The large endogenous plasmid of Anacystis nidulans: Mapping, cloning and localization of the origin of replication

Summary Anacystis nidulans contains two cryptic plasmids of 8.0 (pANS) and 48.5 (pANL) kilobasepairs (kbp). A clone bank of the large plasmid pANL consisting of 7 Bam HI fragments has been established. The cloned fragments were used as radioactive probes to Bam HI, Sal I, Hind III and Eco R1 digests of pANL in blot hybridization experiments to verify the clones and map the restriction fragments. Further size characterization of the physical map was done by restriction analysis of the cloned fragments. The origin of replication has been located in the largest Bam HI fragment of the large plasmid.     

Conjugation in the cyanobacterium Anacystis nidulans

Summary Genetic recombination in the cyanobacterium Anacystis nidulans was first reported by Kumar (1962) and confirmed by Bazin (1968). Although genetic transformation in this organism was demonstrated by Shestakov and Khyen (1970) and Herdman (1973), conjugation does not seem to have been reported so far. In Escherichia coli, one common mode of gene transfer involves conjugation between donor and recipient cells. This conjugation is mediated by the formation of a pilus or conjugation tube through which the chromosome of the male cell passes into the recipient (female) cell (Hayes 1962). Offprint requests to: H.D. Kumar (Bot., B.H.U., Varanasi, India, after November only)     

Stability of cloned Brassica napus chloroplast DNA fragments in the cyanobacterium Anacystis nidulans R2

Summary Brassica napus (cv. Triton) chloroplast (cp) DNA BamHI gragments were inserted into a bacteria-cyanobacteria shuttle vector pCB4. The chloroplast genomic library was screened in Escherichia coli and 28 individual clones, which represent 94% of the total chloroplast genome, were isolated. Cyanobacterium Anacystis nidulans R2 was transformed with each member of the clone bank by selection for ampicillin resistance. A study of transformation efficiency showed dramatic variation (up to 200-fold) among recombinant clones. Furthermore, plasmid DNA reisolated from some cyanobacterial transformants exhibited instability. Variations in transformation efficiency and plasmid instability were shown to be DNA sequence specific. B. napus cpDNA clones were thus classified into three types according to their stability in the cyanobacterial host.     

Self-cloning in the cyanobacterium Anacystis nidulans R2: Fate of a cloned gene after reintroduction

Functional analysis of cloned genes often makes use of complementation after introducing these genes into cells of a mutant strain. Problems with this self-cloning step in the cyanobacterium Anacystis nidulans R2 have been encountered, which were mainly due to recombinational instability of gene and vector after transformation. Therefore, conditions determining the exchange of material between chromosome, insert and plasmids were studied to achieve the necessary stability. The fate of plasmid pME1, containing a wild-type methionine gene from A. nidulans R2, was investigated after its introduction into a Tn901-induced methionine mutant strain as recipient, so that the mutant chromosomal gene could be distinguished from the plasmid-borne wild-type copy. Two different recipients were constructed, one containing and one lacking the resident plasmid pCH1, which is a derivative of the indigenous small plasmid pUH24. When using the pCH1-free strain and with combined selection for both wild-type gene and vector, the original configuration of the genes in chromosome and vector was retained in the majority of the transformed cells, while the remaining transformants were reciprocal recombinants; under conditions of single selection mainly nonreciprocal recombination or loss of the vector was observed. When the recipient strain contained pCH1 additional recombinational events took place. The results show that under appropriate conditions a chromosomal gene cloned on a plasmid vector can be stably maintained in a majority of the transformants, thus making self-cloning experiments feasible in A. nidulans R2. On the other hand, the introduction of foreign DNA into the chromosome can be achieved by deliberately exploiting recombination between chromosome and plasmid.     

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

Summary Eighteen 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.Abbreviations EDTA ethylenediamine-tetraacetic acid - MTA mixed alkyltrimethylammonium bromide - TES N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid - Tricine N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]-glycine - Tris Tris(hydroxymethyl)aminomethane     

Characterization of a mutant of Anacystis nidulans r2 resistant to the natural herbicide, cyanobacterin

Cyanobacterin, a secondary metabolite produced by the cysnobacterium, Scytonema hofmanni, inhibits electron transport at a site in photosystem II. It was previously shown that a DCMU-resistant mutant of A. nidulans R2 was still susceptible to cyanobacterin (Gleason et al., Plant Science, 46 (1986) 5–10). Apparently, cyanobacterin acts at a site different from that of DCMU and similar PS II inhibitors. To confirm this conclusion, a cyanobacterin-resistant strain of A. nidulans R2 was produced by nitrosoguanidine mutagenesis and selected by growth in the presence of 4.7 μM cyanobacterin. Hill activity in mutant thylakoids was compared to that of the wild type membranes in the presence of ferricyanide and silicomolybdate as electron acceptors. Photosynthetic electron transport in the mutant membranes shows a high degree of resistance to cyanobacterin in both reactions. In contrast, the mutant exhibits the same susceptibility to DCMU inhibition as the wild type R2. Cyanobacterin acts at a unique site, inhibiting electron flow from quinone-A to quinone-B.     

Pleiotropic behaviour of a cyanophage AS-1-resistant mutant of Anacystis nidulans

Summary A cyanophage AS-1-resistant mutant strain of Anacystis nidulans exhibited a slow rate of nutrient uptake compared to the wild type. The increased Cu⁺⁺ sensitivity of the mutant could be correlated with higher rates of Cu⁺⁺ uptake. The results are discussed in the light of alterations in the proteins involved in permeability of the outer membrane.     

Regulatory interaction of photosynthetic nitrate utilization and carbon dioxide fixation in the cyanobacterium Anacystis nidulans

The rate of photosynthetic nitrate utilization in Anacystis nidulans is strongly influenced by the availability of carbon dioxide. This dependence can be relieved by inhibiting the metabolism of the ammonium derived from nitrate reduction. Nitrate uptake seems to be modulated through a sensitive regulatory system integrating the photosynthetic metabolism of carbon and nitrogen, with CO₂ fixation products antagonizing the inhibitory effect of ammonium derivatives.     

Effect of chloride ions on the S-state distribution and deactivation kinetics in preparations of the cyanobacterium Anacystis nidulans

The roles of Ca²⁺ and Cl⁻ on the photosynthetic O₂ yield under flash illumination have been examined in EDTA-washed preparations of the cyanobacterium Anacystis nidulans. Especially the effect of Cl⁻ deficiency on the O₂ yield and on the S-state distribution was analyzed. As the results show, omission of both Ca²⁺ and Cl⁻ (Mn²⁺ present) almost totally inhibited O₂ evolution. When Ca²⁺ was replaced by Na⁺, a substantial reduction of the O₂ yield was observed, but only a minor change in the S-state distribution occurred. However, when Cl⁻ was displaced by NO⁻₃, which is equivalent to Cl⁻ deficiency of the water-splitting complex, a substantial reduction of the O₂ yield and in addition a significant change in the S-state distribution was observed. The comparison of deactivation kinetics in NO⁻₃ containing samples with those in control samples indicated that Cl⁻ deficiency allowed accumulation of oxidizing equivalents up to the S₃ state but modified the final step of O₂ evolution. Moreover, those centers which advanced to the S₃ state in the absence of Cl⁻ deactivated in a special way which involved a faster deactivation of S₂ and an increased formation of S₋₁.     

Molecular cloning and characterization of ribosomal RNA genes from a blue-green alga, Anacystis nidulans

Summary Two PstI fragments (5.3x10⁶ and 4.3x10⁶ daltons) coding for Anacystis nidulans rRNA genes were cloned. The cloned rDNAs were characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis and the R-loop technique. The results indicated that both fragments contained 16S, 23S and 5S rRNA genes in this order. A tRNA gene(s) was detected in the spacer region between 16S and 23S rRNA genes. The organization of A. nidulans rRNA genes resembles those of E. coli and of Euglena chloroplasts rather than those of higher plant chloroplasts.     

Frequency of replication from alternative origins in the composite R plasmid NR1.

The relative frequency of initiation of DNA replication within the RTF-Tc and r-determinant components of the composite drug resistance plasmid NR1 in Proteus mirabilis was evaluated. Using fractionated radioactively labeled plasmid DNA, analytical procedures that distinguished between the two components of the composite plasmid were carried out. A mixture of uniformly 14C-labeled and 3H-pulse-labeled plasmid DNA (pulse-labeled origin[s] of replication) was used in each of three experiments. First, shear products of the DNA were analyzed using CsCl density gradient centrifugation. Second, fragmented DNA was hybridized to nonradioactive RTF-Tc and r-determinant DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNA was digested with restriction enzyme (EcoRI), producing a set of RTF-Tc and r-determinant fragments with differing 3H/14C isotpe ratios. The three experiments suggested that under the conditions used to accumulate replicating plasmid DNA molecules (DNA substrate limitation), the r-determinant origin of replication was preferentially utilized in the composite plasmid.     

Nucleotide sequence of the 16S-23S spacer region in the rrnA operon from a blue-green alga, Anacystis nidulans

Summary The nucleotide sequence of an entire spacer region between the 16S and 23S rRNA genes of the rrnA operon from a blue-green alga, Anacystis nidulans, has been determined. The spacer region is 545 base pairs long and encodes tRNA^fle and tRNA^Ala in the order of 16S rRNA-tRNA^fle-tRNA^Ala-23S rRNA. A striking feature is that the A. nidulans tRNA^fle gene contains no 3-CCA sequence while the tRNA^Ala gene does. These spacer tRNA genes show strong sequence homology with those of chloroplasts and bacteria.     

Mechanism of the light-state transition in photosynthesis: V. 77 K linear dichroism of Anacystis nidulans in State 1 and State 2

Linear-dichroism spectra of Anacystis nidulans at 77 K were determined for whole cells chemically fixed in light State 1 and light State 2. Whole cells were oriented by the squeezed gel technique using 5% gelatin 2.2 M sucrose gels. Peaks with positive dichroism were observed at 638 nm and 688 nm with shoulders at approx. 650 nm and 700 nm. The amplitude of the 650 nm shoulder was greater for cells in State 2 than those in State 1, and the State-2-minus-State 1 difference spectrum had a single peak at 656 nm. The linear dichroism spectrum of phycobilisomes isolated from A. nidulans showed peaks at 635 nm (phycocyanin) and 656 nm (allophycocyanin). The spectrum for thylakoid membranes free of phycobilisomes had one peak at 685 nm with a shoulder at 698 nm. We suggest that the change in dichroism at 656 nm between cells in State 1 and State 2 results from a change in orientation of the allophycocyanin core of the phycobilisome. This result is discussed in the context of our model for the light-state transition in phycobilisome-containing organisms.     

Electron-transport reactions in cytoplasmic and thylakoid membranes prepared from the cyanobacteria (blue-green algae) Anacystis nidulans and Synechocystis PCC 6714

Cytoplasmic and thylakoid membranes prepared from the cyanobacteria Anacystis nidulans and Synechocystis PCC 6714 were compared for their electron-transport activities. High cytochrome oxidase activity, which was sensitive to cyanide and azide, was found only in the thylakoid membranes of both strains. Activities of NAD(P)H-cytochrome c and succinate-cytochrome c oxidoreductase were low in both membranes from both strains. The NADH-cytochrome c oxidoreductase activity of the cytoplasmic membranes from Synechocystis was markedly stimulated by quinones, among which 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) was the most effective. High NADH-DBMIB oxidoreductase activity, which was relatively resistant to salt washing, was found in the cytoplasmic membranes from Synechocystis.     

The cloning of Aspergillus nidulans mitochondrial DNA in Escherichia coli on plasmid pBR322

Summary We report the cloning of almost the entire mitochondrial DNA of Aspergillus nidulans on plasmid pBR322 in Escherichia coli. Only one fragment containing about 11% of the mitochondrial genome has not been cloned. We believe that this fragment cannot be maintained in E. coli on the pBR322 plasmid.