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Molecular analysis and characterization of a broad-host-range plasmid, pEP2.

ABSTRACT: Plasmid pEP2 was found to encode a protein, RepA, which is essential and rate limiting for its replication in Escherichia coli and Corynebacterium pseudotuberculosis. Mutations which altered the rate of synthesis of this protein in E. coli affected the copy number and segregational stability of pEP2 in the two hosts. RepA contains 483 amino acid residues and has the calculated molecular weight of 53,925. It shows 45% amino acid residue identity with open reading frame ORF2 of pSR1, a plasmid isolated from Corynebacterium glutamicum (J. A. C. Archer and A. J. Sinskey, J. Gen. Microbiol. 139:1753-1759, 1993). Plasmid pEP2 was shown to accumulate single-stranded DNA corresponding to the RepA coding strand during its replication in E. coli and C. pseudotuberculosis, suggesting that it may replicate by a rolling circle mechanism. However, RepA has no significant sequence homology with the replication initiator proteins of plasmids known to use this mode of replication.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC196776 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Molecular analysis and characterization of a broad-host-range plasmid, pEP2.

Zhang Y Y Praszkier J J Hodgson A A Pittard A J AJ

Journal of bacteriology 19940901 18

Plasmid pEP2 was found to encode a protein, RepA, which is essential and rate limiting for its replication in Escherichia coli and Corynebacterium pseudotuberculosis. Mutations which altered the rate of synthesis of this protein in E. coli affected the copy number and segregational stability of pEP2 in the two hosts. RepA contains 483 amino acid residues and has the calculated molecular weight of 53,925. It shows 45% amino acid residue identity with open reading frame ORF2 of pSR1, a plasmid iso ...[more]

PMID: 7521871

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Project description:The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was use to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil+) phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.

Project description: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, O. 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 vector-independent 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 ColE1 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.

Dataset Information

Molecular analysis and characterization of a broad-host-range plasmid, pEP2.

Publications

Molecular analysis and characterization of a broad-host-range plasmid, pEP2.

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