Characterization of a replication mutant of the bacteriocinogenic plasmid Clo DF13.

In a previous paper (Kool, A.J. and Nijkamp, H.J.J. (1974) J. Bacteriol. 120, 569--578) the isolation of a mutant of the bacteriocinogenic plasmid Clo DF13-Rep3, has been described. It was observed that cells harbouring the wild type plasmid synthesize more plasmid DNA cells harbouring the wild type plasmid. This paper deals with the characterization of the nature of this plasmid-specific mutation. The following properties of the Clo DF13-Rep3 mutant plasmid could be observed: 1. The plasmid-specific mutation did not lead to a significant change in the sedimentation value of Clo DF13 DNA. 2. The specific rate of Clo DF13-Rep3 DNA synthesis (expressed as the number of plasmid DNA molecules synthesized per min) is on average seven times the specific rate of wild type Clo DF13 DNA synthesis. 3. Also chromosomeless minicells, harbouring the Clo DF 13-Rep 3 plasmid, contain about seven times more plasmid DNA as wild type Clo DF13 harbouring minicells. 4. The replication time of the Clo DF13-Rep3 plasmid is approx. 90 s at 30 degrees C and does not differ significantly from the replication time of the wild type plasmid. 5. The Rep3 mutation did not alter the dependence of Clo DF13 plasmid replication on the dnaA and dnaC gene products. 6. The plasmid-specific mutation is cis-dominant over wild type. The data presented in this paper indicate that this mutant plasmid is not affected in the elongation but in the initiation of plasmid DNA replication.

Most AAL toxin-sensitive Nicotiana species are resistant to the tomato fungal pathogen Alternaria alternata f. sp. lycopersici.

The phytopathogenic fungus Alternaria alternata f. sp. lycopersici produces AAL toxins required to colonize susceptible tomato (Lycopersicon esculentum) plants. AAL toxins and fumonisins of the unrelated fungus Fusarium moniliforme are sphinganine-analog mycotoxins (SAMs), which are toxic for some plant species and mammalian cell lines. Insensitivity of tomato to SAMs is determined by the Alternaria stem canker gene 1 (Asc-1), and sensitivity is associated with a mutated Asc-1. We show that SAM-sensitive species occur at a low frequency in the Nicotiana genus and that candidate Asc-1 homologs are still present in those species. In Nicotiana spp., SAM-sensitivity and insensitivity also is mediated by a single codominant locus, suggesting that SAM-sensitive genotypes are host for A. alternata f. sp. lycopersici. Nicotiana umbratica plants homozygous for SAM-sensitivity are indeed susceptible to A. alternata f. sp. lycopersici. In contrast, SAM-sensitive genotypes of Nicotiana spegazzinii, Nicotiana acuminata var. acuminata, Nicotiana bonariensis, and Nicotiana langsdorffii are resistant to A. alternata f. sp. lycopersici infection concomitant with localized cell death. Additional (nonhost) resistance mechanisms to A. alternata f. sp. lycopersici that are not based on an insensitivity to SAMs are proposed to be present in Nicotiana species.

Structure and nucleotide sequence of the region encoding the mobilization proteins of plasmid CloDF13.

Mobilization of the non-conjugative plasmid CloDF13 requires both gene products of a conjugative plasmid and CloDF13-encoded information. About 30% of the CloDF13 genome is involved in plasmid transfer. The CloDF13 mobilization region comprises sequences acting in cis (bom, basis of mobilization) as well as protein-coding sequences (mob). Here we present the nucleotide sequence of the genes encoding the CloDF13 mobilization proteins. We confirmed the previous genetic data that the plasmid CloDF13 encodes two proteins involved in plasmid mobilization. The information for these proteins, designated B and C having Mrs of 57,890 and 15,870, respectively, is located within one operon directed by a promoter at 94% of the CloDF13 genome. The gene encoding the smaller protein is located distally within this operon. Transcription proceeds counter-clockwise and is terminated beyond gene C, although it can not be excluded that attenuation of the transcript occurs in the intergenic region. The role of the CloDF13 mobilization proteins in plasmid transfer will be discussed.

Molecular characterization of an Escherichia coli mutant with a temperature-sensitive malonyl coenzyme A-acyl carrier protein transacylase.

The temperature-sensitive malonyl CoA-ACP transacylase found in the Escherichia coli strain LA2-89, carrying the fabD89 allele, was shown to result from the presence of an amber mutation in the fabD gene, at codon position 257, in combination with the supE44 genotype of this strain. The truncated form of the protein produced as the result of the amber mutation was demonstrated to be enzymatically inactive, whereas amber suppression rendered the resulting enzyme temperature labile. Site-directed mutagenesis of codon 257 revealed a requirement for an aromatic amino acid at this position in the polypeptide chain, to assure temperature stability of the enzyme.

Molecular analysis of a new cytoplasmic male sterile genotype in sunflower.

Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS-associated restriction fragment length polymorphisms (RFLPs) were found in the vicinity of the atpA locus, generated by various restriction enzymes. The organization of the mitochondrial genes 26S rRNA, 18S + 5S rRNA and coxII was investigated by Southern blot analysis. These genes have similar structures in fertile and all studied sterile sources. Using the atpA probe, 5 from the 6 investigated CMS genotypes showed identical hybridization patterns to the Petiolaris CMS line, which is used in all commercial sunflower hybrids. Only 1 cytoplasm derived from an open pollination of Helianthus annuus ssp. texanus, known as ANT1, contained a unique mitochondrial DNA fragment, which is distinguishable from the fertile and sterile Petiolaris genotypes and from all investigated CMS genotypes. Male fertility restoration and male sterility maintenance of the ANT1 line are different from the Petiolaris CMS system, which is a confirmation that a novel CMS genotype in sunflower has been identified.

Regulatory role of adenine nucleotides in the biosynthesis of guanosine 5'-monophosphate.

Derepression of the synthesis of inosine 5'-monophosphate (IMP) dehydrogenase and of xanthosine 5'-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5'-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.

Mutants of the Clo DF13 plasmid in Escherichia coli with a decreased bacteriocinogenic activity.

Three Clo DF13 mutant plasmids (designated as clp03, clp05 and clp21) that show a decreased cloacin activity were isolated. The decreased cloacin activity was not due to a reduced number of Clo DF13 copies per cell. The cloacins produced by the clp03 and the clp21 mutant plasmids have a strongly decreased killing activity in vivo in comparison with the wild type cloacin and the cloacin of the clp05 mutant plasmid. Furthermore no lacunae could be observed from clp03 or clp21 harbouring strains, while strains harbouring the clp05 plasmid showed a 50-100 times decreased frequency of lacunae. In addition the clp05 mutant showed a decreased rate of RNA synthesis in clp05 harbouring Escherichia coli minicells. No complementation between the three mutant plasmids was observed. We suggest that the clp03 and clp21 mutations are located in the gene coding for the cloacin. Since the cloacin produced by the clp05 mutant plasmid has retained all the known wild type cloacin activities, the reduced inhibition zone in the stab test is probably caused by a mutation affecting the expression of the cloacin gene. The nature of this mutation is discussed.

Isolation and characterization of a copy mutant of the bacteriocinogenic plasmid Clo DF13.

After nitrosoguanidine mutagenesis, strain Escherichia coli P678-54, bacteriocinogenic for Clo DF13, yielded a mutant strain that showed an enhanced bacteriocin production. The results from conjugation experiments indicated that the mutation, responsible for the enhanced bacteriocin production, is located on the Clo DF13 plasmid. The following properties of strains harboring the mutant Clo DF13 plasmid could be observed. (i) The bacteriocin production in these strains can be further enhanced at least fourfold by mitomycin C. (ii) The fraction of spontaneously induced cells, as revealed by lacunae experiments, in cultures of these strains is about nine times higher than in cultures of wild-type Clo DF13-harboring strains. (iii) Chromosomeless minicells from strain P678-54 harboring the mutant Clo DF13 plasmid synthesize about six times more deoxyribonucleic acid, ribonucleic acid, and protein as compared to wild-type Clo DF13-harboring minicells. (iv) Analysis of this mutant Clo DF13-specific ribonucleic acid and protein on polyacrylamide gels revealed mainly the same ribonucleic acid and polypeptide species as synthesized by the wild-type Clo DF13 minicells, but in larger amounts (Kool et al., 1974). (v) Segregation experiments, using a strain with temperature-sensitive polymerase I, show that mutant Clo DF13-harboring cells contain an average of 70 Clo DF13 copies per cell, whereas wild-type Clo DF13-harboring cells contain only about 10 Clo DF13 copies per cell. The data presented in this paper indicate that the mutation on the Clo DF13 plasmid leads to an altered control of Clo DF13 replication and results in an enhanced number of Clo DF13 copies per cell. As a secondary effect, this enhanced number of Clo DF13 copies enhances the probability of "spontaneous" induction per cell. Since the mutation is plasmid specific and affects the number of plasmid copies produced, one can conclude that the Clo DF13 plasmid is not dependent solely on chromosomal information, but that at least plasmid base sequences are involved in Clo DF13 plasmid replication.

Replication of the bacteriocinogenic plasmid Clo DF13 in thermosensitive Escherichia coli mutants defective in initiation or elongation of deoxyribonucleic acid replication.

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.

Bacteriocinogenic Clo DF13 minicells of Escherichia coli synthesize a protein that accounts for immunity to bacteriocin Clo DF13: purification and characterization of the immunity protein.

Cloacin DF13 inhibits protein synthesis both in vivo and in vitro by inactivation of ribosomes. In this paper we describe the purification, from purified bacteriocinogenic plasmid DF13 (Clo DF13)-harboring minicells of Escherichia coli, of an acidic protein (molecular weight about 10,000) that prevents the inhibition of in vitro protein synthesis by cloacin DF13. It turned out that this protein is one of three Clo DF13 plasmid-specific polypeptides that are synthesized by the noninduced Clo DF13 plasmid in minicells. Furthermore, we observed that, after induction with mitomycin C, the amount of immunity protein is only slightly increased, whereas the cloacin DF13 synthesis is increased about 40 times. These results imply that the genes that code for immunity protein and cloacin DF13 are not located in the same regulatory unit. Under noninduced conditions, apparently an excess of immunity protein is synthesized, because this amount of immunity protein is even sufficient to neutralize the strongly increased amount of cloacin after induction, since growth of induced cloacinogenic cells is continued.

Bacteriocinogenic Clo DF13 minicells of Escherichia coli synthesize a protein that accounts for immunity to bacteriocin Clo DF16: action of the immunity protein in vivo and in vitro.

The Clo DF13 plasmid-specific immunity protein is able to prevent the inhibitory effect of cloacin DF13 on in vitro protein synthesis. We have shown, by gel filtration, that direct binding of the Clo DF13 immunity protein to cloacin occurs in vitro. This cloacin DF13-immunity protein complex is rather stable, and the cloacin present in the complex is no longer able to cause inhibition of in vitro protein synthesis. The binding of immunity protein to cloacin DF13 is rather specific because the Clo DF13 immunity protein does not bind to in vitro inactive cloacin and binds very poorly to the closely related bacteriocin colicin E3. Furthermore, we present data which strongly suggest that in vitro at least a fourfold excess of immunity protein is required to ensure that every cloacin molecule is inactivated by cloacin-immunity protein complex formation. Only a fraction (an about equimolar amount) of the immunity protein molecules, however, actually binds to cloacin DF13. The existence of an immunity protein-cloacin complex in vivo was concluded from the observation that cloacin, purified by chromatography on diethyl-(2-hydroxypropyl)-aminoethyl Sephadex in the absence of urea, contains an about equimolar amount of a second protein which comigrates with immunity protein on sodium dodecyl sulfate-polyacrylamide and urea-polyacrylamide gels. In an in vitro protein-synthesizing system, this component appeared to behave identical to the Clo DF13 immunity protein. The purified immunity protein-containing cloacin was at least 80 times less active in inhibiting in vitro protein synthesis, compared to cloacin, free of immunity protein. These data imply that few, if any, cloacin DF13 molecules are present in cloacinogenic cells as active, free cloacin molecules.

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host. I. Localisation of the gene and identification and subcellular localisation of the gene H product.

The gene expression of the Clo DF13 "replication region", located between 1.8% and 12% on the plasmid genome, was studied using newly constructed Clo DF13 insertion and deletion mutants. We were able to detect a Clo DF13 specified protein of 6 kilodaltons (kd) by electrophoretic analysis of plasmid proteins, synthesized in Escherichia coli minicells, on 14-25% gradient polyacrylamide gels. The gene encoding this protein was mapped between 1.8% and 12% on the Clo DF13 genome. The nucleotide sequence of this region, as determined by Stuitje et al. (1980), revealed three open reading frames each potentially coding for a protein of 6 kd. Since these three proteins differ in amino acid composition we could distinguish which of these proteins was actually synthesized, by labeling Clo DF13 proteins with specific 14C-labeled amino acids. We found that gene H, located between 9.3% (bp 744) and 11% (bp 893), encodes the observed protein of 6 kd (denominated protein H). With respect to the subcellular localization we observed that protein H, which contains a large hydrophobic region at its C-terminal part, is predominantly present in the bacterial membrane. Although gene H is located close to the region known to be involved in Clo DF13 replication, its gene product, protein H, is not essential for the plasmid DNA replication process. The possibility of the existence of a comparable protein encoded by the related plasmid Col E1 will be discussed.

Isolation, characterization and restriction endonuclease mapping of the Petunia hybrida chloroplast DNA.

A procedure is developed for the isolation of intact chloroplast DNA (ctDNA) from Petunia hybrida. The molecular weight, calculated from contour length measurements, is 96.0 +/- 4.5 x 10(6) daltons. This value is in good agreement with the value of 101.2 x 10(6) daltons that was estimated from the electrophoretic mobilities of restriction endonuclease fragments of ctDNA. Analysis of petunia ctDNA in neutral CsCl gradients revealed the presence of only one type of DNA at a buoyant density of 1.6987 +/- 0.0005 gcm-3. This corresponds with a GC-content of 39.3 +/- 0.5%. A physical map of petunia ctDNA was constructed by using the restriction endonucleases Sal I, Bgl I, Hpa I and Kpn I. The map indicates that petunia ctDNA contains two copies of a sequence in an inverted orientation. The inverted repeat regions have a minimum length of 10 x 10(6) daltons. Hybridization data indicate that part of the inverted repeat regions contain the genes for chloroplast ribosomal RNAs.

Ds read-out transcription in transgenic tomato plants.

To select for Ds transposition in transgenic tomato plants a phenotypic excision assay, based on restoration of hygromycin phosphotransferase (HPT II) gene expression, was employed. Some tomato plants, however, expressed the marker gene even though the Ds had not excised. Read-out transcriptional activity of the Ds element is responsible for the expression of the HPT II gene. Transcription initiation was mapped to multiple positions spanning about 300 bp in the subterminal part of the Ds element. In this respect Ds in tomato resembles the maize element Mu1, which also promotes transcription outward from the element. Transposon read-out transcription might thus supply an additional general mechanism for controlling plant gene expression.

Genetic information of the bacteriocinogenic plasmid Clo DF13 involved in the inhibition of the multiplication of double stranded DNA phages.

The presence of plasmid Clo DF13 in Escherichia coli cells alters the response of these cells to infection with the double stranded DNA phages P1vir, lambda vir or T1. The multiplication of these phages is reduced in Clo DF13 harbouring cells, resulting in an altered burstsize and plaque morphology. The degree of reduction is correlated to the amount of particular Clo DF13 gene product(s) in the cell. The genetic information of Clo DF13 involved in this plasmid-phage interaction could be located, using insertion and deletion mutants of Clo DF13, between 29 an 62% on the Clo DF13 physical map. The genetic analysis of this region shows that at least two different genes, K and L, coding for polypeptides with a molecular weight of respectively 21 KD and 10.5 KD, are located in this region. The results presented, indicate that gene L and not gene K is involved in the interaction of Clo DF13 with the propagation of double stranded DNA phages.

An effective chemical mutagenesis procedure for Petunia hybrida cell suspension cultures.

By using mercury(II)-chloride (HgCl2) and Dl-6-fluorotryptophan (6FT) as positive selection conditions we were able to show that N-methyl-N'-nitro-N-nitrosoguanidine (NG) is an effective mutagen for Petunia hybrida suspension cells.A number of the 205 calli resistant to HgCl2 and 17 calli resistant to 6FT were isolated. The highest mutation frequency was 1.0 x 10(-5) and 2.0 x 10(-6) for HgCl2 and 6FT, respectively. A preliminary characterization of the mutants is presented.A significant increase in the number of drug-resistant calli was only obtained at NG-concentrations (5-40 µg/ml) that had no observable effect on the survival of the mutagenized cultures.