Genetic analysis of a lactococcal plasmid replicon

Xu, Fengji; Pearce, L. E.; Yu, Pak-Lam

doi:10.1007/bf00260703

Cited by 39 publications

(26 citation statements)

References 40 publications

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“…In addition to pLS1, only two other members of the same family are available to us: staphylococcal plasmid pE194 (14) and lactococcal replicon pFX2 (35). We have been unable to establish pE194 in S. pneumoniae, whereas pFX2 was readily transferred to this host (our unpublished observations).…”

Section: Resultsmentioning

confidence: 99%

“…Larger amounts of the protein or prolonged incubation times proved to be ineffective (data not shown). These results were somewhat unexpected, because the predicted nick site of pFX2 is located at an internal loop within a predicted plasmid hairpin (35), whereas that of pE194 should be placed on the terminal loop of one of the putative major secondary structures of the plasmid (32), as in pLS1. The inability of RepB to relax pE194 DNA could be due to lack of exposure of the nick site as single-stranded DNA.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, to determine whether RepB is able to relax supercoiled DNAs of related plasmids, we employed two plasmids of the pLS1 family, namely, pE194 (14) and pFX2 (35). Whereas RepB readily cleaved supercoiled pFX2 DNA, it failed to do so on pE194 unless the degree of DNA supercoiling of pE194 was increased.…”

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confidence: 99%

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In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

1995

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Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.Many multicopy plasmids isolated from gram-positive and -negative bacteria replicate by an asymmetric rolling-circle (RC) mechanism (reviewed in references 10, 13, and 23). Characterization of various staphylococcal replicons has led to the proposal that plasmids replicating by the RC mode are constructed as interchangeable genetic modules (26). Only one of these modules is essential for plasmid replication, the leading-strand initiation and control region (10). On the basis of homologies in the DNA sequence and in the genetic organization of the leading-strand initiation and control region, four families have been defined, and their prototypes are pT181, pSN2, pC194/pUB110, and pMV158 (10, 13, 23). The latter plasmid has been studied in some detail for its derivative pLS1, which lacks the mob gene involved in conjugative mobilization (19,25). The leading-strand initiation and control region includes the plasmid double-strand origin (dso) and the gene encoding the initiator of replication (Rep) protein. The dso can be physically and functionally divided into two regions, (i) bind, where the Rep protein binds, and (ii) nic, which includes the Rep-mediated nick site used to initiate replication (11). The two regions can be contiguous (as in pT181) or separated by dozens of bases (as in pLS1). The nic region contains one or two stem-loop structures which have been mapped by determining the sensitivity of plasmids pLS1 and pT181 to nuclease S1 in vitro and in vivo (21, 27). The DNA seq...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

1995

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“…Single-stranded DNA replicons are classified into families of highly related plasmids isolated from diverse bacteria (11). The plus origin (plus-on) and the Rep protein of pWVO1 (the parent of the Ts plasmid [25]) have homologies with members of the pE194 family, including pADB201 (2), pLB4 (1), pLS1 (19,30), pSH71 (7a), pFX2 (49), and pHPK255 (16). A Ts derivative of pE194 has already been characterized; two mutations were found, one immediately upstream of its start codon, and the other within the Rep ORF (48).…”

Section: Resultsmentioning

confidence: 99%

New thermosensitive plasmid for gram-positive bacteria

et al. 1992

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We isolated a replication-thermosensitive mutant of the broad-host-range replicon pVWVOl. The mutant pVE6002 is fuly thermosensitive above 35°C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon TnlO derivative in BaciUus subtilis, we observed transposition frequencies of about 1%.

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“…The sequence analysis of pSTK1 reveals that open reading frame 1 (ORF1) of this plasmid has a helix-turn-helix motif typical of many DNA-binding proteins , and exhibits a significant degree of similarity with the Cop proteins (formerly RepA proteins) in a lactococcal plasmid pFX2 (Xu et al, 1991) and a streptcoccal plasmid pLS1 (del Solar et at., 1989). Cop protein is considered to control replication of the plus-strand of some rolling circle replication (RCR) plasmids as a transcriptional repressor (del Solar et at., 1993).…”

Section: Introductionmentioning

confidence: 99%

Bacillus stearothermophilus plasmid pSTK1 replicon is functional in Escherichia coli

et al. 1995

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A kanamycin-resistant plasmid possessing a thermostable replicon derived from Bacillus stearothermophilus cryptic plasmid pSTK1 was constructed. The plasmid could transform not only B. stearothermophilus and Bacillus subtilis, but also Gram-negative Escherichia coil The behavior of the plasmid in the hosts was examined. The plasmid was stably maintained even at 67°C in B. stearothermophilus without selective pressure. During the plasmid replication, single-stranded DNA (ssDNA) intermediates were found in E. coli, while these were not found in B. subtilis.

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Genetic analysis of a lactococcal plasmid replicon

Cited by 39 publications

References 40 publications

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

New thermosensitive plasmid for gram-positive bacteria

Bacillus stearothermophilus plasmid pSTK1 replicon is functional in Escherichia coli

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