Early development of bacteriophages SP01 and SP82G in minicells of Bacillus subtilis.

SP01- and SP82G-infected Bacillus subtilis CU403 divIVBI minicells synthesize 13 easily detectable early RNA species with molecular weights ranging from 60 × 10³ to 430 × 10³. Comparison of in vivo and in vitro translation of early messenger RNA indicates that five early mRNAs of SP01 are synthesized but not translated unless protein synthesis has been permitted in the infected minicell, providing evidence for a translation control mechanism. A sequential appearance of 48 polypeptides has been determined in SP01-infected minicells. The polypeptides have been grouped into two classes of early polypeptides, i.e. those encoded by early mRNA and three subsequent classes as demonstrated by the analysis of polypeptides synthesized in minicells infected with the SP01 mutants, susF21, susF4 and susF14. Phage capsid proteins are not synthesized in minicells. RNA synthesized in infected minicells is subject to turnover. The individual mRNA species have differing functional stabilities ranging from a loss of only 50% functional activity, in 20 minutes at 37 °C, to loss of over 99% activity.Infection of anucleate minicells has been shown to be a very simple method for comparison of closely related phages (slight differences are detected between SP01- and SP82G-encoded mRNA and polypeptides), detection of polypeptides affected by amber mutations and the analysis of early events in phage development in the absence of host syntheses.     

Physiological Studies of Bacillus subtilis Minicells

Minicells produced by Bacillus subtilis strains carrying the div IV-B1 mutation, (CU 403 div IV-B1 and CU 403 div IV-B1, tag-1), were purified by a procedure which destroys parental cells with ultrasound, but spares minicells. Such preparations generally contain 10(9) or more minicells/ml and less than 10(4) colony-forming units/ml. Purified minicells are resistant to autolysis in tris(hydroxymethyl)aminomethane buffer, pH 7.5, at 30 C, conditions which result in total lysis of parental cells. Minicells are not completely devoid of autolytic activity, however. The medium in which minicells are produced, the temperature at which purified minicells are incubated, and the genotype of cells from which the minicells are derived all influence the rate of autolysis of purified minicells. These parameters are demonstrated by using minicells obtained from div IV-B1 and div IV-B1, tag-1 strains. Ultrastructural differences have been observed in the products of autolysis of these two minicell strains. Minicells are sensitive to low levels of lysozyme and yield miniprotoplasts when the wall is removed in an osmotically protective environment. Although minicells are unable to grow, they can maintain their integrity over long periods of time, which suggests functional energy metabolism in minicells. Direct measurements of adenosine 5'-triphosphate (ATP) levels by the luciferase assay indicated that minicells can produce ATP. Oxygen consumption, measured by standard respirometry techniques, also indicates functional metabolism in minicells. These findings demonstrate that minicells purified by ultrasound are suitable material for study of physiological processes in anucleate cells.     

Synthesis of Ribonucleic Acid and Protein in Plasmid-Containing Minicells of Escherichia coli K-12

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds

Formed by aberrant cell division, minicells possess functional metabolism despite their inability to grow and divide. Minicells exhibit not only superior stability when compared with bacterial cells but also exceptional tolerance—characteristics that are essential for a de novo bioreactor platform. Accordingly, we engineered minicells to accumulate protein, ensuring sufficient production capability. When tested with chemicals regarded as toxic against cells, the engineered minicells produced titers of C6–C10 alcohols and esters, far surpassing the corresponding production from bacterial cells. Additionally, microbial autoinducer production that is limited in expanding bacterial population was conducted in the minicells. Because bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-responsive gene circuits. When bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-based gene circuits with the minicells. This study demonstrates the potential of minicells as bioreactors suitable for products with known limitations in microbial production, thus providing new possibilities for bioreactor engineering.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Stimulation in dnaB(ts) Donors by Minicells

R64-11(+) donor cells that are thermosensitive for vegetative DNA replication will synthesize DNA at the restrictive temperature when recipient minicells are present. This is conjugal DNA replication because it is R64-11 DNA that is being synthesized and there is no DNA synthesis if minicells that cannot be recipients of R64-11 DNA are used. The plasmid DNA present in the donor cells before mating is transferred to recipient minicells within the first 20 min of mating, but additional copies of plasmid DNA synthesized during the mating continue to be transferred for at least 90 min. However, the transfer of R64-11 DNA to minicells is not continuous because the plasmid DNA in minicells is the size of one R64-11 molecule or smaller, and there are delays between the rounds of plasmid transfer. DNA is synthesized in minicells during conjugation, but this DNA has a molecular weight much smaller than that of R64-11. Thus, recipient minicells are defective and are not able to complete the synthesis of a DNA strand complementary to the single-stranded R64-11 DNA received from the donor cell.     

F- mating materials able to generate a mating signal in mating with HfrH dnaB(Ts) cells.

The purified outer membrane from F- (W1-3) cells was shown to inhibit mating effectively, but the purified cytoplasmic (inner) membrane did not. These membranes, heat-treated minicells, and ultraviolet-irradiated minicells were examined for their ability to generate a mating signal at 43 degrees C in mating with HfrH dnaB(Ts) cells. The outer and inner membranes and heat-treated minicells all failed to stimulate incorporation of radioactive thymine; only ultraviolet-irradiated minicells retained the ability to generate a mating signal for the donor to initiate transfer replication.     

Rapid isolation of Escherichia coli minicells by glass-fiber filtration: study of plasmid-coded polypeptides

A filtration technique is described to purify Escherichia coli chi 1488 minicells much more rapidly than the usual method involving sucrose gradient centrifugation, and to produce minicells that have not been subjected to osmotic stress. The minicells so prepared are metabolically active as indicated by the in vivo incorporation of [35S]methionine into plasmid-coded polypeptides.     

Minicell production and bacteriophage superinducibility of thymidine-requiring strains of Haemophilus influenzae.

Aminopterin- or trimethoprin-resistant thymidine-requiring strains of Haemophilus influenzae produce minicells, and the ratio of minicells to cells increases during the stationary phase of growth. Strain LB11, isolated after mutagenesis of a thymidine-requiring strain (Rd thd), produces more minicells than the parent strain. The mutations involved in high frequency minicell production have been transferred into the wild type (strain Rd) by transformation. The thymidine requirement in the resulting strain, MCl, is essential for minicell production, since spontaneous revertants of MCl to prototrophy do not produce minicells. The ratio of minicells to cells was increased more than 10(3)-fold by differential centrifugation. The minicells contain little or no deoxyribonucleic acid (DNA). Phage HPlcl apparently cannot attach to minicells. Competent cells of LB11 and its thymidine-requiring parent strain produce defective phage as a result of exposure to transforming DNA, whereas only LB11 produces many defective phage in response to the competence regime alone. Competent HP1c1 and S2 lysogens of MC1 and Rd thd are also superinducible by transforming DNA, but competent LB11 lysogens produced about the same amount of HP1c1 or S2 phage with or without exposure to transforming DNA possibly because of competition between the induced defective phage and Hp1c1 or S2 phage.     

Regions on the F plasmid which affect plasmid maintenance and the ability to segregate into Escherichia coli minicells

Certain derivative mini-F plasmids were found to segregate into Escherichia coli minicells, in contrast to the intact mini-F plasmid which does not. Segregation was not related to the presence or absence of the normal origin of vegetative replication, but appeared to be affected by regions of F which encode replication, incompatibility, copy number control, and partitioning functions. Segregation of mini-F plasmids into minicells was not random; the plasmid concentration in minicells did not correlate with the plasmid concentration in cells. Genes, or gene products, of F from the region spanning the sequences 44.1–49.3F appeared to affect the ability of mini-F plasmids to segregate into minicells. Segregation of mini-F plasmids into minicells was not directly related to stable plasmid inheritance. These results argue for the sequestration of mini-F plasmids in host cells.     

Efficiency of phage protein synthesis in lambda-infected E. coli minicells

Phage lambda major head protein, the gene E product, has been identified among other phage proteins synthesized in lambda-infected Escherichia coli minicells, separated by SDS-acrylamide gel electrophoresis. On stained gels, the same protein has also been detected among total (bacterial and phage) proteins of lambda-infected minicells. The contribution of lambda proteins to the total protein content of lambda-infected minicells was found to be about 12% following 30 min lambda-infection. The inhibition of lambda early protein synthesis (shown by other authors in nucleate bacterial cells) practically does not occur in minicells; this may be the reason of the observed high efficiency of phage protein synthesis.     

A coupling process for improving purity of bacterial minicells by holin/lysin

Drug and gene delivery systems using bacterial minicells have attracted much attention. Here we attempted to enhance the yield and purity of the minicells using a novel method in which autolysin induced in actively metabolizing parent cells led to autolysis. A two-step protocol coupling an autolysin (holin/lysin) step with a conventional centrifugation step achieved a purity of the minicells similar to that resulting from successive six- or seven-step methods.     

Proteins synthesized by a non-induced bacteriocinogenic factor in minicells of Escherichia coli

Summary The cloacinogenic factor Clo DF13 from Enterobacter cloacae has been transferred to the minicell-producing strain P678-54 of Escherichia coli K12. The data presented show that this Clo DF13 factor segregates into minicells of P678-54 (Clo DF13) and that this factor is the only plasmid, present in these minicells. Proteins from purified P678-54 (Clo DF13) and P678-54 minicells, previously labelled with ¹⁴C-amino acids, were compared after electrophoresis on SDS-polyacrylamide gels. From this comparison it appeared that a noninduced Clo DF13 factor directs the synthesis of 4 proteins. The molecular weights of these proteins could be estimated to be about 72000, 32000, 18500 and 12000. In P678-54 (Clo DF13) minicells, one additional Clo DF13 protein was found to be unlabelled. Apparently this protein is not synthesized in P678-54 (Clo DF13) minicells, but is segregated into or is attached to the minicells after being synthesized in the P678-54 (Clo DF13) cells. The molecular weight of this protein is about 62000, which corresponds to the molecular weight of cloacin.     

Derepression of anthranilate synthase in purified minicells of Escherichia coli containing the Col-trp plasmid

Purified minicells of Escherichia coli K-12 containing the plasmid Col-trp(+) or Col-trpA2 could be derepressed for the synthesis of anthranilate synthase, the first enzyme encoded in the trp operon. Non-plasmid-containing, deoxyribonucleic acid-deficient minicells could not be derepressed. Derepressed enzyme synthesis was initiated by l-tryptophan starvation. The kinetics of derepression were studied with minicells containing the Col-trpA2 plasmid. The derepression curves were biphasic with a rapid initial rate of enzyme synthesis followed by a slower rate of synthesis. The presence of l-tryptophan (20 to 50 mug/ml) or chloramphenicol (200 mug/ml) abolished enzyme synthesis. The presence of rifamycin SV (280 mug/ml) partially inhibited enzyme synthesis after at least 3.5 min of exposure. The ratio of minicell-to-cell synthetic capacity was 1:2.4 when compared on the basis of derepressed enzyme activity per unit cell volume. This work demonstrates that plasmid-containing minicells are capable of considerable functional protein and messenger ribonucleic acid synthesis and that the regulation of at least the trp operon is similar in minicells to that observed in cells.     

Nucleoside triphosphate pools in minicells of Escherichia coli.

The nucleoside triphosphate pools of Escherichia coli minicells are different from those in parental cells. The growth phase in which minicells accumulate significantly affects the pool sizes.     

A New Method for the Preparation of Minicells for Physiological Studies

A procedure has been developed for preparing minicells that does not rely on sucrose gradients in a rate-zone centrifuge. In the presence of low levels (10 units/ml) of penicillin, the contaminating bacteria present in minicell cultures after low-speed differential centrifugation are turned into long filamentous cells and can be killed by sonic treatment. An additional low-speed centrifugation (2, 000 g for 5 min) yields purified minicells with less than one contaminating cell per 10 minicells.     

Plasmid segregation into minicells is associated with membrane attachment and independent of plasmid replication.

The role of plasmid replication in the segregation of plasmids into Escherichia coli minicells was investigated with temperature-sensitive replication mutants derived from E. coli plasmids ColE1 and pSC101. For as long as six generations of growth, at permissive or nonpermissive temperatures (when greater than 80% of plasmid replication was inhibited), the same amount of previously 3H-labeled plasmid DNA segregated into minicells. Density gradient separations of wild-type and temperature-sensitive plasmid DNA from both replicons segregated into the minicells showed that about 20 to 25% was stably associated with the minicell membrane at both temperatures. Electron microscopy showed this DNA to consist of circular plasmid molecules attached to the minicell membrane. These combined findings suggest that segregation of plasmids into minicells and their association with the minicell membrane are interrelated and independent of plasmid replication.     

The use of bacterial minicells to transfer plasmid DNA to eukaryotic cells

The delivery of DNA to mammalian cells is of critical importance to the development of genetic vaccines, gene replacement therapies and gene silencing. For these applications, targeting, effective DNA transfer and vector safety are the major roadblocks in furthering development. In this report, we present a novel DNA delivery vehicle that makes use of protoplasted, achromosomal bacterial minicells. Transfer of plasmid DNA as measured by green fluorescent protein expression was found to occur in as high as 25% of cultured Cos-7 cells when a novel chimeric protein containing the D2-D5 region of invasin was expressed and displayed on the surface of protoplasted minicells. Based on endoplasmic reticulum stress and other responses, protoplasted minicells were non-toxic to recipient eukaryotic cells as a consequence of the transfection process. Taken together, these results suggest that bacterial minicells may represent a novel and promising gene delivery vehicle.     

Characterization of minicells (nuclei) obtained by cytochalasin enucleation

By centrifuging monolayers of rat L6 myoblasts adhering to plastic discs in the presence of cytochalasin it is possible to enucleate close to 100% of the cells. The nuclei drawn out of the cells are surrounded by a narrow rim of cytoplasm and an intact plasma membrane and therefore are ‘minicells’ stripped of most of the original cytoplasm. In addition to minicells, the pellet material on the bottom of the centrifuge tubes contains fragments of cytoplasm and occasional intact cells. The number of intact cells can be drastically reduced by precentrifugation of the monolayers prior to enucleation so as to remove loosely attached cells from the plastic discs. Biochemical analysis of minicell pellets show that at least 65–75% of the original cytoplasm is removed during enucleation. Microinterferometric dry mass measurements on individual minicells where free cytoplasmic fragments (without nuclei) can be excluded indicate that at least 80–90% of the cytoplasm is removed.Most of the minicells are impermeable to trypan blue and are able for some time after enucleation to incorporate precursors into nucleic acids and proteins. Under normal tissue culture conditions the minicells fail, however, to regenerate a cytoplasm and to multiply. Instead the minicells undergo a gradual decrease in dry mass and most seem to be dead 36 h after enucleation. The minicells can, however, be rescued by fusion with enucleated cytoplasms and may therefore be used to reconstitute nucleated cells.