Plasmid-free Cells Research Articles

Post-segregational killing by restriction modification gene complexes: Observations of individual cell deaths

Through a mechanism known as post-segregational killing, several plasmids mediate their stable maintenance by carrying genes that kill plasmid-free segregant cells. We demonstrated earlier that loss of plasmids carrying type II restriction modification (RM) gene complexes inhibits the propagation of a cell population and causes chromosome breakage. We now show the morphology of individual cells changes following loss of thermosensitive plasmids carrying EcoRI RM or PaeR7I RM after a shift to a non-permissive temperature. After a lag, many cells formed long filaments containing multiple nuclei as detected by DAPI staining. Several hours after the shift, many of these long filaments lacked nuclei. Fragmentation of chromosomal DNA down to 5 kb was detected by electrophoresis. These observations lend strong support to the concept of post-segregational cell killing by type II restriction modification gene complexes.

Post-segregational killing by restriction modification gene complexes: Observations of individual cell deaths

Through a mechanism known as post-segregational killing, several plasmids mediate their stable maintenance by carrying genes that kill plasmid-free segregant cells. We demonstrated earlier that loss of plasmids carrying type II restriction modification (RM) gene complexes inhibits the propagation of a cell population and causes chromosome breakage. We now show the morphology of individual cells changes following loss of thermosensitive plasmids carrying EcoRI RM or PaeR7I RM after a shift to a non-permissive temperature. After a lag, many cells formed long filaments containing multiple nuclei as detected by DAPI staining. Several hours after the shift, many of these long filaments lacked nuclei. Fragmentation of chromosomal DNA down to 5 kb was detected by electrophoresis. These observations lend strong support to the concept of post-segregational cell killing by type II restriction modification gene complexes.

Multiple hok genes on the chromosome of Escherichia coli.

The hok/sok locus of plasmid R1 mediates plasmid stabilization by the killing of plasmid-free cells. Many bacterial plasmids carry similar loci. For example, the F plasmid carries two hok homologues, flm and srnB, that mediate plasmid stabilization by this specialized type of programmed cell death. Here, we show that the chromosome of E. coli K-12 codes for five hok homologous loci, all of which specify Hok-like toxins. Three of the loci appear to be inactivated by the insertion elements IS150 or IS186 located close to but not in the toxin-encoding reading frames (i.e. hokA, hokC and hokE), one system is probably inactivated by point mutation (hokB), whereas the fifth system is inactivated by a major genetic rearrangement (hokD). In the ECOR collection of wild-type E. coli strains, we identified hokA and hokC loci without IS elements. A molecular and a genetic analysis show that the hokA and hokC loci specify unstable antisense RNAs and stable toxin-encoding mRNAs that are processed at their 3' ends. An alignment of the mRNA sequences reveals all the regulatory elements known to be required for correct folding and refolding of the plasmid-encoded mRNAs. The conserved elements include fbi that ensure a long-range interaction in the full-length mRNAs, and tac and antisense RNA target stem-loops that are required for translation and rapid antisense RNA binding of the processed mRNAs. Consistently, we find that the chromosome-encoded mRNAs are processed at their 3' ends, resulting in the presumed translationally active mRNAs. Despite the presence of all of the regulatory elements, the chromosome-encoded loci do not mediate plasmid stabilization by killing of plasmid-free cells. The chromosome-encoded mRNAs are poorly translated in vitro, thus yielding an explanation for the lacking phenotype. These observations suggest that the chromosomal hok-like genes may be induced by an as yet unknown signal.

Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum.

Higher plants efficiently conserve energy ATP in cellulose biosynthesis by expression of sucrose synthase, in which the high free energy between glucose and fructose in sucrose can be conserved and used for the synthesis of UDP-glucose. A mixture of sucrose synthase and bacterial cellulose synthase proceeded to form UDP-glucose from sucrose plus UDP and to synthesize 1,4-beta-glucan from the sugar nucleotide. The mutant sucrose synthase, which mimics phosphorylated sucrose synthase, enhanced the reaction efficiency (Vmax/Km) on 1,4-beta-glucan synthesis, in which the incorporation of glucose from sucrose was increased at low concentrations of UDP. Because UDP formed after glucosyl transfer can be directly recycled with sucrose synthase, UDP-glucose formed appears to show high turnover with cellulose synthase in the coupled reaction. The expression of sucrose synthase in Acetobacter xylinum not only changed sucrose metabolism but also enhanced cellulose production, in which UDP-glucose was efficiently formed from sucrose. Although the level of UDP-glucose in the transformant with mutant sucrose synthase cDNA was only 1.6-fold higher than that in plasmid-free cells, the level of UDP was markedly decreased in the transformant. The results show that sucrose synthase serves to channel carbon directly from sucrose to cellulose and recycles UDP, which prevents UDP build-up in cellulose biosynthesis.

Toxin-antitoxin systems homologous with relBE of Escherichia coli plasmid P307 are ubiquitous in prokaryotes

Toxin-antitoxin systems encoded by bacterial plasmids and chromosomes specify two proteins, a cytotoxin and an antitoxin. The antitoxins neutralize the cognate toxins by forming tight complexes with them. The antitoxins are unstable due to degradation by cellular proteases (Lon or Clp), whereas the toxins are stable. Here we show that orf7 (denoted relBP307)and orf6 (denoted relEP307)of Escherichia coli plasmid P307 are homologous to the relBE genes of E. coli and constitute a two-component toxin-antitoxin system: (i) relEP307 encodes a cytotoxin lethal or inhibitory to host cells; (ii) relBP307 encodes an antitoxin that prevents the lethal action of the relE-encoded toxin; (iii) RelBP307 antitoxin is degraded by Lon protease; (iv) RelBP307 antitoxin autoregulates the relBE operon of P307 at the level of transcription; (v) RelEP307 toxin acts as a co-repressor of transcription; and (vi) the relBE system stabilizes a mini-P307 replicon by the killing of plasmid-free cells. Using database searching, we found relBE homologues on the chromosomes of many Gram-negative and Gram-positive bacteria. Even more surprising, numerous relBE-homologous gene systems are present on the chromosomes of Archae. Thus, toxin-antitoxin systems homologous with relBE of E. coli are ubiquitous in prokaryotic organisms.

A sufficiency analysis of asymptotic stability in continuous recombinant fermentation with auxotrophic mutants

Fermentations employing genetically modified microorganisms are sensitive to the disturbances likely in large bioreactors. Since these disturbances may affect the competition between recombinant cells and plasmid-free cells, with consequent effects on plasmid stability and reactor stability, it is useful to identify regions of asymptotic stability (RAS) which do not vary with shifts in the operating condition. Using a model for continuous fermentation with auxotrophic mutants, sufficient conditions have been derived for such operationally invariant RAS. Plots for Saccharomyces cerevisiae containing the pBR322 plasmid show that improving the segregational stability makes the fermentation more vulnerable to disturbances. Thus, real reactor operation must balance both kinds of stability.

Isolation of a Derivative ofEscherichia coli–Enterococcus faecalisShuttle Vector pAM401 Temperature Sensitive for Maintenance inE. faecalisand Its Use in Evaluating the Mechanism of pAD1par-Dependent Plasmid Stabilization

A derivative of theEscherichia coli–Enterococcus faecalisshuttle vector pAM401 was isolated by mutagenesis in anE. colimutator strain. This plasmid, designated pAM401ts, was more than an order of magnitude less stable at 38°C than at 30°C in theE. faecalishost strain JH2-2. TheE. faecalisplasmid pAD1-encodedparstability locus was cloned onto pAM401ts, and its effects on plasmid stability and host cell viability were assessed. It was found thatparstabilized pAM401ts at 38°C but also caused a substantial drop in cell viability three to four generations after a temperature shift from 30 to 38°C. After a maximum viability drop of 94%, culture growth recovered as plasmid-free cells began to accumulate. Provision of excess RNAII, the putativeparantidote,in transattenuated cell killing. These characteristics support a postsegregational killing mechanism forpar-mediated plasmid stabilization.

Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability.

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.

Characteristics and transformation of Zymomonas mobilis with plasmid pKT230 by electroporation

Transformation of Zymomonas mobilis with plasmid pKT230 by electroporation was achieved with a transformation efficiency of 9.0 ± 1.8 × 103 per μg plasmid DNA. The growing state of the host cells before transformation, the RC time constant for pulsing at the optimal electric field strength (7.5 kV/cm), the plasmid concentration and the post-incubation time prior to outgrowth in RM medium were the sensitive factors influencing the efficiency of the transformation. The data from batch cultures revealed that the plasmid-harboring cells, Z. mobilis (pKT230), had the same growth pattern as plasmid-free cells. The yield factors of biomass production and ethanol formation by Z. mobilis were nearly unchanged after being transformed and grown in the selective medium where the gene for antibiotic resistance was expressed. The results suggested that the plasmid pKT230 was stable in Z. mobilis and qualified for being a cloning vector in the construction of a recombinant ethanol-producer.

Extracellular expression of human epidermal growth factor encoded by an Escherichia coli K-12 plasmid stabilized by the ytl2-incR system of Salmonella typhimurium

A plasmid stabilization system, active in high copy-number plasmids, was cloned from the large resident plasmid, pSLT, of Salmonella typhimurium. The ytl2 gene, together with a 249-bp region (termed incR) downstream of the gene, imparted >104-fold stability to a pBR322-based plasmid. The ytl2-incR region was then used to stabilize a recombinant plasmid carrying the human epidermal growth factor gene (with the Escherichia coli K-12 ompA signal sequence), behind the lacUV5 promoter. In shake flask tests to optimize expression of human epidermal growth factor, loss of recombinant plasmid was <1% when growth (both before and after induction with isopropyl-β-d-galactopyranoside) took place even in the absence of antibiotic selection, and the specific activity of secreted human epidermal growth factor was ca 20 μg per 108 cells at harvest, compared to a figure of ca 3 μg per 108 cells when a comparable plasmid, but devoid of the ytl2-incR region, was employed, as outgrowth of plasmid-free cells after induction severely compromised the specific activity of the secreted product.

The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family.

Toxin-antitoxin systems are defined as a group of plasmid- and chromosome-encoded loci that specify a cell toxin and a protein antitoxin. Plasmid-encoded toxin-antitoxin systems stabilize their replicons by killing plasmid-free cells. Here, we show that the relBE genes of Escherichia coli K-12 have all the basic features previously connected with toxin-antitoxin systems: (i) relE encodes a cytotoxin lethal or inhibitory to host cells; (ii) relB encodes an antitoxin that prevents the lethal action of the relE-encoded toxin; (iii) the relBE genes stabilize a mini-R1 test plasmid; and (iv) the RelB antitoxin autoregulates the relBEF operon at the level of transcription. Using database searching, we found relBE homologues on the chromosomes of E. coli K-12, Haemophilus influenzae and Vibrio cholerae. A fifth relBE homologue was identified on the enterotoxin encoding E. coli plasmid P307. Indirect evidence suggests that the toxicity of RelE may be related to the inhibition of protein synthesis. Based on these observations, we propose a model that explains the delayed relaxed phenotype associated with mutations in relB.

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. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 625–632, 1998.

Plasmid maintenance inEscherichia 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.

Analysis of the effect of interruptions in substrate inflow on a fed-batch fermentation with recombinant bacteria

Fermentations employing recombinant bacteria are sensitive to inflow disturbances. In this study, a fed-batch fermentation using a recombinant Escherichia coli strain is analyzed for its performance under interruptions in the flow rate and concentration of the substrate. To enable separate control of the two variables, the experimental scheme has two inflow streams—a concentrated substrate and a diluent—which can be mixed in any desired proportion. The mathematical model expresses the dynamic variations of the overall concentrations of the plasmid-containing and plasmid-free cells in the bioreactor, as well as the intra-cellular concentrations of the plasmid DNA and the recombinant protein (β-galactosidase). The intra-cellular and the extra-cellular variables follow different patterns with time. While interruptions in any one inflow stream reduce β-galactosidase production, simultaneous failure and restart of both streams promote its formation. This result may be explained by analogy with previous studies with feed cycling, and suggests that short-duration interruptions may have fortuitously beneficial effects.

Plasmid-mediated rifampicin resistance in Pseudomonas fluorescens.

Rifampicin is an antibiotic mostly used to treat tuberculosis and leprosy, and, occasionally, other diseases. Resistance is due to alterations in membrane permeability or to mutation in the rpoB gene coding for mRNA polymerase. Both these mechanisms originate via chromosomal mutation. However, a rifampicin-resistant Pseudomonas fluorescens strain harboured a multiresistance plasmid which transferred rifampicin resistance when transformed into P. putida or Escherichia coli. Rifampicin readily diffused into the sensitive cells of the E. coli and P. putida recipients, but the transformants with the plasmid, pSCL were resistant to the drug and did not accumulate it. Potassium cyanide restored the diffusion of rifampicin into the resistant cells, indicating that an efflux pump was involved in the resistance mechanism. The resistance of the transformants and the wild strain was also abolished in sphaeroplasts generated by EDTA lysozyme treatment. Analysis of membrane proteins by SDS-PAGE revealed the presence of two new proteins in the plasmid-containing cells of E. coli, P. putida and P. fluorescens and not in the plasmid-free cells. These may be involved in the efflux of rifampicin.

Molecular mechanism of peptide-specific pheromone signaling in Enterococcus faecalis: functions of pheromone receptor TraA and pheromone-binding protein TraC encoded by plasmid pPD1.

Conjugative transfer of the Enterococcus faecalis plasmid pPD1 is activated by cPD1, one of several peptide sex pheromones secreted by plasmid-free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1. Using a tritiated pheromone, [3H]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [3H]cPD1. The cell extract but not the membrane fraction of the donor strain exhibited significant [3H]cPD1-binding activity. On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the traA-bearing multicopy plasmid (pDLHH21) also exhibited high [3H]cPD1-binding activity. A recombinant TraA exhibited a dissociation constant of 0.49 +/- 0.08 nM against [3H]cPD1. iPD1 competitively inhibited [3H]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA. A strain carrying the traC-bearing multicopy plasmid (pDLES23) exhibited significant [3H]cPD1-binding activity. A strain carrying traC-disrupted pPD1 (pAM351CM) exhibited lower [3H] cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor strain. Some of the other pheromones and inhibitors inhibited [3H]cPD1 binding to the traC transformant like cPD1 and iPD1 did. These results show that TraC, as an extracellular less-specific pheromone-binding protein, supports donor cells to receive cPD1.

Plasmid stability of recombinantPseudomonas sp. B13 FR1 pFRC20P in continuous culture

Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture. The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate. Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate. Plasmid stability decreased with increasing dilution rate. Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed. After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations. Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells. By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70%. Literature models on plasmid stability could not be applied to describe the experimental data. Therefore, a new but unstructured model was developed to describe the experimental results. The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells.

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

Fermentation strategy to enhance plasmid stability during the cultivation of Escherichia coli for the production of recombinant levansucrase

To produce levansucrase, a fructosyltransferase enzyme, in a recombinant Escherichia coli harboring the levU gene of Zymomonas mobilis, fermentation strategies were examined in terms of induction methods and plasmid stability. Although the recombinant levansucrase was induced rapidly by IPTG, high instability of the plasmid and formation of inclusion bodies were observed. Segregational instability was aggravated by the excretion of β-lactamase into the culture broth during subculturing, which caused an overgrowth of plasmidfree cells. A combination of methicillin (2, 6-dimethoxyphenyl-penicillin) and ampicillin to provide selective pressure was effective in preventing the growth of plasmid-free cells. The population of plasmid-harboring cells was maintained above 95% of the total cells for more than 100 generations under this condition. In order to replace IPTG, which is toxic and too expensive for use in a large scale, d-lactose was tested and found to be favorable as an alternative inducer.

Antisense RNA-regulated programmed cell death.

Eubacterial plasmids and chromosomes encode multiple killer genes belonging to the hok gene family. The plasmid-encoded killer genes mediate plasmid stabilization by killing plasmid-free cells. This review describes the genetics, molecular biology, and evolution of the hok gene family. The complicated antisense RNA-regulated control-loop that regulates posttranscriptional and postsegregational activation of killer mRNA translation in plasmid-free cells is described in detail. Nucleotide covariations in the mRNAs reveal metastable stem-loop structures that are formed at the mRNA 5' ends in the nascent transcripts. The metastable structures prevent translation and antisense RNA binding during transcription. Coupled nucleotide covariations provide evidence for a phylogenetically conserved mRNA folding pathway that involves sequential dynamic RNA rearrangements. Our analyses have elucidated an intricate mechanism by which translation of an antisense RNA-regulated mRNA can be conditionally activated. The complex phylogenetic relationships of the plasmid- and chromosome-encoded systems are also presented and discussed.