Maintenance of multicopy plasmid Clo DF13 III. Role of plasmid size and copy number in partitioning.

To elucidate the mechanisms that operate in plasmid maintenance, we analysed the stability of different combinations of Clo DF13 derivatives present in the same bacterial cell. From the data described in this paper we conclude: (i) each Clo DF13 plasmid molecule has an equal chance of colonizing daughter cells upon cell division, (ii) the Clo DF13 minimal replicon harbours functions involved in plasmid segregation and incompatibility, (iii) in the case of cells harbouring plasmid replicons which differ in size, the smaller plasmid is gradually lost from the cell population, (iv) in the case of cells habouring plasmid replicons which differ in copy number, the lower copy number plasmid is always lost from the cell population. The effect of plasmid copy number is dominant over the effect of plasmid size.

Maintenance of multicopy plasmid Clo DF13 in E. coli cells: evidence for site-specific recombination at parB.

Certain derivatives of copy-control mutants of plasmid Clo DF13 are not stably inherited in E. coli. These plasmids, predominantly present as multimeric DNA molecules, lack a specific region, designated parB. Here we present the nucleotide sequence of this parB region spanning 328 bp between 46% and 49% on the plasmid genome. parB is a noncoding region with extensive internal symmetry. A recA-independent, site-specific resolution process occurs between two intramolecular parB sites present in direct orientation relative to each other. A gene located in the direct vicinity of parB, gene L, is not essential for parB functioning. However, our genetic data indicate that transcription from the gene L-containing operon into parB is required. We conclude that the efficient maintenance of Clo DF13 cop derivatives containing parB is provided by resolution of mutimeric molecules. Because Clo DF13 wt and cop derivatives have a different response to the deletion of parB we postulate that two different recombination systems, a parB-dependent and a parB-independent system, operate in the efficient maintenance of Clo DF13 plasmids.

Structure and regulation of gene expression of a Clo DF13 plasmid DNA region involved in plasmid segregation and incompatibility.

The bacteriocinogenic plasmid Clo DF13 contains genetic information involved in the accurate partitioning of the plasmid (parA and parB) as well as in incompatibility phenomena (incA, B, C and D). In this paper we report on the primary structure and regulation of gene expression of the 29% - 50% part of Clo DF13, containing the DNA regions incA, incB and parB as well as genes K and L. According to the results of our DNA sequence analysis, mapping of transposon insertions, RNA blotting and S1 mapping experiments, we conclude that: a) genes K and L are transcribed as one operon; transcription of this operon is initiated at a promoter (P2) located at 32.5% and proceeds in a clockwise direction. b) treatment of cells with mitomycin-C, significantly enhances transcription from P2, although this promoter is probably not directly repressed by lexA protein. c) Termination of transcription of this operon occurs between genes K and L, as well as distal to gene L. The possible role of gene products and/or sites, located within the 29-50% DNA region, in plasmid incompatibility and segregation is discussed.

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. I. Localisation and mutual interactions of four Clo DF13 incompatibility regions.

The incompatibility properties of the bacteriocinogenic plasmid Clo DF13 have been examined. By using Clo DF13, Clo DF13 deletion, and transposon insertion mutants as well as compatible R plasmids into which Clo DF13 fragments have been cloned, we could identify and localise four different incompatibility regions on the Clo DF13 genome. These regions, designated incA, incB, incC, and incD are located in the following positions: incA about 32%, incB between 45% and 50%; incC about 97% and incD between 1.8% and 9% of the Clo DF13 genome. We studied the contribution of each of the four inc regions, separately and/or in combination with each other, to the incompatibility between two plasmid replicons. Two types of incompatibility can be distinguished: Type I evoked by incD, that overlaps the replication control area of Clo DF13 and type II, caused by incA, B and C. From our observations we present a model for plasmid incompatibility based on a combination of the existing repressor dilution and membrane attachment models.

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.

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host. II. Functions and regulation of synthesis of the gene H product.

We studied the expression of gene H, located between 9.3% and 11% on the CLo DF13 genome, as well as the functions of the gene product. We found that treatment of bacterial cells with mitomycin-C results in the induced synthesis of three Clo DF13 specified proteins namely cloacin DF13, immunity protein and protein H. Evidence was obtained that the genes encoding these proteins form one, mitomycin-C induceable, operon; the promoter at 32% in front of the cloacin gene is essential for the induced expression. Furthermore we could demontrate that protein H is involved in the lethal effect of mitomycin-C treatment of bacteriocinogenic cells. The data in this paper show that a high concentration of protein H in cells, due either to an induced expression of gene H (mitomycin-C induction) or to a gene dosage effect (Clo DF13 copl Ts copy control mutant), results in the lysis of bacterial cells. The implication of these data are discussed.

Subcellular distribution of RNA sequences complementary to sonchus yellow net virus RNA.

RNA isolated from free and membrane-bound polyribosomes of sonchus yellow net virus (SYNV)-infected tobacco was hybridized to SYNV RNA. RNA from free polyribosomes was shown to be complementary to nearly 100% of the SYNV genome, whereas RNA from membrane-bound polyribosomes was complementary to only 40% of the SYNV RNA. When RNA derived from the two classes of polyribosomes was fractionated by chromatography on oligo(dT)-cellulose, sequences complementary to SYNV RNA were present in both bound and unbound fractions. Neither nuclear RNA nor poly(A)-containing nuclear RNA from SYNV-infected plants hybridized to SYNV RNA. The results suggest that SYNV messenger RNAs are selectively partitioned in the cytoplasm and that hybridizing sequences are not abundant in the nuclei of infected cells.

Development of the nitrogen-fixing and protein-synthesizing apparatus of bacteroids in pea root nodules.

Some aspects of root nodule development of Pisum sativum inoculated with Rhizobium leguminosarum were examined. 1. Nitrogenase activity (measured as acetylene reduction) appears to be preceded by leghemoglobin synthesis (measured immunologically). 2. Syntheses of component I and component II of nitrogenase are not strictly coordinated. Synthesis of component I starts before component II. 3. Plant and bacteroid protein synthesis (measured by [35S]sulfate labeling) in root nodules declines rapidly during nodule development. Corresponding with this decline is a decrease in quantity and quality of rRNA.