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.     

Topographical distribution of penicillin-binding proteins in the Escherichia coli membrane.

The penicillin-binding proteins (PBPs) found in the membranes of Escherichia coli X925 minicells (primarily cell ends or septa) were compared with those found in rod-shaped cells (primarily sidewalls) in an effort to determine whether certain PBPs are unevenly distributed over the bacterial cell membrane. The seven major PBPs of E. coli were all present in minicell membranes. PBP 1B was altered in minicells, however, appearing as two bands on sodium dodecyl sulfate-polyacrylamide gels rather than the usual three. PBP 2, which is needed for longitudinal growth of the cell but not for septum formation, was significantly reduced in minicell membranes. This observation is consistent with the fact that minicells contain very little sidewall material and raises the possibility that the specialized function of PBP 2 may be determined or regulated by its uneven topographical distribution in the membrane. None of the PBPs appeared to be selectively enriched in minicell membranes.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R(-) minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Membrane-Associated R Factor Proteins

R factor proteins are synthesized in R factor-containing Escherichia coli minicells. Half of this protein remained associated with the minicell membrane upon lysis of the minicells. Over 90% of the membrane-associated protein was extracted by sodium lauryl sarcosinate, suggesting a location of these proteins in the inner membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane preparations demonstrated the presence of multiple peptides, including a prominent band with a molecular weight of 28,000 to 30,000. A polypeptide of similar size was seen in membrane preparations from minicells harboring R factors from five different compatibility types. This major R factor membrane peptide was seen with R factors repressed or derepressed for pilus synthesis, with and without antibiotic resistances. It was associated with R factor deoxyribonucleic acid in membrane-deoxyribonucleic acid complexes. Its possible role in R factor replication and/or transfer is being investigated.     

Interactions between phage lambda replication proteins, lambda DNA and minicell membrane

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.     

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.     

Cell growth and division in Escherichia coli: a common genetic control involved in cell division and minicell formation

Escherichia coli Div 124(ts) is a conditional-lethal cell division mutant formed from a cross between a mutant that produces polar anucleated minicells and a temperature-sensitive cell division mutant affected in a stage of cross-wall synthesis. Under permissive growth temperature (30 C), Div 124(ts) grows and produces normal progeny cells and anucleated minicells from its polar ends. When transferred to nonpermissive growth temperature (42 C), growth and macromolecular synthesis continue, but cell division and minicell formation are inhibited. Growth at 42 C results in formation of filamentous cells showing some constrictions along the length of the filaments. Return of the filaments from 42 to 30 C results in cell division and minicell formation in association with the constrictions and other areas along the length of the filaments. This gives rise to a "necklace-type" array of cells and minicells. Recovery of cell division is observed after a lag and is followed by a burst in cell division and finally by a return to the normal growth characteristic of 30 C cultures. Recovery of cell division takes place in the presence of chloramphenicol or nalidixic acid when these are added at the time of shift from 42 to 30 C, and indicates that a division potential for filament fragmentation is accumulated while the cells are at 42 C. This division potential is used for the production of both minicells and cells of normal length. The conditional-lethal temperature sensitive mutation controls a step(s) in cross-wall synthesis common to cell division and minicell formation.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

The fate of phage lambda DNA in lambda-infected minicells.

The fate of phage lambda DNA in lambda-infected Escherichia coli minicells harboring the plasmid ColE1, and in plasmid-free minicells, were studied. Binding of lambda DNA to the minicell membrane, and formation of the supercoiled covalently-closed circular structure has been demonstrated. Phage infection abolishes plasmid DNA synthesis. Only a very slight, non-replicative lambda DNA synthesis occurs, soon after infection. This synthesis is associated with fragments of lambda DNA arising during, or soon after its penetration.     

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.     

Use of Genetically Marked Minicells as a Probe in Measurement of Predation on Bacteria in Aquatic Environments

Minicells produced by Escherichia coli M2141 were used as probes to measure predation on pelagic bacteria in situ. The minicells, labeled with [³⁵S]methionine in one specific protein, were shown to disappear in the presence of a microflagellate (Ochromonas sp.), as seen by a decrease in the amount of labeled marker protein with time. Incubation in filtered (pore size, 0.2 μm) and autoclaved seawater did not affect the amount of labeled marker protein in the minicell. The generation time of flagellates feeding on minicells was determined to be similar to that found for flagellates grown on seawater bacteria or living E. coli NC3. Data indicate that minicells are seen as true food particles by the flagellates. The minicell probe was used in recapture experiments, in which predation in situ on pelagic bacteria was demonstrated. The rate of bacterial production showed a clear covariation with the rate of predation, both in different sea areas and in depth profiles. The obtained results (11 field experiments) showed that the rate of predation, on average, accounts for the consumption of 62% of the bacteria produced.     

Minicell formation by Campylobacter jejuni

Campylobacter jejuni sheds its flagella and varying proportions of the poles of the cell late in the growth cycle, resulting in the production of very small flagellated structures 0.1 to 0.3 microM in diameter. Electron microscopy revealed that these structures were minicells possessing outer membrane, cytoplasmic membrane, flagellar basal complex, and polar membrane; nucleoplasms were not seen. The initial event in the formation of these minicells involved a constriction of the cytoplasmic membrane, segregating the polar regions of the cell. The peptidoglycan layer of the cell wall was not visible, but was presumed to lyse at the separation site of minicell formation, and to reform or remain intact along the main length of the cell because the rods did not spheroplast. Finally, rupture and resealing of the outer membrane component of the wall resulted in the release of fully enclosed minicells and nonflagellated rods.     

Physical and Functional Characteristics of R-Factor Deoxyribonucleic Acid Segregated into Escherichia coli Minicells

Both fi(+) and fi(-) type R factors function as intact replicative units when segregated into Escherichia coli minicells. Hybridization studies demonstrate that at least 95% of the deoxyribonucleic acid (DNA) in R(+) minicells is episomal in origin. About half of the DNA can be extracted in a closed circular form and about 75% of the DNA is membrane associated. DNA, ribonucleic acid, and protein synthesis proceeds in R(+) minicells in contrast to R(-) minicell controls. The system offers a unique opportunity to study a relatively small replicative unit in a native cell environment and a simple means of isolating large quantities of episomal DNA.     

Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli

Bacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division. In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function. Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously. In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli. We identified membrane proteins enriched in E. coli minicells using a combination of two-dimensional electrophoresis and mass spectrometry. Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes. The minicell-enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane-associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB. We immunolocalized two of the minicell-enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.     

Subcloning, nucleotide sequence, and expression of trkG, a gene that encodes an integral membrane protein involved in potassium uptake via the Trk system of Escherichia coli.

The trkG gene encodes a component of the K+ uptake system Trk and is located at 30.5 min inside the lambdoid prophage region rac of the Escherichia coli chromosome. trkG was subcloned, its nucleotide sequence was determined, and its product was identified in a minicell system. The open reading frame of 1,455 bp encodes a hydrophobic membrane protein with a calculated molecular weight of 53,493 that is predicted to contain up to 12 transmembrane helices. The trkG gene product behaved as a hydrophobic membrane protein; it was found exclusively in the membrane fraction of the minicells and its migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was anomalous, indicating an apparent molecular weight of 35,000. The trkG gene contains an exceptionally high proportion of infrequently used codons, raising the question of the origin of this gene. trkG does not appear to be a prophage gene since no similarity was observed between the nucleotide sequence of trkG or the amino acid sequence of its product and the sequences of genes or proteins from bacteriophage lambda.     

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.     

Prediction of signal sequence-dependent protein translocation in bacteria: assessment of the Escherichia coli minicell system

The use of phenethyl alcohol (PEA) as a probe for signal sequence-dependent protein translocation in minicells was examined. Processing of beta-lactamases and tonA was inhibited by PEA at concentrations which did not affect production of the alpha and gamma forms of penicillin binding protein (PBP) lb. The PBPlbs are believed to lack leader sequences whereas the other proteins contain them. Processing of a beta-lactamase which shares the murein-lipoprotein export pathway was relatively resistant to PEA, consistent with previous findings in whole bacteria. The results reported here suggest that PEA is a suitable probe for leader sequences in the minicell system. By using PEA we predict that PBP4 does not require a leader sequence for membrane insertion.     

Polypeptides encoded by the mer operon.

HgCl2-induced polypeptides synthesized by Escherichia coli minicells containing recombinant or natural HgR plasmids were labeled with [35S]methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All plasmids examined encoded two heavily labeled, HgCl2-inducible polypeptides of 69,000 and 12,000 daltons. Most plasmids also encoded two additional HgCl2-inducible proteins in the 14,000- to 17,000-dalton range. Antiserum prepared against a purified mercuric ion reductase reacts with the 69,000-dalton polypeptide and a minor 66,000-dalton protein seen in several different HgR minicells. Recombinant plasmids constructed from portions of mer DNA from the IncFII plasmid NR1 were also analyzed in the minicell system. Five HgCl2-inducible polypeptides (69,000, 66,000, 15,100, 14,000, and 12,000 daltons) were synthesized in minicells carrying pRR130, a recombinant derivative containing the EcoRI-H and EcoRI-I restriction fragments of NR1. The EcoRI-H fragment of NR1 encodes the three small mer proteins of 15,100, 14,000, and 12,000 daltons and the amino-terminal 40,000 daltons of the mercuric ion reductase monomer.     

The divIVA minicell locus of Bacillus subtilis.

The Bacillus subtilis divIVA1 mutation causes misplacement of the septum during cell division, resulting in the formation of small, circular, anucleate minicells. This study reports the cloning and sequence analysis of 2.4 kb of the B. subtilis chromosome including the divIVA locus. Three open reading frames were identified: orf, whose function is unknown; divIVA; and isoleucyl tRNA synthetase (ileS). We identified the point mutation in the divIVA1 mutant allele. Inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype. Mutants with mutations at both of the minicell loci of B. subtilis, divIVA and divIVB, possess a minicell phenotype identical to that of the DivIVB- mutant. The DivIVA-mutants, but not the DivIVB- mutants, show a decrease in sporulation efficiency and a delay in the kinetics of endospore formation. The data support a model in which divIVA encodes the topological specificity subunit of the minCD system. The model suggests that DivIVA acts as a pilot protein, directing minCD to the polar septation sites. DivIVA also appears to be the interface between a sporulation component and MinCD, freeing up the polar septation sites for use during the asymmetric septation event of the sporulation process.