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A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.

To determine possible functions of the Edwardsiella ictaluri plasmids, pEI1 and pEI2, we analyzed the sequence of both plasmids. Plasmid pEI1 is 4807 bp, with 51% G + C, and 23 possible open reading frames of 40 amino acids or greater. Plasmid pEI2 is 5643 bp, with 51% G + C, and 24 possible reading frames. Database searches indicated that pEI1 contains an insertion element and a ROM analog. In addition, pEI1 possesses an open reading frame with strong homology to SlrP, SspH1, and SspH2 of Salmonella typhimurium and IpaH of Shigella flexnari, which have leucine-rich repeat regions and are components of type III secretory systems. pEI2 has a frame with weak homology to Spa15 of S. flexnari 5 and InvB of S. sonnei and S. typhimurium, which are also type III secretory system components, three origins of replication, a Rep analog, and a multimer resolution site.

Pleiotropic chromosomal mutations were earlier identified in saprophytic associative bacterium Agrobacterium radiobacter 5D-1. The mutations changed nitrogen metabolism, disturbed synthesis of indolylacetic acid (IAA), and conferred the ability to sustain replication of ColE1 plasmid derivatives, which are not normally maintained in bacteria other than Escherichia. The mutations were designated Nr (Nitrogen metabolism) and assigned to a single cluster on an A. radiobacter genetic map. A 420-bp fragment AGH23.1.1 was cloned from an agrobacterial genomic library. Introduced in the Nr mutants as a part of a pUC18-based recombinant plasmid, the AGH23.1.1 fragment complemented the Nr mutations with respect to nitrogen metabolism and IAA biosynthesis, but transformants still sustained replication of ColE1 plasmids. Transformation with the linear AGH23.1.1 fragment was due to substitution of a mutant allele of the nr gene with its wild-type counterpart as a result of recombination and completely restored the wild type in the Nr mutants, including the inability to maintain ColE1 plasmids. The AGH23.1.1 fragment and its flanking regions were sequenced. The established sequence was shown to contain two open reading frames (ORFs) coding for proteins with unknown functions. Thus, the cloned fragment contained a gene(s) that controls nitrogen metabolism and IAA synthesis and prevent replication of ColE1 plasmids in A. radiobacter cells. Possible variants of the genetic control of these processes are considered.

The interaction of distamycin with ColE1 DNA was examined by using differential scanning calorimetry (DSC) taking the helix-coil transition theory of DNA into consideration. Our results here strongly indicate that the affinity of distamycin to DNA, at a low distamycin concentration, depends highly on the DNA sequence, and preferential binding occurs to the sites of four to six successive A-T pairs having two or more successive G-C pairs on both their ends.

Escherichia coli PepA is a hexameric aminopeptidase that is also endowed with a DNA-binding activity that functions in transcription control and plasmid dimer resolution. To gain further insight into the functioning of PepA, mutants were selected on the basis of reduced repressibility of a genomic carA-lacZ fusion and studied for the various cellular processes requiring PepA, i.e. repression of the carAB operon, autoregulation, resolution of ColE1 multimers, and peptide proteolysis. The methylation status of the carAB control region was analysed in several pepA mutants and purified proteins were assayed in vitro for car operator DNA binding. This study provides a critical test of predictions advanced on the basis of the structural analysis of PepA and demonstrates the importance for DNA binding of several secondary structural elements in the N-terminal domain and near the very C terminus. By analysis of single amino acid substitutions, we could distinguish the mode of PepA action in car regulation from its action in plasmid resolution. We demonstrate that mere binding of PepA to the car control region is not sufficient to explain its role in pyrimidine-specific regulation; protein-protein interactions appear to play an important role in transcriptional repression. The multifunctional character of PepA and of an increasing number of transcriptional regulators that combine catalytic and regulatory properties, of which several participate in the metabolism of arginine and of the pyrimidines, suggests that enzymes and DNA (RNA) binding proteins fulfilling an essential primeval function may have been recruited in evolution to fulfil an additional regulatory task.

Small particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used for biotechnological purposes. In such applications, tungsten was observed to affect the integrity of plasmid DNA. Here we present evidence that interaction between tungsten particles and intact circular plasmids pU19, pUC119, and ColE1 may result in generation of a limited number of single-strand DNA breaks. As a consequence, supercoiled DNA is converted into its open circular form and no fragmentation products can be detected. The rate of the tungsten-mediated reaction depends on pH but is not influenced by ascorbate, Tris, or EDTA. No DNA nicking can be observed when the tungsten particles are replaced by substances that can be leached out from these particles with water or incubation buffers. Likewise, commercial sodium tungstate, tungsten (VI) oxide, and tungsten (VI) chloride and products of its decomposition remain DNA undamaged. Native plasmid DNA molecules, upon adsorption on the surface of tungsten microparticles, may undergo some nicking without a need for participation of external catalysts.

A detailed analysis of the mobilizable, ColE1-like resistance plasmid, pUB2380, is reported. The 8.5-kb genome encodes six (possibly seven) major functions: (1) a ColD-like origin of replication, oriV, with associated replication functions, RNAI and RNAII; (2) a set of active mobilization functions highly homologous to that of ColE1, including the origin of transfer, oriT; (3) a ColE1-like multimer resolution site (cer); (4) a kanamycin-resistance determinant, aph, encoding an aminoglycoside-3'-phosphotransferase type 1; (5) an insertion sequence, IS1294; and (6) two genes, probably cotranscribed, of unknown function(s). The GC content of the various parts of the genome indicates that the plasmid is a hybrid structure assembled from DNA from at least three different sources, of which the replication region, the mobilization functions, and the resistance gene are likely to have originated in the enterobacteriaceae.

A plasmid with a size of 2682 base pairs isolated from the Yersinia enterocolitica biogroup 1A strain #29807 was characterized in respect to its suitability as a basic replicon for cloning vectors. The copy number of the plasmid was determined to be approximately 14 copies per cell and it was shown to be compatible with vectors with an origin of replication derived from ColE1 and p15A. The replication region of the plasmid encodes a primer RNAI and countertranscript RNAII. Two vectors, pIV1 and pIV2, containing a kanamycin resistance gene and the lacZα fragment with the multiple cloning site of pBluescriptSK+ were constructed. A mobilizable derivative was successfully introduced into different bacteria belonging to the family Enterobacteriacea . To prove the applicability of the novel vectors for cloning purposes, a 13 kb hemolysin operon of Escherichia coli was inserted into pIV1, and the resulting recombinant plasmid was stably maintained and expressed in E. coli and Y. enterocolitica .

The plasmid pJHCMW1 encodes resistance to several aminoglycosides and β-lactams and consists of a copy of the transposon Tn1331, a region including the replication functions, and a sequence with homology to ColE1 cer, designated mwr. In this work, the role of this cer-like site in ensuring the stable inheritance of pJHCMW1 by multimer resolution was studied. The Escherichia coli Xer site-specific recombination system acts at sites such as ColE1 cer to resolve plasmid multimers formed by homologous recombination, thereby maintaining plasmids in a monomeric state and helping to ensure stable plasmid inheritance. Despite its high similarity to ColE1 cer, the pJHCMW1 mwr was a poor substrate for Xer recombination in E. coli and did not contribute significantly to plasmid stability. Instead, the Tn1331 co-integrate resolution system was highly active at resolving pJHCMW1 multimers and ensured the stable inheritance of pJHCMW1. Although Xer recombination at pJHCMW1 mwr was inefficient in E. coli, the recombination that did occur was dependent on ArgR, PepA, XerC and XerD. A supercoiled circular DNA molecule containing two pJHCMW1 mwr sites in direct repeat yielded Holliday-junction-containing product when incubated with ArgR, PepA, XerC and XerD in vitro, confirming that pJHCMW1 mwr is a functional recombination site. However, unlike cer, some Holliday-junction-containing product could be detected for mwr in the absence of ArgR, although addition of this protein resulted in formation of more Holliday junctions. Binding experiments demonstrated that XerD bound to pJHCMW1 mwr core with a high affinity, but that XerC bound to this site very poorly, even in the presence of XerD.

SSOR, a clinical isolate of Shigella sonnei which exhibits a Salicin-negative phenotype, is unable to mutate to give rise to Sal+ derivatives although a homolog of the Escherichia coli bgl operon is retained by the strain. This was correlated to the presence of an endogenous plasmid in the strain. A plasmid-cured derivative, AK711, could give rise to Sal+ mutants in two steps. Introduction of the plasmid DNA, extracted from SSOR, into various strains of E. coli and S. sonnei, resulted in ampicillin resistant transformants. Interestingly, the presence of the plasmid suppressed the mutational activation of the bgl operon in the transformants. This was further substantiated by the observation that, transformants that have lost the plasmid regained the ability for mutational activation of the bgl operon. Preliminary characterisation of the plasmid indicated a size of 3.8 kb with an origin of replication resembling that of ColE1 replicons and the bla gene homolog of Tn3. Observations of the mutation frequency at the srl and lac loci in the presence of the plasmid indicate that there is a reduction in the mutation frequency, suggesting an antimutator activity associated with the plasmid.

The structures of two RNA duplexes, whose sequences correspond to portions of the ColE1 plasmid copy control RNA I and RNA II, have been determined. Crystals containing the 18mers 5'-CA CCGUUGGUAGCGGUGC-3' and 5'-CACCGCUACCAACGGUGC-3' diffract to 1.20 A resolution while those containing the 19mers 5'-GCACCGUUGGUAGCGGUGC-3' and 5'-GCACCGCUACCAACGGUGC-3' diffract to 1.55 A resolution. Both duplexes are standard A form, with Watson-Crick base pairing throughout. Use of anisotropic atomic displacement factors in refinement of the 1.20 A structure dramatically improved refinement statistics, resulting in a final R(free) of 15.0% and a crystallographic R-factor of 11.6%. Perhaps surprisingly, these crystals of the 18 base pair RNA exhibit a 36-fold static disorder, resulting in a structure with a single sugar-phosphate backbone conformation and an averaged base composition at each residue. Since the sugar-phosphate backbone structure is identical in the 36 different nucleotides that are superimposed, there can be no sequence-dependent variation in the structure. The average ribose pucker amplitude is 45.8 degrees for the 18 base pair structure and 46.4 degrees for the 19 base pair structure; these values are respectively 19% and 20% larger than the average pucker amplitude reported from nucleoside crystal structures. A standard RNA water structure, based on analysis of the hydration of these crystal structures and that of the TAR RNA stem [Ippolito, J. A., and Steitz, T. A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9819-9824], has been derived, which has allowed us to predict water positions in lower resolution RNA crystal structures. We report a new RNA packing motif, in which three pro-S(p) phosphate oxygens interact with an ammonium ion.

The dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. Until now, this hypothesis has been investigated using multicopy ColE1 plasmids. However, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the same mechanism of replication regulation. Here we used a modified lambda plasmid, pTC lambda 1. The advantage of this plasmid is that it can be maintained at different copy numbers depending on the concentration of an inducer which stimulates the initiation of plasmid replication. Results obtained with this plasmid in recombination proficient and deficient cells generally support the dimer/multimer catastrophe hypothesis, but also suggest some modification in the model.

Summary A method is described for the in situ detection of individual whole fixed cells of Escherichia coli containing ColE1-related plasmids. It makes use of fluorescence in situ hybridization (FISH) and the regulatory RNA II as a target molecule for both, Cy3- and HRP-labeled olinucleotide probes. Various methods for signal amplification were compared. Probes targeting the regulatory RNA I did not result in the in situ detection of plasmid-bearing cells.

Effect of copy number on the expression of bovine growth hormone gene (bGH) was investigated using the copy number mutants such as pKBJ10, pBJ(Δ tet)10, pUBJ10-1, and pUBJ10 plasmids. The cells harboring plasmids below 84 copies/cell did not produced detectable levels of bGH. When the ColE1 replicon was replaced with the mutated ColE1 replicon originated from pUC19 plasmid, the copy number was increased to about 300 copies/cell and bGH production was enhanced by 11.5% (pUBJ10-1) and 12.3% of total cell protein (pUBJ10). A large amount of mRNA caused by increment of copy number would be needed to overcome some inhibitory threshold and might be an important factor for regulating bGH expression.

Plasmid pDB31 is a ColE1-compatible expression vector based on the p15A origin of replication. It is designed to express His-tagged fusion proteins in cells co-hosting a compatible expression vector. It was constructed by assembling the operator/promoter region plus the 6xHis and the multiple cloning site of pQE31 (QIAGEN) with the p15A origin of replication plus KanR of pGP1-2. The plasmid was found to be stable in Escherichia coli strains BL21 and DH11S. It was used to produce and purify the ferredoxin reductase component of Comamonas testosteroni B-356 biphenyl dioxygenase inside a clone hosting the remaining dioxygenase genes on a compatible plasmid.

A ColE1-compatible vector was constructed to expressed His-tagged fusion protein in Escherichia coli cells. The vector designated pYH31 is derived from pREP4 and pQE31. It was designed to express simultaneously in the same cell, a His-tagged fused protein and a non His-tagged protein from separate plasmids. The vector was used to express both subunits of the biphenyl dioxygenase oxygenase component together in the same E. coli clone.

A modified low copy number plant binary vector pUN has been constructed and successfully used to clone unstable DNA sequences. The vector pUN comprises of low copy number, broad host range RK2 replicon from pBin19 and of multiple cloning site (MCS) and T-DNA region, both from a pBINPLUS-derived pLV06 vector. The absence of the ColE1 replicon in the backbone of the binary vector significantly contributed to stability of hardly clonable DNA sequences and enabled their transfer into the tobacco plants through Agrobacterium-mediated transformation.

Summary The field of gene regulation underwent a major revolution with the discovery of small non-coding RNAs (sRNAs) and the various roles they play in organisms from bacteria to man. Escherichia coli has more than 60 sRNAs that are transcribed primarily from intergenic regions. They usually target the leader region of mRNAs and prevent their translation. Protein targets are relatively rare. In this issue of Molecular Microbiology, Chant and Summers provide an example of a totally unexpected protein target. They show that dimers of plasmid ColE1 make an sRNA that interacts directly with the enzyme tryptophanase and enhances its affinity for its substrate, tryptophan. A breakdown product, indole, then arrests cell division until the dimers are resolved to monomers. The monomerization helps to prevent plasmid loss. Targeting a catabolic enzyme to buy time for recombination is an amazing example of adaptation, which illustrates the power of a selfish element (a plasmid in this case) to exploit the host cell machinery to its advantage.

ColE1-derived plasmids containing different recombinant genes which are controlled by the tac promoter were amplified following induction with IPTG, but no amplification occurred if product formation was not induced. The plasmid copy number of recombinant E. coli increased three- to sixfold within a period of about 6 h in shake flask experiments, batch cultures, and glucose-limited fed-batch cultivations. Plasmid amplification occurred in E. coli B strains as well as in K-12 strains with different plasmids (rop+ and rop-) coding for various heterologous proteins. The amplification was not caused by a toxic effect of IPTG, but was related to a strong inhibition of translation and chromosomal replication after the induction of heterologous gene expression. Similar to the amplification after chloramphenicol addition, plasmid replication proceeded even if oriC replication and translation were inhibited following strong induction of a recombinant gene. In accordance with the effect of chloramphenicol, the level of ppGpp, which is a negative regulator of ColE1 derived plasmid replication, decreased after induction.

Multicopy plasmids of Escherichia coli are distributed randomly at cell division and, as long as copy number remains high, plasmid-free cells arise only rarely. Copy number variation is minimized by plasmid-encoded control circuits, and the limited data available suggest that deviations are corrected efficiently under most circumstances. However, plasmid multimers confuse control circuits, leading to copy number depression. To make matters worse, multimers out-replicate monomers and accumulate clonally within the culture, creating a subpopulation of cells with a significantly increased rate of plasmid loss. Multimers of natural multicopy plasmids, such as ColE1, are resolved to monomers by a site-specific recombination system (Xer-cer) whose activity is limited to intramolecular recombination. Recombination requires the heterodimeric XerCD recombinase plus two accessory proteins (ArgR and PepA), which activate recombination and prevent intermolecular events. Evidence is accumulating that Xer-cer recombination is relatively slow, and there is a risk that cells might divide before multimer resolution is complete. The Rcd transcript encoded within cer may solve this problem by preventing the division of multimer-containing cells. Working in concert, the triumvirate of copy number control, multimer resolution and cell division control achieve an extremely high fidelity of plasmid maintenance.

The Xer site-specific recombination system is involved in the stable maintenance of replicons (certain plasmids and chromosomes) in Escherichia coli and other bacteria by converting multimers into monomers. This system requires a cis-acting DNA sequence (the chromosomal dif site or the ColE1 cer site) and two trans-acting factors: the XerC and XerD recombinases, which belong to the lambda integrase family of tyrosine site-specific recombinases. In addition, in order to resolve plasmid multimers into monomers, two additional factors are required: the ArgR and PepA proteins. We have previously shown the presence of xerC and xerD genes (and their function) by Southern hybridisation and by in vivo recombination in a wide variety of Enterobacteriaceac. We have now cloned and sequenced the xerD gene of Proteus mirabilis using degenerate and inverse PCR methods. This gene encodes a tyrosine recombinase which is highly similar to the E. coli XerD recombinase, is capable of complementing an E. coli xerD mutant, and displays sequence-specific DNA binding activity.

A Tn5-derived mobile element has been constructed to identify genes and promoters related to pathogenesis and virulence in Pseudomonas syringae pv. phaseolicola. To enhance the rate of mutation this Tn5 derivative was constructed carrying a mutant transposase which was placed in cis to the transposable element, but just outside the inverted repeats, therefore eliminating secondary transposition and increasing the stability of the insertion. The new element also contains a promoterless cat (chloramphenicol acetyltransferase) gene as reporter to allow for positive selection of promoters being expressed under specific conditions. To facilitate cloning and manipulations in Escherichia coli, a ColE1 origin of replication has been included within the transposable element as well as the Mob region from the broad-host-range plasmid RP4, which allows this element to be efficiently mobilized by a triparental mating or by using an E. coli strain such as S17-1 to provide the tra functions. Sites for the rare cutters PacI and PmeI have also been included to facilitate locating the insertions on a PacI and/or PmeI physical map. This construction combines the properties of both a mobilizable plasmid and a transposon and therefore has been termed pTn5cat. It is almost the same size as the wild-type Tn5, 5877 bp, and has successfully been tested in P.s. phaseolicola and Xanthomonas campestris pv. campestris.

The random distribution of ColE1 plasmids between the daughter cells at cell division introduces large copy number variations. Statistic variation associated with limited copy number in single cells also causes fluctuations to emerge spontaneously during the cell cycle. Efficient replication control out of steady state is therefore important to tame such stochastic effects of small numbers. In the present model, the dynamic features of copy number control are divided into two parts: first, how sharply the replication frequency per plasmid responds to changes in the concentration of the plasmid-coded inhibitor, RNA I, and second, how tightly RNA I and plasmid concentrations are coupled. Single (hyperbolic)- and multiple (exponential)-step inhibition mechanisms are compared out of steady state and it is shown how the response in replication frequency depends on the mode of inhibition. For both mechanisms, sensitivity of inhibition is “bought” at the expense of a rapid turnover of a replication preprimer, RNA II. Conventional, single-step, inhibition kinetics gives a sloppy replication control even at high RNA II turnover rates, whereas multiple-step inhibition has the potential of working with unlimited precision. When plasmid concentration changes rapidly, RNA I must be degraded rapidly to be “up to date” with the change. Adjustment to steady state is drastically impaired when the turnover rate constants of RNA I decrease below certain thresholds, but is basically unaffected for a corresponding increase. Several features of copy number control that are shown to be crucial for the understanding of ColE1-type plasmids still remain to be experimentally characterized. It is shown how steady-state properties reflect dynamics at the heart of regulation and therefore can be used to discriminate between fundamentally different copy number control mechanisms. The experimental tests of the predictions made require carefully planned assays, and some suggestions for suitable experiments arise naturally from the present work. It is also discussed how the presence of the Rom protein may affect dynamic qualities of copy number control.

A model of ColE1 copy number control has been developed where molecular details of replication are connected both to segregational stability and metabolic burden. Efficient replication control reduces copy number variation and increases segregational stability for a given average copy number. Copy number variation is predicted to depend on the type of inhibition mechanism as well as RNA I and RNA II turnover rate constants. It is shown that when both RNA I and RNA II transcription frequencies and the rate constant for degradation of free RNA I are very large, a hyperbolic inhibition mechanism must compensate with a 1.4 times greater average copy number to obtain the same segregational stability as an exponential inhibition mechanism. How sensitively the replication frequency responds to changes in RNA I concentration depends on the type of inhibition mechanism and the number of attempts to form an RNA II replication primer per plasmid and cell cycle. If RNA I is too stable, it will not follow changes in plasmid concentration closely, and when the transcription frequency for RNA I is only slightly higher than for RNA II, RNA I concentration becomes randomized. In both these cases, the proportionality between the single cell RNA I and plasmid concentrations is lost and this impairs copy number control. Thresholds in the rate for degradation of free RNA I as well as in RNA I and RNA II transcription frequencies have been computed, where an increase in these rate constants has a negligible effect on segregational stability but a corresponding decrease leads to segregational disaster. This indicates that there exists a well defined optimal set of rate constants where the regulation system works well without excessive metabolic load. A number of new experiments are suggested to address features of particular importance for the evolution of ColE1 copy number control.

Pyren-1-ylboronic acid 4 and pyrene-1,6-diyldiboronic acid 5 are bound to supercoiled double-strand circular DNA plasmid ColE1 (colDNA) and effect its efficient photocleavage under visible light irradiation. Mechanistic studies show that the cleavage occurs according to the radical mechanism. The cleavage activity of monoacid 4 could be suppressed by the addition of D-fructose because of boronic acid–saccharide complexation. Diacid 5 is bound to colDNA more strongly than is monoacid 4 and effects its efficient photocleavage to yield Form III. The results show that diradical-forming diacid 5 is able to cleave DNA at two reaction sites, mimicking the cleavage mechanism of enediyne antitumor antibiotics.

In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori.

The Xer site-specific recombination system of Escherichia coli resolves both chromosome dimers and multimers of certain plasmids including those of ColE1. In this manner, Xer site-specific recombination contributes to the accurate distribution of circular chromosomes at cell division. Two related site-specific recombinases, XerC and XerD, are required for this process. The xerC and xerD genes of Salmonella typhimurium LT2 were isolated from libraries of LT2 genomic DNA by genetic complementation of E. coli Xer mutants. The putative proteins specified by the S. typhimurium genes can substitute for and are highly homologous to the corresponding proteins in E. coli. The distribution of amino acid dissimilarities differs, however, between pairs of cognate Xer proteins. The immediate genetic contexts of equivalent xer genes, i.e., in operons with genes of apparently unrelated function, are conserved between the two bacteria. This is the first description of the identification of a pair of functional homologues of the xerC and xerD genes of E. coli.

Departamento de Microbiologi´a II, Facultad de Farmacia, Universidad Complutense, E28040 Madrid, SpainReceived 10 December 1996; accepted 11 March 1997; Received by A.M. CampbellAbstractThe multicopy plasmid pFM366 was isolated from a virulent Salmonella enteritidis strain and was found to code for DNAmethylase activity (Iba´n˜ez and Rotger, 1993). The present work was aimed at characterizing the genetic organization andfunctional features of this 5.6 kb plasmid. We found pFM366 almost identical to the plasmid P4 isolated from Shigella sonnei,that encodes the SsoII restriction-modification system (Karyagina et al., 1993), and related to other ColE1-type plasmids.Examination of these plasmids revealed a common organization which suggests they were the result of similar recombinationalevents. The cytosine methylase of pFM366 is nearly identical to M·SsoII, whereas the gene encoding the restrictase homologousto R·SsoII is truncated and its product is inactive. The expression of the cytosine methylase encoded by pFM366 is stronglyaected by deletion of regions located upstream and downstream of its ORF, and is negatively controlled by the rpoS gene inEscherichia coli. The methylase activity encoded by pFM366 induces the SOS response, which could be responsible for theobserved delay in the growth of E. coli. © 1997 Elsevier Science B.V.Keywords: RNA I; Incompatibility; rom; cer; SsoII; SOS response; rpoS

The PriA protein of Escherichia coli provides a vital link between recombination and DNA replication. To establish the molecular basis for this link, we investigated the ability of PriA to target DNA substrates modelled on D-loops, the intermediates formed during the early stages of RecA-mediated recombination. We show that PriA binds D-loops and unwinds the DNA in reactions that rely on its ability to function as a helicase. The minimal structure that binds PriA is a duplex DNA molecule with unpaired single strands at one end, an arrangement likely to occur at a D-loop. It resembles features of the stem-loop formed by primosome assembly site (PAS) sequences in the DNA of bacteriophage phiX174 and plasmid ColE1, and which enable PriA to assemble active primosomes for the initiation of lagging strand synthesis. We suggest that PAS sequences may have evolved to mimic the natural D-loop target for PriA formed in the chromosome of E. coli during recombination and DNA repair. Genetic studies have revealed an interaction between PriA and RecG, a DNA helicase that drives branch migration of recombination intermediates. We therefore compared PriA and RecG for their ability to bind and unwind DNA. RecG, like PriA, binds D-loops and unwinds the DNA. However, it prefers branched structures with at least two duplex components. The possibility that it competes with PriA for binding recombination intermediates is discussed.

1-Pyrenylboronic acid is bound to supercoiled double strand circular DNA plasmid ColE1 and effects its efficient photocleavage under visible light irradiation: the cleavage reaction, which occurs according to the radical mechanism, can be suppressed by the addition of D-fructose because of boronic acid-saccharide complexation. © 1997 Elsevier Science Ltd.

Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein. The kil gene was fused with the stationary-phase promoter of the fic gene of E. coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed. Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase. Quasi-lysis and lethality were not observed. The primary effect of the induction of the kil gene was the overproduction of beta-glucanase. The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control. The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective. This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene.

Summary: The primary structure of a 2671 bp DNA fragment between the pla gene (encoding plasminogen activator) and the origin of replication of the wild-type Yersinia pestis plasmid pYP358 was determined. Two ORFs of 1074 and 426 bp with opposite transcription polarities were identified on both strands. They encode a 357 aa pesticin activity protein (Pst) and a 141 aa pesticin immunity polypeptide (Pim). A GC-rich palindromic structure located between pst and pim can form a hairpin loop and serve as a rho-independent transcription terminator sequence for both genes. The site for the interaction with the LexA repressor of the SOS system was found in another palindromic structure preceding the pst structural gene. A deduced 39.9 kDa Pst polypeptide is devoid of a signal peptide, indicating a Sec-independent mode of export. Pst carries a pentapeptide typical of TonB-dependent colicins (TonB box) that is necessary for the interaction with the yersiniabactin/pesticin receptor and for active energy-dependent transport through the outer membrane. The substitution of the last five C-terminal amino acids did not significantly influence the bactericidal activity of the truncated pesticin. The pesticin lost its ability to kill sensitive bacteria and to bind to a pesticin receptor after deletion of the last 57 C-terminal amino acids. A deduced 16 kDa Pim protein has an N-terminal hydrophobic amino acid stretch with features typical of prokaryotic signal peptides. Pim is a slightly hydrophilic protein with a basic pl. The immunity protein was localized in the periplasmic space and in the outer-membrane fraction after its overexpression under the polymerase T7 promoter. Several other ORFs were identified on the sequenced 2671 bp fragment, but none of them seemed to encode a typical lysis peptide, which is necessary for the release of the pesticin. In the promoter region and in the regions preceding and following the pst operon, the DNA sequence has high (< 70%) identity with other colicin genes. The DNA sequence located 284 bp upstream of the pim gene showed more than 90% similarity to antisense RNA I of the ColE1 replicon. This defined the location of the pYP358 origin of ColE1-type replication.

Production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation. The combined effect of the single base-pair change and the elevated growth temperature produced an overall yield of 2.2 grams of plasmid DNA available for recovery from a 10-liter fed-batch fermentation compared to 0.03 grams from a 10-liter batch fermentation, a 70-fold increase in yield. The plasmid DNA isolated from this process contained lower levels of RNA and chromosomal DNA contaminants, simplifying downstream processing.

The cer-Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat. Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA. The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity. This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint. We describe here the characterization of a novel class of "conditionally constrained' multimer resolution sites whose properties support this hypothesis. In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen. A mutant ArgR derivative (ArgR110), which is defective in cer-mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site. This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution. When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non-integral number of helical turns, the site became unconstrained. Constraint was restored by the insertion of a full helical turn.

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5'-end, and a 31-mer which has 11 5'-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5' end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32 degrees C and 71 degrees C clearly discernible. In conjunction with NMR melting study the major transition at 71 degrees C is assigned to the overall melting of the stem region and the 32 degrees C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.

Initiation of replication of the plasmid ColE1 is primed by the cis-acting RNA II. Copy numbers are regulated by inhibition of RNA II by the antisense RNA I, whose concentration is proportional to the plasmid concentration. This inhibition is enhanced by a protein. Rom, and takes place during a time set by the transcription of 250 bases of the gene for RNA II. When this transcription is dominated by several steps of about equal duration, the probability for RNA II to prime DNA replication is approximately determined by e-constant[RNA I]. For large values of the "constant" small changes in [RNA I] give large variations in the priming probability. It is shown, first, that this type of mechanism can reduce the rate of plasmid loss and enable single copies of ColE1 to duplicate at a well-defined time in the cell cycle; second, that when the rate of initiation of transcription of RNA II increases, plasmid losses decrease and the distribution of single copy duplication times becomes narrower; third, that the action of Rom may further reduce plasmid losses and further narrow the distribution of duplication times in the single-copy case.

To detect entry exclusion of the ColE1 plasmid, we established an assay system that was not influenced by incompatibility of extant plasmids in the recipient cells or by the viability of the cells due to the killing action of colicin E1 protein. The assay revealed that exc1 and exc2, assigned as genes directing entry exclusion, had no exclusion activity. Instead, mbeD, which had been characterized as a gene for plasmid mobilization, directed the exclusion activity. MbeD was overexpressed and identified as a 35S-labeled protein, which was recovered in both the soluble and membrane fractions, particularly in the inner membrane fraction. An amphipathic helical structure was predicted in the N-terminal region of MbeD as well as in the corresponding homologous proteins of ColA and ColK. These proteins may bind to the inner membrane via the N-terminal amphipathic helix and function in entry exclusion.

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.

The nucleotide sequence of the smallest plasmid ofErwinia stewartiiSW2 was determined. This plasmid, pSW100 (4272 bp), consists of a 702-bp region homologous to the origins of replication of plasmids p15A, ColE1, and ColA. Plasmid pSW100 also contains sequences homologous to thebomregion andmobCABDgenes of ColE1, except that a single-base deletion inmobAhas been detected. This deletion did not affect the mobilization ability of pSW100 by an endogenous conjugative plasmid ofE. stewartiiSW2, pDC250. Plasmid pSW100 also has a 1596-bp open reading frame with five 132-bp perfect repeats. However, this open reading frame and the repeats are not required for plasmid replication. Furthermore, curing of pSW100 did not cause any phenotypic change, suggesting that this plasmid is not essential for the survival of the organism.

Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, P E , a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. Hy R transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT , the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Streptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.

The initial interaction between the ColE1 plasmid specific transcripts RNA I and RNA II, which function as antisense regulators of plasmid replication, comprises a transient complex between complementary loops found within the RNA secondary structures. Multidimensional heteronuclear magnetic resonance spectroscopy was used to characterize complexes formed between model RNA hairpins having seven nucleotide complementary loops. Seven base pairs are formed in the loop-loop helix, with continuous helical stacking of the loop residues on the 3' side of their helical stems. A sharp bend in the loop-loop helix, documented by gel electrophoresis, narrows the major groove and allows bridging of the phosphodiester backbones across the major groove in order to close the hairpin loops at their 5'-ends. The bend is further enhanced by the binding of Rom, a ColE1 encoded protein that regulates replication.

RNAII, an RNA species encoded by ColE1-type plasmids, serves as a primer for plasmid DNA replication. Previous work has shown that overproduction of RNAII transcribed by Escherichia coli RNA polymerase results in elevated plasmid copy number. To produce a plasmid in which the elevation of its copy number is inducible, we placed transcription of RNAII under the control of a bacteriophage T7 late promoter regulated by IPTG-inducible T7 RNA polymerase. During induction of T7 RNA polymerase by IPTG, we found that RNAII was overexpressed, but that, surprisingly, this increase in RNAII did not result in elevation of plasmid copy number. These results suggest that RNAII transcribed by T7 RNA polymerase does not function as a primer for plasmid DNA replication. Since RNAII function requires folding of its multiple stem-loop structures in a precise conformation and folding of RNAII can be influenced by its rate of transcription, the extremely rapid rate of travel of the T7 RNA polymerase may preclude proper folding of RNAII during its elongation.

Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRm1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction of the ori T region from pRK2, a broad-host-range plasmid. The vector plasmids transferred readily into a wide range of gram-negative bacteria and had relatively low copy number in R. meliloti; two constructs, pFD1001 and pFD1212, were completely stable in R. meliloti isolated from nodules of alfalfa (Medicago sativa). A representative of the vector constructs (pFD1001) could be maintained in R. meliloti in the presence of the broad-host-range shuttle plasmid pRK290. These two vector plasmids could be introduced into R. meliloti, either simultaneously or singly when pRK290 was the resident plasmid; however, entry of pRK290 was blocked when pFD1001 was the resident plasmid. The cloning vectors constructed in this study should prove to be useful for the genetic manipulation of Rhizobium.

Two quantitative models of plasmid ColE1 copy number control arecompared with respect to mathematical logic of derivation and application to experimental observations. Explanatory background material and clarifications are supplied for selected aspects of each model. Contrasting features are emphasized and experiments are suggested to distinguish between predictions of the models.

A three-dimensional 1H, 13C, 31P triple resonance experiment, HCP-CCH-TOCSY, is presented which provides unambiguous through-bond correlation of all 1H ribose protons on the 5' and 3' sides of the intervening phosphorus along the backbone bonding network in 13C-labeled RNA oligonucleotides. The correlation of the complete ribose spin system to the intervening phosphorus is obtained by adding a C,C-TOCSY coherence transfer step to the triple resonance HCP experiment. The C,C-TOCSY transfer step, which utilizes the large and relatively uniform 1J(C,C) coupling constant (approximately 40 Hz for ribose carbons), efficiently correlates the phosphorus-coupled carbons observed in the HCP correlation experiment (i.e., C4' and C5' in the 5' direction and C4' and C3' in the 3' direction) to all other carbons in the ribose spin system. Of the additional correlations observed in the HCP-CCH-TOCSY, that to the relatively well-resolved anomeric H1',C1' resonance pairs provides the greatest gain in terms of facilitating assignment. The gain in spectral resolution afforded by chemical shift labeling with the anomeric resonances should provide a more robust pathway for sequential assignment over the intervening phosphorus in larger RNA oligonucleotides. The HCP-CCH-TOCSY experiment is demonstrated on a uniformly 13C, 15N-labeled 19-nucleotide RNA stem-loop, derived from the antisense RNA I molecule found in the ColE1 plasmid replication control system.

Bacterial plasmids play important role in gene transport between cells both vertically and horizontally, and are also versatile tools for gene engineering. Their maintenance of inherence in the host cells is believed to depend on the delicate quantity control through the replication origin and the effective management of partition. Without active partition system, high-copy number plasmids are suggested to be distributed as multiple foci but randomly partitioned during cell division. Lack of the direct evidence, the detail mechanism and dynamics of plasmid location and partition in cell is unknown. Taking the advantage of temporary tolerance on the incompatibility of replication origin, two ColE1 derivative plasmids with different antibiotic resists and repressible replication origin are co-transformed and their distributions and motions are observed at single cell level simultaneously. Based on the nature of competition between plasmids, it also provides the opportunity to actually reveal a single plasmid translocation between multiple foci, which is affected by the nucleoids of the dividing cells. Also, the variance of the inhered number of copies for both of two plasmids from parent to daughter cells provides the insight of partition mechanism.

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. C. burnetii 's former obligate intracellular nature significantly impeded the genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the present study, we describe two methods for generating targeted gene deletions in C. burnetii that exploit pUC/ColE1 ori -based suicide plasmids encoding sacB for positive selection of mutants. As proof of concept, C. burnetii dotA and dotB , encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre- lox- mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5′ and 3′ flanking regions of dotA . Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in the deletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop-out strategy to delete dotA and dotB. A single suicide plasmid was first integrated into 5′ or 3′ target gene flanking regions. Resolution of the plasmid cointegrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. Δ dotA and Δ dotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of C. burnetii genetic tools now allows ready fulfillment of molecular Koch's postulates for suspected virulence genes.

Salmonella enterica serovar Typhimurium JOL401 was isolated from chicken in South Korea. A novel plasmid, pSW, harbored in JOL401, consists of 2,216 base pairs (bps) and three putative open reading frames (ORFs). pSW was 86 and 82% homologous to pSGI15 from Salmonella enterica and pJHCMW1 from Klebsiella pneumonia, respectively, both of which are ColE1-type plasmids that undergo theta-type replication. ORF1 of pSW exhibited 98% identity and 99% positive to ORF1 of pJHCMW1, which encodes a putative transmembrane protein. ORF2 of pSW was homologous to sequences that encode replication initiators in pSGI15 and pJHCMW1, exhibiting a high degree of homology to the putative replication initiator of pJHCMW1. Thus, ORF2 appeared to encode the pSW replication initiator. ORF3 of pSW, which exhibited similarity to ORF2 of pSGI15, remains undefined. Newly isolated pSW is a member of the ColE1-type family of plasmids and consists of three putative ORFs, of which one, ORF2, is a strong candidate for the gene encoding the pSW replication initiator. Key words: pSW, plasmid, ColE1, replication initiator, pSGI15, pJHCMW1

We examined DNA sequence polymorphism for the colicin gene clusters of seven ColE1 and six ColIa plasmids obtained from natural isolates of Escherichia coli. These gene clusters harbor levels of nucleotide diversity ranging from 0.006 (ColIa) to 0.054 (ColE1). This level of diversity is similar to that observed for chromosomally encoded E. coli genes. However, the variance associated with these estimates is severalfold higher for the plasmid-encoded genes. This increased variance may be due to the differing plasmid population sizes. The pattern of colicin gene cluster polymorphism suggests that the two colicins are evolving in different fashions. ColE1 accumulates polymorphism at an elevated rate in the central domain of the colicin protein, while ColIa polymorphism is distributed evenly along the gene cluster. Comparison of the patterns of divergence between colicin and related proteins of ColIa and Ib and patterns of polymorphism within ColIa suggest that this gene cluster is not evolving in a neutral fashion. These data lend support to the hypothesis that colicin gene clusters may evolve under the influence of diversifying selection.

A new family of T7-based expression plasmids with unique features is described. The plasmid origin of replication (ori), derived from P15A, is compatible with that of ColE1-derived plasmids, which facilitates the co-production of proteins from these vectors and from ColE1-derived T7 expression vectors in the same cell. The plasmids are medium-copy-number and also carry the M13 ori. Consequently, both double- and single-stranded DNA can be easily obtained. The plasmids encode KmR, thus avoiding the potential for plasmid loss associated with ApR-based systems. One of the plasmids carries the lacI gene, to allow for more stringent regulation of the production of potentially toxic proteins. When the plasmids are introduced into an Escherichia coli strain such as BL21(DE3), which contains the T7 polymerase-encoding gene under control of the lacUV5 promoter, addition of IPTG initiates the production of high levels of the recombinant protein.