Effect of the natural algicide,cyanobacterin, on a herbicide-resistant mutant of anacystis nidulans R2

Cyanobacterin, a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni, inhibits the growth of algae and plants. This compound is a potent inhibitor of photosynthetic electron transport and acts at a site in photosystem II (PS II). To further define the site of action of cyanobacterin, the effects of this natural product were investigated in a herbicide-resistant mutant of the cyanobacterium, Anacystis nidulans R2D2-X1. A. nidulans R2D2-X1 was reported to grow and maintain photosynthetic electron transport in the presence of 20 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 6.0 μM atrazine. Resistance was attributed to an altered 32 kDa (quinone-binding, QB) protein [6]. In the presence of Hill electron acceptors, K₃Fe(CN)₆ and dichlorophenol-indophenol (DCPIP), spheroplasts of A. nidulans R2D2-X1 were inhibited by cyanobacterin at the same concentration as wild type spheroblasts. Under these same conditions, spheroplasts of the mutant maintained their resistance to DCMU. Similar results were obtained with isolated thylakoid membranes. In contrast, silicomolybdate reduction, which is resistant to DCMU inhibition, was very sensitive to cyanobacterin. We conclude that cyanobacterin inhibits electron transport in PS II at a unique site which is different from that of DCMU.     

The natural herbicide, cyanobacterin, specifically disrupts thylakoid membrane structure in Euglena gracilis strain Z

Abstract Cyanobacterin is a natural product produced by the cyanobacterium (blue-green alga), Scytonema hofmanni . The compound has been chemically characterized and shown to inhibit electron transprot in photosystem II. Although the herbicide is lethal to photoautotrophs, photoheterotrophically-grown organisms such as Euglena gracilis can survive and grown in saturating concentrations of cyanobacterin. Electron micrographs of treated E. gracilis cells show extensive damage to the thylakoid membranes of the chloroplasts, similar to the effects observed with 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU). Unlike the synthetic herbicide, cyanobacterin specifically disrupts thylakoid membranes and does not affect other cellular membranes or heterotrophic growth.     

Activity of the natural algicide, cyanobacterin, on angiosperms

Cyanobacterin is a secondary metabolite produced by the cyanobacterium (blue-green alga) Scytonema hofmanni. The compound had previously been isolated and chemically characterized. It was shown to inhibit the growth of algae at a concentration of approximately 5 micromolar. Cyanobacterin also inhibited the growth of angiosperms, including the aquatic, Lemna, and terrestrial species such as corn and peas. In isolated pea chloroplasts, cyanobacterin inhibited the Hill reaction when p-benzoquinone, K₃Fe(CN)₆, dichlorophenolindophenol, or silicomolybdate were used as electron acceptors. The concentration needed to inhibit the Hill reaction in photosystem II was generally lower than the concentration of the known photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. Cyanobacterin had no effect on electron transport in photosystem I. The data indicate that cyanobacterin inhibits O₂ evolving photosynthetic electron transport in all plants and that the most probable site of action is in photosystem II.     

Site of action of the natural algicide,cyanobacterin, in the blue-green alga,Synechococcus sp.

Cyanobacterin is a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni. Highly purified cyanobacterin was found to inhibit the growth of many cyanobacteria at a minimum effective dose of 2 g/ml (4.6 M). The antibiotic had no effect on eubacteria including the photosynthetic Rhodospirillum rubrum. The site of action of cyanobacterin was further investigated in the unicellular cyanobacterium, Synechococcus sp. Electron micrographs of antibiotic-treated Synechococcus cells indicated that cyanobacterin affects thylakoid membrane structure. The antibiotic also inhibited light-dependent oxygen evolution in Synechococcus cells and in spheroplasts. These data support our conclusion that cyanobacterin specifically inhibits photosynthetic electron transport. This activity is similar to herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). The anhydro analog of cyanobacterin had no biological activity.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DCPIP dichlorophenolindophenol     

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).     

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

Antimycin-insensitive mutants of Candida utilis. II. The effects of antimycin on cytochrome b

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutant. A similar reoxidation is observed in the wild type in the presence of low concentrations of antimycin.

2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steadystate reduction; reduction in the presence of substrate, cyanide and oxygen; the ‘red shift’ and lowering of E′₀ of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable.

3. The red shift in the mutant is more extensive than in the wild type.

4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes.

5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant.

6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH₂-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycinbinding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Functional analysis of photosystem II in a PsbO-1-deficient mutant in Arabidopsis thaliana

The Arabidopsis thaliana mutant psbo1 (formerly the mutant LE18-30), which contains a point mutation in the psbO-1 gene leading to defective expression of the PsbO-1 protein, has recently been described [Murakami, R. et al. (2002) FEBS Lett. 523, 138-142]. This mutant completely lacks the PsbO-1 protein and overexpresses the PsbO-2 protein. To further study the effect of PsbO-1 deficiency on the function of photosystem II, the polyphasic chlorophyll a fluorescence rise and flash fluorescence induction and decay of the relative fluorescence quantum yield were measured in whole leaves from wild type and the psbo1 mutant. Additionally, flash oxygen yield experiments were performed on thylakoid membranes isolated from wild type and the psbo1 mutant. The results obtained indicate that during fluorescence induction the psbo1 gene exhibited an enhanced O to P transition. Additionally, while the J to I transition in wild type accounted for more than 30% of the total fluorescence yield, in the mutant it accounted for less than 2% rise in the total. Analysis of the flash-induced fluorescence rise in the presence of DCMU indicated that in wild type the ratio of PS IIalpha to PS IIbeta reaction centers was approximately 1.2 while in the mutant the ratio was approximately 0.3. Fluorescence decay kinetics in the absence of DCMU indicated that electron transfer to QB was significantly altered in the mutant. Fluorescence decay kinetics in the presence of DCMU indicated that the charge recombination between QA- and the S2 state of the oxygen-evolving complex was retarded. Furthermore, flash oxygen yield analysis indicated that both the S2 and S3 states exhibited significantly longer lifetimes in the psbo1 mutant than in wild type. Our data indicate that while PsbO-1-deficient plants can grow photoautotrophically (although at a reduced growth rate) the photochemistry of PS II is significantly altered.     

The photosynthetic electron transport chain of a mutant strain of Chlamydomonas reinhardi lacking P700 activity

Components and reactions of the photosynthetic electron transport chain were investigated in a mutant strain of the unicellular green alga Chlamydomonas reinhardi which is virtually devoid of the System I reaction center pigment, P700. The plastocyanin and ferredoxin isolated from this mutant strain are both qualitatively and quantitatively indistinguishable from that isolated from the wild-type strain. Cytochromes with absorption maxima at 553 and 559 nm cannot be oxidized by far-red light in the mutant strain, but they are reduced by red light. The Fe(CN)₆³⁻-Hill reaction in the mutant strain is about 50% of that of wild type at high light intensities; however, at low light levels, it is not significantly different from the rate of wild type. These results are interpreted to indicate that P700 is not so closely involved or complexed with adjacent electron carriers or with the reaction center of System II that destruction of P700 necessarily leads to alteration of these other components of the electron transport chain. It is suggested that the Hill reaction data can be explained by the existence of two separate sites for photoreduction of Fe(CN)₆³⁻ in wild type, whereas only one remains operative in the mutant strain.     

A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition.

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

Photooxidation reactions of diphenylcarbazide and their DCMU-sensitivity in thylakoids of the blue-green alga Oscillatoria chalybea

Thylakoids of Oscillatoria chalybea are able to split water. The Hill reaction of these thylakoids is sensitive to DCMU. Diphenylcarbazide can substitute for water as the electron donor to photosystem II with these fully functioning thylakoids. However, the diphenylcarbazide photooxidation is completely insensitive to 3-(3,4-dichlorophenyl)-N-N-dimethyl urea (DCMU) at high diphenylcarbazide concentrations. In with Tris-treated Oscillatoria thylakoids the water splitting capacity is lost and diphenylcarbazide restores electron transport through photosystem II as occurs with higher plant chloroplasts. However, also these photoreactions are insensitive to DCMU. If diphenylcarbazide acts in Oscillatoria as an electron donor to photosystem II the result suggests that diphenylcarbazide feeds in its electrons behind the DCMU inhibition site. This in turn indicates that in Oscillatoria the site of inhibition of DCMU is on the donor side of photosystem II.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-N-N-dimethyl urea - DPC diphenylcarbazide - DCPiP 2,6-dichlorophenol indophenol - TMB tetramethyl benzidine - A-2-sulf anthraquinone-2-sulfonate     

Production of the antibiotic-algicide cyanobacterin LU-2 by a filamentous cyanobacterium Nostoc sp.]

A strain of cyanobacterium of Nostoc has been isolated, and found to produce a new antibiotic cyanobacterin LU-2. The antibiotic is synthesized by the cyanobnacterium under intensive cultivation conditions in a liquid mineral medium. Cyanobacterin LU-2 is an exometabolite; its maximum accumulation in the medium is achieved at 34 degrees. Cyanobacterin LU-2 is active against many cyanobacteria tested, including those of Microcystis and Aphanizomenon which are principals to give rise to blooms in fresh water supplies. It is poorly active against green algae and inactive against fungi and bacteria. The antibiotic hinders cell division in Synechococcus sp. R-2 (PCC 7942). It causes compression of the cytoplasm and exfoliation of the cell contents from cell wall; the distance between tylacoids is increased and their destruction is observed. The antibiotic hinders markedly light-dependent oxygen evolution. Cyanobacterin LU-2 is substance of a phenolic nature containing amino-sugar.     

Stimulation of Photosystem I Electron Transport by High Concentration of 3-(3,4-Dichlorophenyl)-1,1-dimethyl Urea in Uncoupled Chloroplasts

The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol → methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate-limitation imposed by energy coupling was not affected. Using N,N,N′,N′-tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol-indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts.     

On two photoreactions in System II of plant photosynthesis

Light-induced absorbance changes of cytochrome b₅₅₉ and C₅₅₀ in chloroplasts indicate that noncyclic electron transport from water to ferredoxin (Fd)-NADP⁺ is carried out solely by System II and includes not one but two photoreactions (IIa and IIb) that proceed effectively only in short-wavelength light. (C₅₅₀ is a new chloroplast component identified by spectral evidence and distinct from cytochromes.) The evidence suggests that the two short-wavelength light reactions operate in series, being joined by a System II chain of electron carriers that includes (but is not limited to) C₅₅₀, cytochrome b₅₅₉, and plastocyanin (PC).

H₂O → IIb_hv → C₅₅₀ → cyt. b₅₅₉ → PC → IIa_hv → Fd → NADP⁺

Photoreaction IIb involves an electron transfer from water to C₅₅₀ that does not require plastocyanin and is the first known System II photoreaction resistant to inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and o-phenanthroline. Cytochrome b₅₅₉ is reduced by C₅₅₀ in a reaction that is readily inhibited by DCMU or o-phenanthroline. Thus, the site of DCMU (and o-phenanthroline) inhibition of System II appears to lie between C₅₅₀ and cytochrome b₅₅₉. Photoreaction IIa involves an electron transfer from cytochrome b₅₅₉ and plastocyanin to ferredoxin-NADP⁺.     

Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (SQDG)-deficient mutant (hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q(A) to Q(B). This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas.