Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution.     

The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division

The Bacillus subtilis divIVA gene, first defined by a mutation giving rise to anucleate minicells, has been cloned and characterized. Depletion of DivIVA leads to inhibition of the initiation of cell division. The residual divisions that do occur are abnormally placed and sometimes misorientated relative to the long axis of the cell. The DivIVA phenotype can be suppressed by disruption of the MinCD division inhibitor, suggesting that DivIVA controls the topological specificity of MinCD action and thus septum positioning. A DivIVA–GFP fusion targets to new and used sites of cell division, consistent with it having a direct role in topological specification.     

Exopolysaccharides of the phytopathogen Pseudomonas syringae pv. glycinea.

Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS-dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division. This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content. Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments). Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC. In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production.     

Molecular cloning using immune sera of a 22-kDal minor outer membrane protein of Vibrio cholerae

Using antisera prepared against live Vibrio cholerae we have selected several recombinant DNA clones, plasmids pPM440, pPM450 and pPM460, encoding the gene for a 22-kDal V. cholerae peptidoglycan-associated-outer-membrane protein. This is a minor protein in V. cholerae but is expressed in large amounts when the cloned gene is present in Escherichia coli K-12, where it is exposed on the cell surface as judged by ELISA. We have localized the gene within the cloned DNA by transposon mutagenesis and deletion analysis followed by analysis of whole cells and minicells to identify the plasmid-encoded proteins. The DNA region encoding the protein seems to be conserved between El Tor and Classical strains as judged by Southern DNA hybridization.     

Purification of the 25-kDa Vibrio cholerae major outer-membrane protein and the molecular cloning of its gene: ompV

The 25-kDa peptidoglycan-associated outer-membrane protein and most likely porin of Vibrio cholerae is a major immunogenic species. It has been purified by ion-exchange elution on hydroxyapatite followed by gel filtration on Bio-Gel P150 both in the presence of sodium dodecyl sulfate. This protein, of greater than 90% purity as judged by Western blotting, has been used to raise antibodies in rabbits. The antisera were then used to screen V. cholerae gene banks, constructed in Escherichia coli K12, and this has enabled us to isolate several colonies harbouring the cloned gene. The plasmids in these colonies have been designated pPM451, pPM455 and pPM472. These plasmids have a 5.3 X 10(3)-base BamHI fragment of V. cholerae DNA in common. Restriction endonuclease mapping of these plasmids has been performed and the protein identified both by Western blot analysis and in E. coli K12 minicells. The protein is not efficiently expressed in E. coli K12. It is proposed to use the name ompV to describe the structural gene, present in the cloned DNA, for this V. cholerae outer membrane protein.     

Membrane Association of Conjugally Transferred Deoxyribonucleic Acid in Escherichia coli Minicells

The conjugally acquired deoxyribonucleic acid (DNA) of small, anucleate cells ("minicells") of a mutant strain of Escherichia coli K-12 was found to be predominantly associated with the bacterial membrane. Evidence from X-irradiation studies in vivo shows that there is no decrease in DNA-membrane association under conditions which reduce the DNA to one-sixth its original size and suggests the possibility of multiple DNA-membrane association sites. Preliminary enzymatic studies indicate the involvement of protein, DNA, and lipids in the membrane association of the DNA.     

Role of the C Terminus of FtsK in Escherichia coli Chromosome Segregation

FtsK is essential for Escherichia coli cell division. We report that cells lacking the C terminus of FtsK are defective in chromosome segregation as well as septation, often exhibiting asymmetrically positioned nucleoids and large anucleate regions. Combining the corresponding truncated ftsK gene with a mukB null mutation resulted in a synthetic lethal phenotype. When the truncated ftsK was combined with a minCDE deletion, chains of minicells were generated, many of which contained DNA. These results suggest that the C terminus of FtsK has an important role in chromosome partitioning.     

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.     

Sequence of an osmotically inducible lipoprotein gene.

The osmB gene of Escherichia coli, whose expression is induced by elevated osmolarity, was cloned and physically mapped to a 0.65-kilobase-pair NsiI-HincII DNA fragment at 28 min on E. coli chromosome. The OsmB protein was identified in minicells expressing the cloned gene. The nucleotide sequence of a 652-base-pair chromosomal DNA fragment containing the osmB gene was determined. The open reading frame encodes a 72-residue polypeptide with an Mr of 6,949. This reading frame was confirmed by sequencing the fusion joint of an osmB::TnphoA gene fusion. The amino-terminal amino acid sequence of the open reading frame is consistent with reported signal sequences of exported proteins. The sequence around the putative signal sequence cleavage site, Leu-Ser-Ala-Cys-Ser-Asn, is highly homologous to the consensus sequence surrounding the processing site of bacterial lipoproteins. The presence of a lipid moiety on the protein was confirmed by demonstrating the incorporation of radioactive palmitic acid and inhibition of processing by globomycin. Preliminary localization of the authentic OsmB protein was determined in minicells harboring a plasmid that carries the NsiI-HincII fragment; it was primarily in the outer membrane. Surprisingly, an osmB mutant carrying the osmB::TnphoA insertion mutation was more resistant to the inhibition of metabolism by high osmolarity than the parent strain was.     

Cloning and expression of Thiobacillus versutus aspartate-semialdehyde dehydrogenase gene in Escherichia coli

The Thiobacillus versutus asd gene coding for aspartate-semialdehyde dehydrogenase was cloned in Escherichia coli cells using pBR322 as a vector. The gene was expressed independently of its orientation, suggesting that E. coli RNA polymerase recognized T. versutus promoter sequence. The T. versutus DNA coded protein, of the molecular weight 44,000, was identified by the analysis of the proteins produced by minicells.     

Expression of cloned plasmid regions encoding colonization factor antigen I (CFA/I) in Escherichia coli

Molecular cloning from a plasmid encoding colonization factor antigen I (CFA/I) and heat-stable enterotoxin isolated two regions, 1 and 2, that are required for the production of CFA/I fimbriae. The level of CFA/I synthesis measured by ELISA was similar in an Escherichia coli K12 strain carrying regions 1 and 2 cloned separately on compatible plasmid vectors to that in the same strain containing the parental plasmid. The structural gene for the CFA/I fimbrial subunit was within region 1. This region directed production in E. coli minicells of at least six independent polypeptides, of which the fimbrial subunit and at least three others appeared to be synthesized as precursor molecules that underwent processing. Cloned DNA containing CFA/I region 2 specified three polypeptides in minicells. Attempts to reduce the size of the cloned region 1 resulted in a derivative plasmid that carried the CFA/I structural gene but did not complement a region-2 recombinant plasmid to restore production of CFA/I fimbriae.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

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.     

Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis

Expression of the cloned levansucrase gene (sacB) was demonstrated in E. coli minicells by assay of the enzyme in crude extracts, SDS-polyacrylamide gel electrophoresis and immunoblotting. The existence of a precursor form of the enzyme of MW 53000 was also demonstrated and confirmed by the DNA sequence corresponding to the NH2 terminal region of the protein.