Relationship of Group P1 Plasmids Revealed by Heteroduplex Experiments: RP1, RP4, R68 and RK2 Are Identical

Burkardt, Hans Joachim; Riess, G.; Pühler, Alfred

doi:10.1099/00221287-114-2-341

Cited by 167 publications

(41 citation statements)

References 12 publications

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“…Nevertheless, studies focusing on the self-transmissible IncP plasmid RK2 (27) and the closely related plasmids RP1, RP4, R18, and R68 (9,84) have revealed a replicon of intriguing genetic and regulatory complexity (17,18,77).…”

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kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus

1993

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The kil-kor regulon of IncP plasmid RK2 is a complex regulatory network that includes genes for replication and conjugal transfer, as well as for several potentially host-lethal proteins encoded by the kiL4, kiB, and kilC loci. While kilB is known to be involved in conjugal transfer, the functions of kiL4 and kUC are unknown. The coregulation of kiL4 and kUC with replication and transfer genes indicates a possible role in the maintenance or broad host range of RK2. In this work, we found that a fourth kil locus, designated kilE, is located in the kb 2.4 to 4.5 region of RK2 and is regulated as part of the kil-kor regulon. The cloned kilE locus cannot be maintained in Escherichia coli host cells, unless korA or korC is also present in trans to control its expression. The nucleotide sequence of the kilE region revealed two potential multicistronic operons. The kleA operon consists oftwo genes, kHeA and kieB, predicted to encode polypeptide products with molecular masses of 8.7 and 7.6 kDa, respectively. The kkeC operon contains four genes, kHeC, kieD, kleE, and kieF, with predicted products of 9.2, 8.0, 12.2, and 11.3 kDa, respectively. To identify the polypeptide products, each gene was cloned downstream of the phage T7 +10 promoter and expressed in vivo in the presence of T7 RNA polymerase. A polypeptide product of the expected size was observed for all six Hde genes. In addition, kdeF expressed a second polypeptide of 6 kDa that most likely results from the use of a predicted internal translational start site. The kkA and kieC genes are each preceded by sequences resembling strong &o70 promoters. Primer extension analysis revealed that the putative keA and ideC promoters are functional in E. coli and that transcription is initiated at the expected nucleotides. The abundance of transcripts initiated in vivo from both the klA and kieC promoters was reduced in cells containing korA or korC. When korA and korC were present together, they appeared to act synergistically in reducing the level of transcripts from both promoters. The kieA and kdeC promoter regions are highly homologous and contain two palindromic sequences (A and C) that are the predicted targets for KorA and KorC proteins. DNA binding studies showed that protein extracts from korA-containing E. colt cells specifically retarded the electrophoretic mobility of DNA fragments containing palindrome A. Extracts from korC-containing cells altered the mobility of DNA fragments containing palindrome C. These results show that KorA and KorC both act as repressors of the kHeA and kdeC promoters. In the absence of korA and korC, expression of the cloned kleA operon was lethal to E. colt cells, whereas the cloned kieC operon gave rise to slowly growing, unhealthy colonies. Both phenotypes depended on at least one structural gene in each operon, suggesting that the operons encode genes whose products interact with critical host functions required for normal growth and viability. Thus, the kiL4, kilC, and kilE loci of RK2 constitute a cluster of at least 10 genes th...

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kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus

1993

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“…At such a copy number, it is likely that this plasmid encodes one or more elements responsible for ensuring its maintenance within the bacterial population (reviewed in references 2 and 29). Recent studies have shown that RK2 and the homologous isolate from Pseudomonas sp., RP4 (7), do contain information that promotes plasmid stabilization. A region within the PstI C fragment of these plasmids, from approximately 32.6 to 35.8 kb, has been shown to efficiently stabilize plasmids in a broad-host-range, vector-independent manner (18,31,33).…”

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Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2

Roberts

Helinski

1992

J Bacteriol

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The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, 0. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vectorindependent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColEl cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.RK2 is a large (60-kb), broad-host-range plasmid of the IncPl family. It is stably maintained in Escherichia coli, in spite of its relatively low copy number of approximately 5 to 8 copies per chromosome (for a review, see reference 40). At such a copy number, it is likely that this plasmid encodes one or more elements responsible for ensuring its maintenance within the bacterial population (reviewed in references 2 and 29). Recent studies have shown that RK2 and the homologous isolate from Pseudomonas sp., RP4 (7), do contain information that promotes plasmid stabilization. A region within the PstI C fragment of these plasmids, from approximately 32.6 to 35.8 kb, has been shown to efficiently stabilize plasmids in a broad-host-range, vector-independent manner (18,31,33). This region, termed the RK2 or RP4par region, encodes several genes that have been p...

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“…Naturally occurring IncP elements (as illustrated by RK2, RP1, and R68 [1]) are large (>50 kb), stable, self-transferable plasmids which exist in four to seven copies per chromosome equivalent (7). The IncP plasmid consists of a contiguous block of conjugal transfer functions joined to a block of replication and maintenance functions.…”

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High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor

Butler

Gotschlich

1991

J Bacteriol

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Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5 _MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GG'CC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.In nature, conjugation appears to play an important role in the mobilization of antibiotic resistance plasmids into various species of the genus Neisseria. Resistance plasmids can be transferred, for example, from enteric bacteria to Neisseria gonorrhoeae, as has been hypothesized by Roberts and Falkow (22). Likewise, other investigators have demonstrated that the broad-host-range incompatibility group P (IncP) plasmid pUB307 can mobilize pLES2 from Escherichia coli to N. gonorrhoeae (18). Thus, IncP elements may contribute to the establishment of resistance plasmids in Neisseria spp., although IncP elements have not been found in Neisseria organisms. It is assumed that this group of plasmids cannot be maintained in N. gonorrhoeae because of plasmid replication deficiencies (18). In addition to the 3-lactamase plasmids, plasmids having homology to the broad-host-range incompatibility group Q (IncQ) plasmids, namely, RSF1010, have been found in a variety of commensal Neisseria species and in Neisseria meningitidis (5,19,23,24). These RSF1010-like plasmids are the first multiresistant plasmid group found in Neisseria spp., having resistance not only to sulfonamide and streptomycin but also to ampicillin (23). The possibility exists for dissemination of these plasmids into N. gonorrhoeae.Naturally occurring IncP elements (as illustrated by RK2, RP1, and R68 [1]...

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Relationship of Group P1 Plasmids Revealed by Heteroduplex Experiments: RP1, RP4, R68 and RK2 Are Identical

Cited by 167 publications

References 12 publications

kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus

kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus

Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor

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