Site-specific recombination system from Tn1000(γδ) stabilises recombinant plasmids in Escherichi coli

The resolution system from Tn1000 was cloned into a pBR322 recombinant plasmid carrying E. coli threonine operon. The resulting plasmid displayed a significant increase in segregational stability with respect to the original plasmid, either in a rec or a rec E. coli strain (100% of plasmid-containing cells versus 64% and 1% respectively). The Tn1000 resolution system proved to be responsible for the stabilising effect exerted by the transposon when present in recombinant plasmids.     

Persistence of pBR322-related plasmids in Escherichia coli grown in chemostat cultures

Abstract The stability of plasmid pBR322 and a number of close derivatives was examined by continuous culture of Escherichia coli . Cultures were subjected to either glucose, phosphate or magnesium limitation in non-selective medium at a dilution rate of 0.1/h. Under these conditions pBR322 was eventually lost from the population, but only after a distinct lag period. The closely related plasmids pBR325 and especially pBR327 and pBR328, but not pAT153, were lost more rapidly. Three cosmids pHC79, pSJ55 and pJB8 were generally found to be less stable than the pBR322-type plasmids from which they were derived. Chimaeric plasmids containing DNA from yeast and from a thermophilic bacillus were also unstable in E. coli .     

Mutagenesis of dimeric plasmids by the transposon gamma delta (Tn1000).

The Escherichia coli F factor mediates conjugal transfer of a plasmid such as pBR322 primarily by replicative transposition of transposon gamma delta (Tn1000) from F to that plasmid to form a cointegrate intermediate. Although resolution of this cointegrate always yields a plasmid containing a single gamma delta insertion, the occasional recovery of transposon-free plasmids after conjugal transfer has led to alternative hypotheses for F mobilization. We show here that gamma delta-free plasmids are found after F-mediated conjugal transfer only when the donor plasmid is a dimer and the recipient is Rec+.     

Improvement in segregational stability of recombinant plasmids by retransformation of E. coli host cells

Summary Strains of E. coli JM103 harboring recombinant pUC8 plasmids were found to exhibit severe segregational instability in the absence of antibiotic selection. This was reversed by retransformation into fresh JM103. pUC8 was considerably more stable than its recombinant derivatives.     

Ribitol dehydrogenase overproduction and maintenance of plasmids pBR322 and pACYC184 in chemostat cultures of Escherichia coli

Summary The maintenance of multicopy plasmids pBR322 and pACYC184 was studied in chemostat cultures subjected to limitation by glucose, ribitol or xylitol at D of 0.1 per h. While carbon source-dependent segregational stability of pBR322 was observed, no dependence and greater instability of pACYC184 was found. Plasmid-free and plasmid-bearing strains overproducing chromosomally coded ribitol dehydrogenase were found in pentitol-limited chemostat cultures.     

Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000

A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.     

Analysis of Tn5 inversion events inEscherichia coli plasmids

The ability of the bacterial transposon Tn5 to undergo sequence inversion in Rec⁺ Escherichia coli cells as a result of recombination between its duplicated IS50 elements was examined using specially designed plasmid constructs. Surprisingly, recombination events in the IS50 elements that led to crossover and therefore Tn5 inversion could be detected at a frequency of only 10^?5. This was approximately an order of magnitude lower than the frequency of IS50 recombination that led to conversion events (i.e. non-reciprocal recombination) without crossover, and at least two orders of magnitude lower than the frequency of intermolecular recombination between IS50 elements on two different plasmids. These rare conversion and inversion events in Tn5 appeared to be due to intramolecular recombination and not simply to multiple rounds of reciprocal crossing over, since the heterodimeric intermediates that would be generated during the latter process could be readily isolated but were shown to yield a completely different set of plasmid products upon resolution.     

Polarity of Tn5 insertion mutations in Escherichia coli.

We assessed the effect of insertions of the kanamycin resistance transposon Tn5 in the lac operon of Escherichia coli on the expression of distal genes lacY and lacA (melibiose fermentation at 41 degrees C and thiogalactoside transacetylase synthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 in lacY) was strongly polar. However, approximately one-third of the insertion mutants expressed distal genes at low levels due to a promoter associated with Tn5. To localize this promoter, we (i) reversed the orientation of Tn5 at several sites and (ii) replaced wild-type Tn5 with several substitution derivatives which lack Tn5's central region. Neither alteration changed the expression of distal genes. Thus, in contrast to transposons IS2 and TnA. Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon. Our results suggest that the promoter is within 186 base pairs of the ends of Tn5. It is possible that the promoter is detected in only a fraction of insertions because it overlaps Tn5-target sequence boundary.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

Increased stability of pBR322-related plasmids in Escherichia coli W3101 grown in carrageenan gel beads

Abstract The stability and the copy number of pBR322, pBR325 and pBR328 were studied during continous cultures of free and immobilized E. coli W3101 without selective pressure. In the free-cell system, it was found that pBR328 and pBR325-free E. coli cells appeared after a lag period. They rapidly overgrew the cultures and the plasmid copy number subsequently declined. On the other hand, an increase in the proportion of pBR322- carrying cells during a free continuous culture was observed. This increase correlated with that of plasmid copy number. By contrast, in the immobilized- cell system, plasmid free segregants were not detected in all the cases even after 250 generations. We have also shown that plasmid copy number remained constant and phenomena such as fluctuations or genetic modifications which occured after long term growth of bacteria in a free continuous culture could be avoided throughout cell immobilization.     

Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat

Summary The maintenance and genetic stability of the vector plasmids pBR322 and pBR325 in two genetically different Escherichia coli hosts were studied during chemostat cultivation with glucose and ammonium chloride limitation and at two different dilution rates. The plasmid pBR322 was stably maintained under all growth conditions tested. However pBR325 segregated from both hosts preferentially during glucose limitation and at low dilution rate. In addition to this general segregation process a separate loss of tetracycline resistance was observed. The remaining plasmid conferred resistance to ampicillin and chloramphenicol only, without any remarkable alteration of its molecular weight.Cultivation conditions in the chemostat were found that allowed the stable genetic inheritance of both plasmids in the hosts studied.     

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.     

Regulation of bistability in glucose metabolism of Escherichia coli ML 30 chemostat cultures by cyclic AMP

Evidence is presented that cyclic AMP is engaged in the regulation of a bistability in the glucose and energy metabolism of NH3-limited chemostat cultures of Escherichia coli ML 30. Cyclic AMP probably reverses the repression of the citric acid cycle by glucose favouring the state of glycogen and energy overproduction.     

The use of the immobilization of whole living cells to increase stability of recombinant plasmids in escherichia coli

Immobilization of whole living cells was used as an experimental approach to enhance plasmid stability in cultured recombinant micro-organisms. pTG201 plasmid which is very unstable in continuous cultures with free cells, was found to be extremely stable in continuous cultures with immobilized cells.To elucidate the mechanism by which immobilization increases the plasmid stability, we analyzed the growth of pTG201-containing E. coli W3101 cells within the gel beads. We found that in immobilized continuous culture, plasmid-free segregants were not detected even after 240 generations. This appears to be due to the mechanical properties of the gel-bead system that allow only a limited number of cell divisions (10–16) to occur in each clone of cells before the clone escapes from the gel bead. This number of generations is not sufficient for the plasmid-free cells to appear within the cavities compared to what was observed in a free-system (plasmid-free segregants were detected after a lag period of approximately 25–30 generations). Even when they appear, they cannot overcome the culture. From the data described in this paper we conclude that cells released from the gel beads at any time during continuous culture are cells which are issued from cells grown in the cavities for only 10–16 generations.