Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs

The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R. EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M(r) of 35,554. The convergently oriented EcoVIII methyltransferase (M. EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M(r) of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5'-AAGCTT-3'. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R. EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5' protruding ends. M. EcoVIII functions as a monomer and modifies the first adenine residue at the 5' end of the specific sequence to N(6)-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M. EcoVIII with M. HindIII and M. LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m(6) N-adenine beta-class methyltransferases. The deduced amino acid sequence of R. EcoVIII shows weak homology with its two isoschizomers, R. HindIII (26%) and R. LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R. EcoVIII (D(108)X(12)DXK(123)), as well as in the primary structures of R. LlaCI and R. HindIII. Polyclonal antibodies raised against R. EcoVIII did not react with R. HindIII, while anti-M. EcoVIII antibodies cross-reacted with M. LlaCI but not with M. HindIII. R. EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M. EcoVIII enzyme. The biological implications of this finding are discussed.     

Studies on a Vibrio vulnificus Functional Ortholog of Escherichia coli RNase E Imply a Conserved Function of RNase E-like Enzymes in Bacteria

RNase E (Rne) plays a key role in the processing and degradation of RNA in Escherichia coli. In the genome of Vibrio vulnificus, one open reading frame potentially encodes a protein homologous to E. coli RNase E, designated RNase EV, which N-terminal (1-500 amino acids) has 86.4% amino acid identity to the N-terminal catalytic part of RNase E (N-Rne). Here, we report that both the full-length and the N-terminal part of RNase EV (N-RneV) functionally complement E. coli RNase E and their expression consequently supports normal growth of RNase E-depleted E. coli cells. E. coli cells expressing N-RneV showed copy numbers of ColE1-type plasmid similar to that of E. coli cells expressing N-Rne, indicating in vivo ribonucleolytic activity of N-RneV on RNA I, an antisense regulator of ColE1-type plasmid replication. In vitro cleavage assays further showed that N-RneV has cleavage activity and specificity of RNase E on RNase E-targeted sequence of RNA I (BR13). Our findings suggest that RNase E-like proteins have conserved enzymatic properties that determine substrate specificity across species.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.     

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.     

Alteration of RNA I metabolism in a temperature-sensitive Escherichia coli rnpA mutant strain.

E. coli strain A49 carries the themosensitive mutation in the rnpA gene encoding the protein component of RNase P, a tRNA-processing enzyme. Two small RNAs were highly accumulated in the A49 carrying derivatives of ColE1-type plasmids, at nonpermissive temperature. Characterization of these RNAs showed that they were the processed or degraded products derived from RNA I, which is the negative controller of ColE1-type plasmid replication. These derivatives of RNA I only differ in size at the 5' ends. The data of their degradation and synthesis kinetics suggest that they are intermediates of RNA I metabolism.     

Nucleotide sequence and gene organization of ColE1 DNA

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).     

Lateral transfer of rfb genes: a mobilizable ColE1-type plasmid carries the rfbO:54 (O:54 antigen biosynthesis) gene cluster from Salmonella enterica serovar Borreze.

Plasmid pWQ799 is a 6.9-kb plasmid isolated from Salmonella enterica serovar Borreze. Our previous studies have shown that the plasmid contains a functional biosynthetic gene cluster for the expression of the O:54 lipopolysaccharide O-antigen of this serovar. The minimal replicon functions of pWQ799 have been defined, and a comparison with nucleotide and protein databases revealed this replicon to be virtually identical to ColE1. This is the first report of involvement of ColE1-related plasmids in O-antigen expression. The replicon of pWQ799 is predicted to encode two RNA molecules, typical of other ColE1-type plasmids. RNAII, the putative replication primer from pWQ799, shares regions of homology with RNAII from ColE1. RNA1 is an antisense regulator of DNA replication in ColE1-related plasmids. The coding region for RNAI from pWQ799 shares no homology with any other known RNAI sequence but is predicted to adopt a secondary structure characteristic of RNAI molecules. pWQ799 may therefore represent a new incompatibility group within this family. pWQ799 also possesses cer, rom, and mob determinants, and these differ minimally from those of ColE1. The plasmid is mobilizable in the presence of either the broad-host-range helper plasmid pRK2013 or the IncI1 plasmid R64drd86. Mobilization and transfer of pWQ799 to other organisms provides the first defined mechanism for lateral transfer of O-antigen biosynthesis genes in S. enterica and explains both the distribution of related plasmids and coexpression of the O:54 factor with other O-factors in different Salmonella serovars. The base composition of the pWQ799 replicon sequences gives an average percent G+C value typical of Salmonella spp. In contrast, the percent G+C value is dramatically lower with rfb0:54, consistent with the possibility that the cluster was acquired from an organism with much lower G+C composition.     

Sequence analysis and characterization of plasmid pSFD10 from Salmonella choleraesuis

The nucleotide sequence of a small plasmid, designated pSFD10, is isolated from the vaccine strain Salmonella choleraesuis C500 in China, has been determined. This plasmid is 4091 bp long with a total G+C content of 51.4%, which is in the range of Salmonella genomic DNA. Analysis of the complete nucleotide sequence reveals that pSFD10 has a high degree of similarity to ColE1-type plasmid, having the possible cer and rom genes, and a putative mobilization origin of ColE1-type. Plasmid pSFD10 possesses six main open reading frames (ORFs), five of which have a very high degree of amino acid identity to ColE1-type plasmid gene products involved in mobilization and copy number control. The other ORF (ORF6) encodes a putative protein, which has 49% homology to the invasion plasmid antigen J protein (IpaJ) secreted by the type III secretion apparatus of Shigella flexneri. In addition, pSFD10 belongs to a different incompatibility group than ColE1-type and pMB1-type to which it is related. Plasmid pSFD10 can be mobilized by the plasmid RP4 in E. coli.     

Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase

Mutations affecting a region of the Escherichia coli RNA polymerase have been isolated that specifically reduce the copy number of ColE1-type plasmids. The mutations, which result in a single amino acid alteration (G1161R) or a 41-amino acid deletion (Delta1149-1190) are located near the 3'-terminal region in the rpoC gene, which encodes the largest subunit (beta ') of the RNA polymerase. The rpoC deletion and the point mutation cause over 20- and 10-fold reductions, respectively, in the copy number of ColE1. ColE1 plasmid numbers are regulated by two plasmid-encoded RNAs: RNA II, which acts as a preprimer for the DNA polymerase I to start initiation of replication, and RNA I, its antisense inhibitor. Altered expression from the RNA I and RNA II promoters in vivo was observed in the RNA polymerase mutants. The RNA I/RNA II ratio is higher in the mutants than in the wild-type strain and this is most probably the main reason for the reduction in the ColE1 copy number in the two rpoC mutants.     

Plasmid pEC156, a Naturally Occurring Escherichia coli Genetic Element That Carries Genes of the EcoVIII Restriction-Modification System,Is Mobilizable among Enterobacteria

Type II restriction-modification systems are ubiquitous in prokaryotes. Some of them are present in naturally occurring plasmids, which may facilitate the spread of these systems in bacterial populations by horizontal gene transfer. However, little is known about the routes of their dissemination. As a model to study this, we have chosen an Escherichia coli natural plasmid pEC156 that carries the EcoVIII restriction modification system. The presence of this system as well as the cis-acting cer site involved in resolution of plasmid multimers determines the stable maintenance of pEC156 not only in Escherichia coli but also in other enterobacteria. We have shown that due to the presence of oriT-type F and oriT-type R64 loci it is possible to mobilize pEC156 by conjugative plasmids (F and R64, respectively). The highest mobilization frequency was observed when pEC156-derivatives were transferred between Escherichia coli strains, Enterobacter cloacae and Citrobacter freundii representing coliform bacteria. We found that a pEC156-derivative with a functional EcoVIII restriction-modification system was mobilized in enterobacteria at a frequency lower than a plasmid lacking this system. In addition, we found that bacteria that possess the EcoVIII restriction-modification system can efficiently release plasmid content to the environment. We have shown that E. coli cells can be naturally transformed with pEC156-derivatives, however, with low efficiency. The transformation protocol employed neither involved chemical agents (e.g. CaCl₂) nor temperature shift which could induce plasmid DNA uptake.     

Isolation and Characterization of a ColE1-like Plasmid fromEnterobacter agglomeranswith a Novel Variant ofromGene

Complete nucleotide sequence of a plasmid isolated fromEnterobacter agglomeranshas been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring thecatgene (pBW4), which could be introduced intoEscherichia colicells, and demonstrating that pBW4 cannot replicate in the absence of thepolAfunction and that its copy number is significantly decreased in thepcnBmutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starvedrelAmutants but not inrelA⁺bacteria. Inactivation of the putativeromgene by insertion of an ampicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number inE. colidespite the fact that amino acid sequence of the putative RNA I modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derivedrom-like coding sequence is also homologous to therom-like gene present in theProteus vulgarisplasmid pPvu1. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) inE. coli,one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the newrom-like gene coding for a protein useful in investigation of RNA–protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.     

Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target

Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3' end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I-the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI(-5)) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI(-5) with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI(-5) decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number.     

Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication.

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.     

The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

A functional map of Streptomyces coelicolor plasmid SCP2* was deduced from derivatives constructed by in vitro deletions. Functions were analyzed on bifunctional shuttle plasmids that contained pBR322 for selection and replication in Escherichia coli and fragments of SCP2* for replication in Streptomyces griseofuscus C581 and strains of Streptomyces lividans. The aph gene for neomycin resistance from Streptomyces fradiae and the tsr gene for thiostrepton resistance from Streptomyces azureus were incorporated as selectable antibiotic resistance markers in streptomycetes. An 11.8-kb sequence bounded by EcoRI and KpnI restriction sites contains the information for self-transfer and normal replication of the plasmid. A 5.9-kb EcoRI-SalI fragment contains all of the information for normal replication. Partial digestion generated a 2.2-kb Sau3A fragment that is sufficient for replication but it produces ten times higher plasmid copy number than the basic replicon. pHJL400 and PHJL401 are useful shuttle vectors containing the moderate-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. A 1.4-kb BclI-Sau3A fragment with an additional internal BclI site contains the minimal replicon but it produces 1000 times higher plasmid copy number than the basic replicon. pHJL302 is a useful shuttle vector containing the ultrahigh-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19.     

Transformation of Coxiella burnetii to ampicillin resistance.

A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.