Effect of cycling on the stability of plasmid-bearing microorganisms in continuous culture

The implication of the possible existence of differences in the times required for plasmid-bearing and non-plasmid-bearing microorganisms to adjust their metabolic activities to step changes in their environment is examined. This adaptability difference suggests the possibility of maintaining an engineered strain in continuous culture by transient operation. It is shown for the case where adaptability is neglected that no cycling strategy will prevent the washout of the engineered strain, but the addition to the model of a time delay in substrate utilization can result in coexistence upon cycling. Numerical simulations of cycling in feed substrate concentration are carried out to illustrate the concept Operating diagrams are also constructed to indicate the conditions under which washout of the plasmid bearing strain can be prevented.     

Analysis of continuous cultures of recombinant methylotrophs

Static and dynamic characteristics of continuous cultures of recombinant methylotrophs, which are designed to improve the selectivity of plasmid-bearing cells and the plasmid stability, are investigated in detail. Operational regions in which coexistence (survival of plasmid-bearing and plasmid-free cells) operation is feasible have been identified in the entire space of kinetic parameters and operating variables. The stability characteristics of each steady state are examined. The existence of oscillatory states around the coexistence steady state is investigated using the dynamic (Hopf) bifurcation analysis. For proper startup of the continuous culture operation, it is critical to identify the sets of initial conditions, if any, which lead to transients that ultimately result in washout of plasmid-bearing cells and avoid such conditions. For the numerical illustrations presented, the coexistence steady state happens to be locally stable over much of its region of existence, particular for the operating conditions corresponding to maximum productivity.     

Estimating the instability parameters of plasmid-bearing cells. I. Chemostat culture

What determines the stability of plasmid-bearing cells in natural and laboratory conditions? In order to answer this question in a quantitative manner, we need tools allowing the estimation of parameters governing plasmid loss in different environments. In the present work, we have developed two methods for the estimation of the instability parameters of plasmid-bearing cells growing in chemostat. These instability parameters are: (i) selection coefficient (or cost of the plasmid)alpha and (ii) the probability of plasmid loss at cell division tau(0). We have found that generally selection coefficient alpha changes during elimination of plasmid-bearing cells due to changes in substrate concentration; hence, methods which assume constancy of alpha are intrinsically imprecise. Instead, one can estimate selection coefficient at the beginning and the end of cultivation when the substrate concentration is approximately constant. Applying developed techniques to two sets of experimental data, we have found that (i) the cost of the plasmid pBR322 depended on the dilution rate in chemostat and was higher at low dilutions; (ii) high levels of plasmid gene expression led to a high cost of the plasmid pPHL-7; (iii) the probability of plasmid loss was lower at high levels of plasmid gene expression and independent of the dilution rate. We have also discussed the application of our results to understanding the basic biology of bacterial plasmids.     

Plasmid maintenance and protein overproduction in selective recycle bioreactors

A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid-bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid-encoded protein beta-lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid-bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid-bearing cells by separating flocculent, plasmid-bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid-bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions.     

Continuous recombinant bacterial fermentations utilizing selective flocculation and recycle.

Selective recycle has successfully been used to maintain an unstable plasmid-bearing bacterial strain as dominant in a continuous reactor, whereas the culture reverts to 100% segregant cells when selective recycle is not used. The plasmid-bearing strain is slower growing and flocculent; however, when the cells lose their plasmid, the resulting segregant cells are nonflocculent and grow at a faster rate due to their decreased metabolic burden. Both types of cells exit a chemostat and enter an inclined settler where the flocculent plasmid-bearing cells are separated from the nonflocculent segregant cells by differential sedimentation. The underflow from the cell separator, which is enriched with plasmid-bearing cells, is recycled back to the chemostat, while the segregant cells are withdrawn off the top of the settler and discarded. The experimental results agree well with selective recycle reactor theory. On the basis of the theory, a criterion is presented that has been shown to successfully predict whether or not a selective recycle reactor can maintain a plasmid-bearing strain.     

A mathematical method for analysing plasmid stability in micro-organisms

A mathematical model describing the instability of plasmids in micro-organisms has been developed. The model is based on the assumption that the overall causes of plasmid instability are described by the segregational instability of the plasmid, R (i.e. the rate at which plasmid-free cells are generated from plasmid-bearing cells), and the growth rate difference, d mu (i.e. the difference in growth rate between plasmid-free and plasmid-bearing cells). A method for determining the values of R and d mu (accompanied by 95% confidence limits) for any plasmid-bearing micro-organism is described. This method is based on the observation that, depending on the plasmid, various exponential patterns of plasmid instability are observed. The stability of Escherichia coli 1B373(pMG169), where d mu much greater than R, and E. coli RV308(pHSG415), where R much greater than d mu, are analysed in order to demonstrate the method.     

Plasmid maintenance in Escherichia coli recombinant cultures is dramatically, steadily, and specifically influenced by features of the encoded proteins

A set of eight closely related plasmid constructs carrying CI857-controlled recombinant genes has been used as a model to study plasmid stability in Escherichia coli, in the absence of antibiotic selection. Plasmid loss rates and relative interdivision times of plasmid-bearing cells and plasmid-free cells have been analyzed throughout prolonged cultures. Whereas the calculated plasmid loss rates are not consistent for a given plasmid and set of conditions, the relative growth fitness of plasmid-bearing cells is highly reproducible. In the absence of gene expression, plasmid maintenance is influenced by the length of the cloned segment, the growth temperature, and the plasmid copy number, but not by the plasmid size. At high, inducing temperatures, the effects of the metabolic burden are eclipsed by the toxicity exhibited by the different proteins produced, which is determined by structural features. Despite the multifactorial nature of the negative pressures acting independently on plasmid-bearing cells, the relative cell fitness in a mixed cell population is very reproducible for a given vector, resulting in a monotonous spread of the plasmid-free cells in recombinant cultures.     

Induction of CAT gene expression on a plasmid vector (L factor) by retinoic acid in mouse embryonal carcinoma (F9) cells

We reported previously that composite DNA constructed from a mammalian plasmid (L factor) and foreign gene can be reestablished as a plasmid in mouse embryonal carcinoma (F9) cells after transfection and the plasmid-bearing F9 cells undergo normal in vitro differentiation in response to retinoic acid, an inducer for F9 cell differentiation. We constructed F9 cells bearing plasmidal L factor DNA in which a reporter (chloramphenicol acetyltransferase; CAT) gene was placed under the control of a differentiation-responsive viral (Moloney murine leukemia virus or simian virus 40) enhancer-promoter. When such plasmid-bearing cells were treated with retinoic acid, the CAT gene was inducibly expressed. These results indicate that mammalian gene expression can be studied with the plasmidal expression vector which is structurally dissociated from complex chromosomes.     

Experimental Infection of Mice with Yersinia enterocolitica Serotype O9 by Oral and Parenteral Routes: Spreading and Enterotropism of Virulent Yersiniae

An isogenic pair of Yersinia enterocolitica serotype O9 strains, with and without virulence plasmid, was used to study the plasmid role in the infection of BALB/c mice by oral, intraperitoneal, and intravenous routes. The plasmid-bearing strain, but not its plasmid-less derivative, caused enteric infection after challenge by all three routes. The virulence plasmid did not influence the peritoneal clearance of yersiniae, but only the plasmid-bearing yersiniae were able to move from the peritoneal cavity to the bloodstream, and thus they spread to spleen and liver. Moreover, plasmid-bearing yersiniae were able to move from the liver to the gallbladder, and they shed in bile into the intestine. Western blot analysis of antibody responses to chromosomally encoded outer membrane proteins revealed similar patterns with sera from mice challenged with each one of the two strains by intraperitoneal route. In contrast, only the plasmid-bearing strain elicited an antibody response to these antigens in mice challenged by oral route. Although mice experimentally infected with plasmid-bearing O9 yersiniae developed an enteric infection, irrespective of the inoculation route, differences between the first steps in infection by oral and parenteral routes may be important, especially when the infection model is used as an approach to study the yersinia-host interactions. Received: 31 August 1998 / Accepted: 13 November 1998     

Identification of environmental plasmid-bearing Vibrio species isolated from polluted and pristine marine reserves of Hong Kong, and resistance to antibiotics and mercury

Fifty environmental isolates of Vibrio species were isolated from water samples of Mai Po Nature Reserve and the Cape d’Aguilar Marine Reserve in Hong Kong and screened for the presence of plasmid. Mai Po is a wastewater-impacted area while the Cape d’Aguilar Marine Reserve is pristine natural marine water. Plasmid was found in Vibrio isolates from both sites at similar frequencies and each site showed distinctive plasmid profiles. These plasmid-bearing Vibrio isolates were identified as different species of the Vibrio genus by both biochemical test and subsequently full-length 16S rRNA sequences. Antibiotic resistance test showed that all these plasmid-bearing Vibrio isolates showed multiple resistance to 21 antibiotics tested. In addition, selective isolates also showed tolerance to 10 M Hg²⁺ in culture medium and they generally harbored large plasmid(s) (>‰30 kb). Our results show that the high frequency of plasmid in Vibrio species of both polluted and pristine environments may be ecologically important to the survival of these bacteria in the environment. The specific functioning of the cryptic plasmids remains the focus of current investigations.     

Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.     

Plasmid dynamics in a model soil column

Continuous-flow column reactors were used to study the dynamics of plasmid exchange in a structured, thermodynamically open system containing either Enterobacter cloacae or Pseudomonas cepacia , both carrying the transmissible recombinant plasmid R388::Tn1721. Plasmid transfer rates were higher in vermiculite and sterile soil columns supplied with nutrient solution than those in sterile and non-sterile soil columns without input of nutrient solution. For both species, donor and recipient strains took about 5 days to reach their maximum densities in effluents from the columns supplied with nutrient solution. After about 8 day s the donor and transconjugant populations of P. cepacia in the effluent solution decreased exponentially, whereas E. cloacae donor, recipient and transconjugant strains maintained steady-state concentrations. The difference between plasmid stability in the two species may have significant consequences in terms of releasing plasmid-bearing genetically modified microorganisms into the natural environment. The plasmid is persistent in E. cloacae in non-sterile soil even though its transfer to the marked recipient in non-sterile soil was minimal.     

Homology among nearly all plasmids infecting three Bacillus species.

We have surveyed naturally occurring plasmids in strains of Bacillus subtilis and the closely related species B. mojavensis and B. licheniformis. Previous studies have failed to find host-benefitting functions for plasmids of these species, suggesting that these plasmids are nonmutualistic. Only one type of plasmid was found in each plasmid-bearing strain, suggesting that most of the plasmids infecting these Bacillus species are in the same incompatibility group. A sample of 18 plasmids from these species ranged in size from 6.9 to 16 kb, with all but 6 plasmids falling into three size groups. These groups differed in the sizes of their host ranges and geographical ranges. All but 1 of the 18 plasmids from these three host species are homologous with one another. The cryptic plasmids from these three species are far less diverse than are plasmids (from other species) that are known to benefit their bacterial hosts. The low-level diversity among these cryptic plasmids is consistent with the hypothesis that host-benefitting adaptations play an important role in fostering the coexistence of plasmid populations, but other explanations for the low-level plasmid diversity are possible. Comparison of the phylogenies of the plasmids with those of their hosts suggests that Bacillus plasmids are horizontally transferred in nature at a low rate similar to that found for the colicin plasmids of Escherichia coli.     

Production of an immunoglobulin gene product by the plasmid expression vector L factor in mouse myeloma cells.

L factor, originally discovered in a subclone of mouse L cells, is a multicopy mammalian plasmid whose structure is related to that of polyoma. When a composite DNA consisting of L factor, pBR, bacterial neo, and an immunoglobulin (kappa) gene was introduced into mouse myeloma cells, the DNA was established as plasmids in the cells without rearrangement or integration into the chromosomes. The plasmid-bearing myeloma cells produced kappa mRNA and the gene product, kappa immunoglobulin, which were apparently derived from the gene located on plasmid L factor. These results suggest that L factor can be used as a plasmid expression vector for studies on gene expression and production of biologically active substances in mammalian cells.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Survival of genetically-engineered and wild-type strains of the yeast Saccharomyces cerevisiae under simulated environmental conditions: a contribution on risk assessment

H. FUJIMURA, Y. SAKUMA AND E. AMANN. 1994. A genetically-engineered strain of Saccharomyces cerevisiae employed for the industrial production of the human coagulation Factor XIIIa (rhFXIIIa) was used for a survival study under simulated environmental conditions. The homologous strain devoid of the recombinant plasmid and the homologous strain bearing the 2 μm-based vector plasmid without the rhFXIIIa-encoding DNA insert were compared. The strains were introduced into natural soil/water suspension, into soil/medium suspension and into waste water. After intervals, samples of cell suspensions were taken and viable cell numbers were determined by plating on antibiotic-containing medium. In addition, a non-radioactive technique involving enhanced chemiluminescence was employed to detect plasmid-bearing yeast cells. The rhFXIIIa expression plasmid showed a high stability during the simulated environmental condition. No differences in survival rates, however, could be detected for the plasmid-bearing and plasmid-less strains under the three conditions tested, suggesting that the presence of plasmid does not confer selective advantages on the survival of the yeast cells. It is concluded that, even after accidental release of the engineered yeast cells into the environment, elimination rates would be comparable to those for non-recombinant yeast strains.     

Physical evidence for plasmids in autotrophic,especially hydrogen-oxidizing bacteria

Agarose gel electrophoresis of crude lysates from 23 species of autotrophic bacteria revealed plasmids of various sizes in 12 species. The plasmid pattern varied considerably. While the majority of the plasmid-bearing species harbored one or two plasmids, one species, Alcaligenes latus, exhibited more than six ccc-DNA bands. With one exception the molecular masses of the plasmids were 50×10⁶ or higher. In Achromobacter carboxydus, Alcaligenes latus, Derxia gummosa and three strains of Paracoccus denitrificans large plasmids of molecular masses higher than 300×10⁶ were resolved. The examination of Thiobacillus A2 resulted in the discovery of two plasmids while Pseudomonas oxalaticus was apparently free of resident plasmid DNA. So far these plasmids can only be characterized as cryptic. Future studies may allow to correlate them with specific metabolic activities of their hosts such as the ability to grow on carbon monoxide or thiosulfate, to fix molecular nitrogen and to form soluble NAD-reducing and/or membrane-bound hydrogenases.     

The effect of temperature and selective agents on the growth of Yersinia enterocolitica serotype O:3 in pure culture

This study examined the individual and combined effects of the selective agents normally present in Yersinia-selective agar (i.e. cefsulodin, irgasan and novobiocin) on the growth kinetics of plasmid-bearing (P+) and plasmid-cured (P-) Yersinia enterocolitica serotype O:3 at 25 and 37 degrees C. Growth studies were carried out in pure culture, and the data obtained were subjected to linear regression analysis to determine lag phase duration(s) and growth rates of the examined strains. In general, the presence of selective agents increased the duration of the lag phase at 37 degrees C, with longer lag phases noted in all cases in which two or more selective agents were present. Growth rates in CIN broth base (CIN NA) and CIN NA plus commercial supplement (SR 109) (CIN) were faster at 37 than 25 degrees C, but in some cultures of incomplete CIN NA broth with less than three supplements added, growth tended to be faster at 25 than 37 degrees C. Generally, plasmid-bearing strains grew slower than plasmid-cured strains in most media at 37 degrees C due to virulence plasmid expression retarding growth. In some instances at 37 degrees C, it was observed that the growth rates of both plasmid-bearing and plasmid-cured strains were comparable, indicating the influence of added selective agent/s negating any effects associated with virulence plasmid expression. The effects of selective agents, incubation temperature and virulence plasmid carriage on the growth kinetics of Y. enterocolitica are discussed.     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.