W. E. Borrias

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.

Homology of plasmids in strains of unicellular cyanobacteria

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 106 and 32.7 × 106. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 106 and 24 × 106. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 106 and 2.0 × 106, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.

Vectors for cloning in cyanobacteria: Construction and characterization of two recombinant plasmids capable of transformation to Escherichia coli K12 and Anacystis nidulans R2

SummaryTwo plasmids were constructed consisting of the E. coli vector pACYC184 and the cyanobacterial plasmid pUC1. These recombinants, designated pUC104 and pUC105, can be transformed to E. coli K12 as well as to the cyanobacterium Anacystis nidulans R2 and in both hosts they express their antibiotic markers. pUC104 and pUC105 differ with respect to the location and the orientation of the pACYC184 segment in pUC1. pUC104 was found to be stable under all circumstances. Transformation of pUC105 to A. nidulans R2 gave intact plasmids when chloramphenicol was the selective agent, but upon ampicillin selection a deletion derivative was produced identical to pUC1. Further characteristics of pUC104 and pUC105 are described and their usefulness as cloning vectors is discussed.

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

SummaryEighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.

Complementation experiments between conditional lethal mutants of bacteriophage ϕX174

SummaryComplementation experiments with temperature sensitive (ts) and suppressor sensitive (sus) mutants of bacteriophage ϕX174 unambiguously revealed five cistrons on the basis of a clear bipartition of burst sizes.A new group of sus mutants (emeralds) was found, defective in a function essential for growth in Shigella sonnei V64.The complementation between ts and sus mutants was in general asymmetric in that the yield of ts particles was lower than that of the sus particles. The mutants of one cistron (defective in RF-replication) showed a completely asymmetric complementation behaviour both of ts and sus mutants. The ts mutants of this group, which show to be early, appear to be defective in two functions.The possibility is discussed that in each cell only one phage genome is replicated. This would explain both kinds of asymmetric complementation and the low burst sizes that were obtained when mutants of particular genes were complemented.

Induction and isolation of mutants in a specific region of gene A of bacteriophage ∅X174

Abstract A new method is described to isolate mutants located in any desired region of the genome of small DNA bacteriophages. The method is based on the induction of mutations on specific DNA fragments obtained after cleavage of double-stranded DNA molecules with restriction enzymes.

Genetic complementation between bacteriophage ϕX174 DNA molecules in spheroplasts

SummaryComplementation experiments were performed with ss and RF DNA1 of bacteriophage ϕX174. Spheroplasts of Escherichia coli K12W6 were mixedly infected under restrictive conditions witha)RF DNAs of mutants in different cistrons,b)ss DNAs of mutants in different cistrons,c)RF DNA of a mutant in one cistron and ss DNA of a mutant in another cistron. In case a) complementation could be demonstrated unambiguously. The phage yield after complementation amounts to about 1–5 percent of the yield of each mutant under permissive conditions. In case b) the ratio of the total yield over the yield of the selfed controls was the same as in case a) but amounts to only 0.5 percent of the yield under permissive conditions. Moreover, a rather high percentage of wild type particles appeared in the burst. Therefore we could not distinguish between rescue of mutant DNA and complementation. Case c) was intermediate between a) and b). These complementation patterns are discussed.