The ssb gene of plasmid ColIb-P9.

The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12. The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein. Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region. High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA. A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth. Hence, no biological role of ColIb SSB protein was detected. However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins.     

Organization of the replication control region of plasmid ColIb-P9.

We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc. The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon. The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases. This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.     

Direction of conjugative transfer of IncI1 plasmid ColIb-P9.

The origin-of-transfer region of ColIb-P9 was inserted into a lambda prophage to give a bacterial chromosome mobilizable by the parental conjugative plasmid. The polarity of mobilization of chromosomal genes indicated that ColIb-P9 transfer is unidirectional, such that the transfer genes adjacent to oriT enter the recipient cell last.     

Organization and maintenance features of IncP-7 naphthalene degradation plasmid pFME5 basic replicon

A basic replicon of the naphthalene degradation plasmid pFME5 (80 kb, IncP-7) has been constructed and sequenced. The nucleotide sequence of pFME5mini is almost identical to replicons of the pND6-1 subgroup, which was separated based on the repA-oriV homology in our previous work. The basic replicon of pFME5 is capable of replication and stable maintenance exclusively in Pseudomonas species. An analysis of the deletion mutation indicated that, in contrast to the parWAB region, the parC gene is not essential for the stability of pFME5mini and this can be a common feature of IncP-7 replicons. We revealed that par-defective mutants of pFME5mini were slowly eliminated from the bacterial population in a nonselective medium compared to their pCAR1-based counterparts. Designed primers specific to the repA and parC genes can be used to detect IncP-7 plasmids, while primers specific to two variants of parA can be used for intragroup classification.     

IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions.

The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.     

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair

Summary The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5.While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc ^- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.Abbreviations MC mitomycin C - ICS induction of colicin synthesis     

Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid

The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene. To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage. Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein. The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY. We also isolated mutants that suppressed the rep57 phenotype. These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ. Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression. We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal. The positive and negative regulations of repZ expression were discussed. The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication.     

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.     

Molecular cloning of plasmid ColIb-P9 genes responsible for its mutator function

The localization of plasmid ColIb-P9 muc genes mediating the plasmids protective and mutagenesis-increasing activity has been determined. The increase of muc genes dose by cloning them within the multicopy vector has been shown to repress the mutator function of the plasmid. No essential homology has been revealed between ColIb-P9 muc gene nucleotide sequences, pKM101 muc genes with a similar function, and umuDC chromosome genes. It has been shown that the synthesis of 38 KD protein is essential for the manifestation of the mutator function of the plasmid.     

Transfer of tra proteins into the recipient cell during bacterial conjugation mediated by plasmid ColIb-P9.

Selective transfer of the two products of the ColIb primase gene, sog, from donor to recipient cell during conjugation was demonstrated by two independent methods. The transfer of these tra proteins was unidirectional and dependent on DNA transfer. The Sog polypeptides were localized to the cytoplasm of the donor cell, but they appeared to interact with other tra gene products located in the inner membrane. After cell mating, the transferred polypeptides were found to be in the cytoplasm of the recipient cell, and it is estimated that as many as 500 Sog polypeptides were transferred per round of conjugation. It is proposed that these proteins are transferred as a result of an interaction with the single-stranded DNA and that the transferred strand may be coated with Sog polypeptides.     

Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin.

The 4.2-kb cryptic plasmid pVA380-1 has been used as a vector for the cloning of antibiotic resistance genes directly in streptococci, and in the construction of Escherichia coli/Streptococcus shuttle vectors. The results of subcloning experiments located the basic replicon of pVA380-1 within a 2.5-kb region. The nucleotide base sequence of this region was determined and contained a single complete open reading frame (ORF) encoding a 237-amino-acid peptide with a predicted size of 29 kDa. This peptide and a region of the DNA molecule 5' to the ORF encoding it shared homology with the replication protein and plus origin, respectively, of the Staphylococcus aureus plasmid pUB110. Data from Tn5 mutagenesis and complementation studies indicated that the protein product of the ORF was required for pVA380-1 replication in streptococci. Deletion of a region of the basic replicon distal to the plus origin and ORF produced an unstable derivative, and resulted in the accumulation of single-stranded replicative intermediates, consistent with the loss of a minus origin. All of these results suggest that pVA380-1 replicates by a rolling circle mode, and is most closely related to the pC194 family of single-stranded DNA plasmids.     

Bacillus stearothermophilus plasmid pSTK1 replicon is functional in Escherichia coli

Summary 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 coli. 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.     

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi.

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G+C-rich and A+T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5' end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis. repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical sigma 70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.     

Organization and regulation of the conjugation genes of IncI1 plasmid ColIb-P9

The IncI₁ plasmid ColIb-P9 is among a group of related plasmids that encode the I₁ type of conjugation system. The I₁ system is known to include two morphologically distinct types of pilus, a DNA primase gene (sog) and an exclusion determinant (exc). Transposon mutagenesis and analysis of cloned fragments of ColIb were used to identify the location of these determinants with respect to an EcoRI restriction map. Also identified were the location of the origin of transfer (oriT) and a gene determining an EDTA-resistant nuclease, which is coordinately regulated with the transfer genes. The results indicate that the ColIb tra genes are separated into at least three Tra regions. The pleiotropic nature of transposon insertion mutations in two of these regions suggests that two positive regulators are required for expression of the transfer genes and evidence is also found for a trans-acting repressor. It is suggested that the I₁ conjugation system may have evolved following fusion of two distinct types of conjugative plasmid.     

Reactivation of ultraviolet-irradiated phage lambda and recombination in Escherichia coli K-12 cells containing plasmid ColIb-P9

It was shown that the presence of colicinogenis plasmid ColIb-P9 increased the survival of UV-irradiated bacteriophage lambda cI857 in non-irradiated cells of Escherichia coli K-12. The effect of this plasmid was retained in the polA and recB mutants, being sharply reduced in the uvrA and recB recC sbcB recF mutants. This effect strongly depended on recA+ and lexA+ genotype. The W-reactivation efficiency was slightly higher in the cells containing ColIb-P9 than in those lacking the plasmid. No significant effect of the plasmid on recombination during transduction, after conjugation under usual conditions and in the case when a conjugation mixture or recipient cells were irradiated, was observed. The data demonstrate that the effect of ColIb-P9 plasmid on DNA repair is not mediated by its influence on recombination.     

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1

Summary Specialized transducing lambda phages, oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA ⁺, but sensitive to the conserted action of Pst1 fragments F₁ and F₂. This mutant was previously assumed to be of the dominant type. Physical mapping of the oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%–2% per cell generation, which is consistent with their lack of the R1par region.