10.1007/bf00392189

Cited by 15 publications

(5 citation statements)

References 38 publications

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“…In the oriT region of pCU1, there are also sequences showing partial homology to oriT of ColEl and oriT of CloDF13 (nucleotides 426 to 450 and 467 to 489, respectively) (Fig. 3 and 4) (1,22). Within the 65-bp sequence that is proposed here as the location of nic of oriT of pCU1 (and by implication, oriT of R46), there is a 7-bp inverted repeat proximal to an 81-bp AT-rich region ( Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Iyer

1992

J Bacteriol

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The oriT region of the conjugative IncN plasmid pCUl has been localized to a 669-bp sequence extending from pCUl coordinates 8.48 to 9.15 kb. The nucleotide sequence of this region was determined. The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues. Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence. Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated. This specificity is encoded by the region lying clockwise of the BgIII site at coordinate 3.3 on the pCUl map. Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCUl or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.The origin of conjugative DNA transfer, oriT, of conjugative plasmids of the gram-negative eubacteria is a cis-acting region that is the focus of several coordinated events, resulting in the acquisition by the recipient of one plasmid duplicate and the retention in the donor of the other (31). There are several transfer (tra) systems, each with its own oriT and oriT specificity (25). One such distinct system is the N tra system, so-called because it is usually associated with plasmids of the incompatibility group N, such as plasmid pCU1 (11).Previous studies have mapped a tra region of approximately 17 kb on the plasmid pCU1. The region is composed of a segment sufficient for the syntheses and assembly of an N pilus that can function to confer sensitivity to N pilusspecific bacteriophages and a second segment believed to specify functions related to conjugative DNA metabolism and plasmid DNA transfer (23,24). This second segment includes its oriT region. Plasmid trans-acting elements that are believed to act at oriT are collectively referred to as mob (mobilization) elements. It is believed that specificity between oriT and mob can determine the difference in specificity between different tra systems.To begin to study interactions between oriT and mob elements or host factors, we have delineated the oriT of pCU1, determined its nucleotide sequence, and constructed and analyzed deletions into and within it. This enables speculations on the possible functions of different domains of oriT. To study the specificity of oriT and mob of the N tra system, we have examined the ability of different conjugative plasmids to mobilize derivatives containing different parts of the mob region of pCU1.(The studies reported here form part of the graduate dissertation of S. Paterson.) * Corresponding author. MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. The strains, plasmids, and phages used are listed with their sources and relevant properties in Tables 1 a...

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Section: Discussionmentioning

confidence: 99%

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

Iyer

1992

J Bacteriol

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“…Surprisingly, pSU4822 (Fig. 2), which contains the same DNA fragment proposed by Snijders et al . (1983) to contain the CloDF13 oriT , was not mobilized.…”

Section: Resultsmentioning

confidence: 99%

“…This structural feature could have implications in the specific interactions with CloDF13 Mob proteins. Snijders et al . (1983) proposed that oriT was located in a 264 bp fragment in the vicinity of the unique Hpa I site.…”

Section: Discussionmentioning

confidence: 99%

Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation

Núñez

Cruz²

2001

Molecular Microbiology

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SummaryThe mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT±mobB±mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5 H -GGGTG/GTCGGG-3 H by primer extension and sequencing reactions. Analysis of Mob 2 insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PIL W . However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which nic cleavage results in a free nicked-DNA intermediate.

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“…DNase I (EC 3.1.21 .I); micrococcal nuclease (EC 3.1.31.1); nuclease P1 (EC 3.1.30.1);, snake venom phosphodiesterase I (EC 3.1.4.1); alkaline phosphatase (EC 3.1.3.1); RNase I (EC 3.1.27.5); pronase (EC 3.4.24.4); trypsin (EC 3.4.21.4). lication [8] or transcription [9]. This is supported by the fact that in the case of blocked vegetative replication of similar plasmids they cannot be transferred to the recipient cell [8].…”

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confidence: 99%

“…Upon relaxation of the ColEl relaxation complex the 60-kDa protein becomes tightly (covalently?) linked to the 5' terminus of the nick [6, 71. In vivo the relaxation may also be induced by the conformational changes of the oriT-oriV site caused by the DNA-protein or DNA-RNA interactions during plasmid rep- lication [8] or transcription [9]. This is supported by the fact that in the case of blocked vegetative replication of similar plasmids they cannot be transferred to the recipient cell [8].…”

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confidence: 99%

A protein covalently bound to ColEl DNA

Drygin

Zuklys²,

Terskich

et al. 1988

European Journal of Biochemistry

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ColEl DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or ['4C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColEl DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethanetreated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. 0-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColEl DNA via a phosphodiester bond between dC and the serine residue.ColEl DNA belongs to a group of non-conjugative mobilizable plasmids which are not capable of independent transfer during bacterial conjugation but are transferred in the presence of a conjugative plasmid. Genetic studies have confirmed that the following sites and proteins are needed to ensure ColEl DNA mobilization: (a) the ColEl born sequence, which includes the nic site (oriT); (b) the ColEl mob region, where at least one ColEl-specified gene product, needed for mobilization, is encoded; (c) the tra-gene products of the donating plasmid, which recognize the oriT sequence in ColEl DNA and may specifically interact with the proteins of the ColEl relaxation complex, inducing relaxation and mobilization of the plasmid [l, 21. In vitro treatment of the relaxation complex, either with protein-denaturing agents such as SDS or with pronase, results in conversion of the DNA in the complex from a supercoiled to an open-circular configuration Abbreviations. PhMeS02F, phenylmethylsulfonyl fluoride; TosPheCH2C1, tosylphenylalanylchloromethane.Enzymes. DNase I (EC 3.1.21 .I); micrococcal nuclease (EC 3.1.31.1); nuclease P1 (EC 3.1.30.1);, snake venom phosphodiesterase I (EC 3.1.4.1); alkaline phosphatase (EC 3.1.3.1); RNase I (EC 3.1.27.5); pronase (EC 3.4.24.4); trypsin (EC 3.4.21.4). lication [8] or transcription [9]. This is supported by the fact that in the case of blocked vegetative replication of similar plasmids they cannot be transferred to the recipient cell [8]. Also, ethidium bromide intercalation with the plasmid DNA in vitro induces the complex relaxation followed by sitespecific nicking at oriT [lo].The site-specific nicking of the transferred DNA strand by the relaxation protein and the formation of an active and specific intermediate covalent complex between the ColEl DNA and the relaxation protein seem to be the key point in the initiation of pla...

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Localization and nucleotide sequence of the bom region of Clo DF13

Cited by 15 publications

References 38 publications

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

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

Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation

A protein covalently bound to ColEl DNA

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