xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli. The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd). The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.     

Derivatives of ColE1 cer show altered topological specificity in site-specific recombination.

ColE1 contains a 250-bp sequence (cer) which is required in cis for the conversion of plasmid dimers to monomers. Recombination between cer and parB (a dimer resolution site from plasmid CloDF13) occurs in vivo at low frequency. The properties of the resulting hybrid sites have been studied. The type I hybrid closely resembles wild-type cer. It supports intramolecular recombination and requires the products of the chromosomal xerA, xerB and xerC genes together with the 250-bp site. In contrast, the type II hybrid (although differing from type I by only 2 bp) functions independently of the topological relationship of the participating sites, supporting both inter- and intramolecular recombination. Furthermore, recombination between type II sites is independent of the products of the xerA and xerB genes and requires a site of less than 50 bp.     

The arginine repressor is essential for plasmid-stabilizing site-specific recombination at the ColE1 cer locus.

The heritable stability in Escherichia coli of the multicopy plasmid ColE1 and its natural relatives requires that the plasmids be maintained in the monomeric state. Plasmid multimers, that arise through recA-dependent homologous recombination, are normally converted to monomers by a site-specific recombination system that acts at a specific plasmid site (cer in ColE1). No plasmid functions that act at this site have been identified. In contrast, two unlinked E.coli genes that encode functions required for cer-mediated site-specific recombination have been identified. Here we describe the isolation and characterization of one such gene (xerA) and show it to be identical to the gene encoding the repressor of the arginine biosynthetic genes (argR). The argR protein binds to cer DNA both in vivo and in vitro in the presence of arginine. We believe this binding is required to generate a higher order protein-DNA complex within the recombinational synapse. The argR gene of Bacillus subtilis complements an E.coli argR deficiency for cer-mediated recombination despite the two proteins having only 27% amino acid identity.     

Escherichia coli XerC recombinase is required for chromosomal segregation at cell division

XerC is a site-specific recombinase of the bacteriophage lambda integrase family that is encoded by xerC at 3700 kbp on the genetic map of Escherichia coli. The protein was originally identified through its role in converting multimers of plasmid ColE1 to monomers; only monomers are stably inherited. Here we demonstrate that XerC also has a role in the segregation of replicated chromosomes at cell division. xerC mutants form filaments with aberrant nucleotides that appear unable to partition correctly. A DNA segment (dif) from the replication terminus region of the E. coli chromosome binds XerC and acts as a substrate for XerC-mediated site-specific recombination when inserted into multicopy plasmids. This dif segment contains a region of 28 bp with sequence similarity to the crossover region of ColE1 cer. The cell division phenotype of xerC mutants is suppressed in strains deficient in homologous recombination, suggesting that the role of XerC/dif in chromosomal metabolism is to convert any chromosomal multimers (arising through homologous recombination) to monomers.     

Site-specific recombination between ColE1 tcer and NTP16 nmr sites in vivo

The site-specific recombination system used by multicopy plasmids of the ColE1 family uses two identical plasmid-encoded recombination sites and four bacterial proteins to catalyze the recombination reaction. In the case of the Escherichia coli plasmid ColE1, the recombination site, cer, is a 280 by DNA sequence which is acted on by the products of the argR, pepA, xerC and xerD genes. We have constructed a model system to study this recombination system, using tandemly repeated recombination sites from the plasmids ColE1 and NTP16. These plasmids have allowed us precisely to define the region of strand exchange during site-specific recombination, and to derive a model for cer intramolecular site-specific recombination.     

Site-specific recombination between ColE1 tcer and NTP16 nmr sites in vivo

The site-specific recombination system used by multicopy plasmids of the ColE1 family uses two identical plasmid-encoded recombination sites and four bacterial proteins to catalyze the recombination reaction. In the case of the Escherichia coli plasmid ColE1, the recombination site, cer, is a 280 by DNA sequence which is acted on by the products of the argR, pepA, xerC and xerD genes. We have constructed a model system to study this recombination system, using tandemly repeated recombination sites from the plasmids ColE1 and NTP16. These plasmids have allowed us precisely to define the region of strand exchange during site-specific recombination, and to derive a model for cer intramolecular site-specific recombination.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI I. Isolation and Specificity of Host and Plasmid Mutations

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.     

Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination

Summary The heritable stability of the multicopy plasmid ColE1 and its natural relatives, requires the presence in the plasmid of a site (cer in ColE1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state. Multimerization, promoted by homologous recombination, leads to plasmid loss. Here we show that the Escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific recombination. One of these functions is encoded by a gene residing on a cosmid that also contains the argI and pyrB genes, mapping it to the 96–97 min region of the E. coli map.     

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI II. Properties of Host and Plasmid Mutations

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.     

Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.     

RecA independent,site-specific recombination between ColE1 or ColK and a miniplasmid they complement for mobilization and relaxation: Implications for the mechanism of DNA transfer during mobilization

We report a novel type of recA independent recombination between plasmids ColE1 or ColK and a naturally occurring miniplasmid (pLG500). This miniplasmid can be complemented for mobilization and relaxation in the presence of ColE1 or ColK. Recombination between ColE1 and pLG500, or ColK and pLG500, was site-specific, and was only detected following the mobilization of these plasmids. The composite plasmids thus formed were stable, but recombination (resulting in dissociation of their component replicons) was again detected following mobilization. For ColE1, the site at which cointegration with pLG500 occurred was mapped to within 47 base pairs of the relaxation nicking site; for ColK, the recombination site was localized to the same region as its genetically defined transfer origin. The generation of these cointegrate plasmids is consistent with the hypothesis that mobilization entails relaxation nicking, transfer of the nicked single strand of DNA, and recircularization of the transferred single strand by ligation of 3′ and 5′ termini by the relaxation protein bound to the 5′ nick terminus. Since both plasmids are mobilized by the same proteins, their cointegration can be explained as a consequence of the ligation of the 5′ end of one plasmid to the 3′ end of the other, and vice versa.     

PepA and ArgR do not regulate Cre recombination at the bacteriophage P1 loxP site

In the lysogenic state, bacteriophage P1 is maintained as a low copy-number circular plasmid. Site-specific recombination at loxP by the phage-encoded Cre protein keeps P1 monomeric, thus helping to ensure stable plasmid inheritance. Two Escherichia coli DNA-binding proteins, PepA and ArgR, were recently reported to be necessary for maintenance or establishment of P1 lysogeny. PepA and ArgR bind to regulatory DNA sequences upstream of the ColE1 cer recombination site to regulate site-specific recombination by the XerCD recombinases. This recombination keeps ColE1 in a monomeric state and helps to ensure stable plasmid maintenance. It has been suggested that ArgR and PepA play a similar role in P1 maintenance, regulating Cre recombination by binding to DNA sequences upstream of loxP. Here, we show that ArgR does not bind to its proposed binding site upstream of loxP, and that Cre recombination at loxP in its natural P1 context is not affected by PepA and ArgR in vitro. When sequences upstream of loxP were mutated to allow ArgR binding, PepA and ArgR still had no effect on Cre recombination. Our results demonstrate that PepA requires specific DNA sequences for binding, and that PepA and ArgR have no direct role in Cre recombination at P1 loxP.     

Isolation and characterization of replication-deficient mutants of ColE1 plasmids.

Replication-defective mutants of plasmid ColE1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid. The replication-defective ColE1 mutants isolated were all spontaneous deletion mutants that had lost the ColE1 replication origin and regions adjacent to it. The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColE1 plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis. None of the chimeras containing the replication-defective ColE1 mutants was able to replicate in the presence of chloramphenicol. The expression of ColE1 incompatibility was either markedly reduced or not detectable in the replication mutants isolated.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination,DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites. The hexameric wild-type protein shows L -arginine-dependent DNA binding. In this work, ArgR mutants that are defective in trimer–trimer interactions and bind DNA as trimers in an L -arginine-independent manner are isolated and characterized. Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination. In the absence of L -arginine, residual wild-type ArgR-binding occurs as trimers. The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.     

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.