Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152.

A strain of the filamentous cyanobacterium Nostoc sp. isolated from a lake in Finland was found to produce at least nine hepatotoxic peptides with chemical and toxicological properties similar to those of the hepatotoxic hepta- and pentapeptides produced by other cyanobacteria. Toxins were isolated and purified by high-performance liquid chromatography. Amounts available for five of the purified toxins (P6, P14, P15, P16, and P18) were adequate for high-performance liquid chromatography amino acid analysis and determination of molecular weight by fast-atom bombardment-mass spectrometry (FAB-MS). Quantities of three toxins (P14, P15, and P16) were adequate for further analysis by high-resolution FAB-MS, FAB-MS/MS, and 1H-nuclear magnetic resonance. Analysis showed that the toxins are new types of microcystin-LR homologs. Microcystin-LR contains equimolar amounts of D-alanine, L-leucine, D-erythro-beta-methylaspartic acid, L-arginine, ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), D-glutamic acid, and N-methyldehydroalanine (molecular weight [MW], 994). Nostoc sp. strain 152 was found to produce the following microcystin-LR homologs: (i) P6 contains an extra methylene group most probably due to the presence of N-methyldehydrobutyrine instead of N-methyldehydroalanine (MW, 1,008); (ii) P14 is O-acetyl-O-demethyl ADDA-microcystin-LR (MW, 1,022); (iii) P15 is 3-demethyl-O-acetylADDA-homoarginine-microcystin-LR (MW, 1,036); (iv) P16 is 3-demethyl-O-acetyl-O-demethylADDA-microcystin-LR (MW, 1,008); (v) P18 is 3-demethyl-O-acetyl-O-demethylADDA-homoarginine-microcystin- LR (MW, 1,022). The toxicities of the new microcystin homologs were not significantly different from those of microcystin-LR or demethylmicrocystin-LR.     

Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.     

D-Leu1]Microcystin-LR, from the cyanobacterium Microcystis RST 9501 and from a Microcystis bloom in the Patos Lagoon estuary, Brazil

[D-Leu1]Microcystin-LR was identified as the most abundant microcystin from a laboratory strain of the cyanobacterium Microcystis sp. isolated from a hepatotoxic Microcystis bloom from brackish waters in the Patos Lagoon estuary, southern Brazil. Toxicity of [D-Leu1]microcystin-LR, according to bioassay and protein phosphatase inhibition assay, was similar to that of the commonly-occurring microcystin-LR, which was not detectable in the Patos Lagoon laboratory isolate. This is the first report of a microcystin containing [D-Leu1] in the cyclic heptapeptide structure of these potent cyanobacterial toxins.     

Differentiation of free-living Anabaena and Nostoc cyanobacteria on the basis of fatty acid composition.

The cellular fatty acids of free-living, nitrogen-fixing cyanobacteria belonging to the genera Anabaena and Nostoc were analyzed to differentiate the genera. The fatty acid compositions of 10 Anabaena strains and 10 Nostoc strains that were grown for 12 days on BG-11o medium were determined by gas-liquid chromatography-mass spectroscopy. Of the 53 fatty acids detected, 17 were major components; the average level for each of these 17 fatty acids was at least 0.9% of the total fatty acids (in at least one of the genera). These fatty acids included (with mean percentages in the Anabaena and Nostoc strains, respectively) the saturated fatty acids 16:0 (30.55 and 23.23%) and 18:0 (0.77 and 1.27%); several unsaturated fatty acids, including 14:1 cis-7 (2.50 and 0.11%), 14:1 cis-9 (3.10 and 3.41%), a polyunsaturated 16-carbon (sites undetermined) fatty acid with an equivalent chain length of 15.30 (1.20 and 1.03%), 16:4 cis-4 (0.95 and 0.87%), 16:3 cis-6 (2.16 and 1.51%), 16:1 cis-7 (1.44 and 0.36%), 16:1 cis-9 (6.53 and 18.76%), 16:1 trans-9 (4.02 and 1.35%), 16:1 cis-11 (1.62 and 0.42%), 18:2 cis-9 (10.16 and 12.44%), 18:3 cis-9 (18.19 and 17.25%), 18:1 cis-9 (4.01 and 5.10%), and 18:1 trans-9 (0.92 and 1.94%); and the branched-chain fatty acids iso-16:0 (2.50 and 1.14%) and iso-15:1 (0.34 and 2.05%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Colorimetric immuno-protein phosphatase inhibition assay for specific detection of microcystins and nodularins of cyanobacteria

A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R2 = 0.94, P < 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.     

Secondary metabolites from a Streptomyces strain isolated from Livingston Island, Antarctica

The producing strain Streptomyces sp. 1010 was isolated from a shallow sea sediment from the region of Livingston Island, Antarctica. From the culture broth of this strain naturally active secondary metabolites were isolated identical to phthalic acid diethyl ester (C12H14O4, MW. 222); 1, 3-bis (3-phenoxyphenoxy)benzene (C30H22O4, MW.446); hexanedioic acid dioctyl ester (C22H42O4, MW.370) and the new substance 2-amino- 9, 13 -dimethyl heptadecanoic acid (C19H39NO2, MW.313). These compounds represent diverse classes of chemical structures and provide evidence for the untapped biosynthetic potential of marine bacteria from Antarctica.     

Molecular characterization of hot spring cyanobacteria and evaluation of their photoprotective compounds

Phylogenetic analysis of 4 cyanobacterial strains isolated from hot springs in Rajgir, India, was carried out using the 16S rRNA gene (1400 bp). These strains were identified as members of Chroococcales ( Cyanothece sp. strain HKAR-1) and Nostocales ( Nostoc sp. strain HKAR-2, Scytonema sp. strain HKAR-3, and Rivularia sp. strain HKAR-4). Furthermore, we evaluated the presence of ultraviolet-screening and (or) photoprotective compounds, such as mycosporine-like amino acids (MAAs) and scytonemin, in these cyanobacteria by using high-performance liquid chromatography. Well-characterized MAAs, including the critical and highly polar compounds shinorine, porphyra-334, and mycosporine-glycine, as well as several unknown MAAs, were found in these hot-spring-inhabiting microorganisms. The presence of scytonemin was detected only in Scytonema sp. strain HKAR-3 and Rivularia sp. strain HKAR-4. The results indicate that hot spring cyanobacteria, namely Cyanothece, Nostoc, Scytonema, and Rivularia, belonging to different groups possess various photoprotective compounds to cope up with the negative impacts of damaging radiations.     

Inhibition of several protein phosphatases by a non-covalently interacting microcystin and a novel cyanobacterial peptide, nostocyclin

Microcystins produced by cyanobacterial 'blooms' in reservoirs and lakes pose significant public health problems because they are highly toxic due to potent inhibition of protein serine/threonine phosphatases in the PPP family. A dehydrobutyrine (Dhb)-containing microcystin variant [Asp3, ADMAdda5, Dhb7]microcystin-HtyR isolated from Nostoc sp. was found to potently inhibit PP1, PP2A, PPP4 and PPP5 with IC50 values similar to those of microcystin-LR. However, in contrast to microcystin-LR, which forms a covalent bond with a cysteine residue in these protein phosphatases, Asp,ADMAdda,Dhb-microcystin-HtyR did not form any covalent interaction with PP2A. Since the LD50 for Asp,ADMAdda,Dhb-microcystin-HtyR was 100 microg kg(-1) compared to 50 microg kg(-1) for microcystin-LR, the data indicate that the non-covalent inhibition of protein phosphatases accounts for most of the harmful effects of microcystins in vivo. A 3-amino-6-hydroxy-2-piperidone containing cyclic peptide, nostocyclin, also isolated from Nostoc sp., was non-toxic and exhibited more than 500-fold less inhibitory potency towards PP1, PP2A, PPP4 and PPP5, consistent with the conclusion that potent inhibition of one or more these protein phosphatases underlies the toxicity of microcystins, both lacking and containing Dhb.     

First report of microcystins from a Brazilian isolate of the cyanobacteriumMicrocystis aeruginosa

This is the first report on microcystins, cyclic heptapeptide hepatotoxins, from Brazilian water supplies. A colony isolate (NPJB-1) of the colonial cyanobacteriumMicrocystis aeruginosa from Lagoa das Garças, São Paulo, was cultured under non-axenic conditions. Exponential phase cells were harvested, concentrated and lyophilized for mouse bioassays and toxin extraction. The LD₁₀₀ of lyophilized cell suspensions was approximately 31 mg kg^–1 (dry cell weight/animal weight). Isolation, purification and characterization of the toxins were carried out by reversed phase HPLC, HPLC amino acid analysis and fast atom bombardment mass spectrometry. Strain NPJB-1 produces two different hepatotoxic heptapeptide microcystins. The main one was microcystin-LR, the most commonly reported microcystin from cyanobacteria. The other was microcystin-LF, the phenylalanine variant of microcystin-LR. This is the first published report for microcystin-LF.     

Biotransformation and volatilization of arsenic by three photosynthetic cyanobacteria

Arsenic (As) is a pervasive and ubiquitous environmental toxin that has created worldwide human health problems. However, there are few studies about how organisms detoxify As. Cyanobacteria are capable of both photolithotrophic growth in the light and heterotrophic growth in the dark and are ubiquitous in soils, aquatic systems, and wetlands. In this study, we investigated As biotransformation in three cyanobacterial species (Microcystis sp. PCC7806, Nostoc sp. PCC7120, and Synechocystis sp. PCC6803). Each accumulated large amounts of As, up to 0.39 g kg(-1) dry weight, 0.45 g kg(-1) dry weight, and 0.38 g kg(-1) dry weight when treated with 100 μM sodium arsenite for 14 d, respectively. Inorganic arsenate and arsenite were the predominant species, with arsenate making up >80% of total As; methylated arsenicals were detected following exposure to higher As concentrations. When treated with arsenate for 6 weeks, cells of each cyanobacterium produced volatile arsenicals. The genes encoding the As(III) S-adenosylmethionine methyltransferase (ArsM) were cloned from these three cyanobacteria. When expressed in an As-hypersensitive strain of Escherichia coli, each conferred resistance to arsenite. Two of the ArsM homologs (SsArsM from Synechocystis sp. PCC6803 and NsArsM from Nostoc sp. PCC7120) were purified and were shown to methylate arsenite in vitro with trimethylarsine as the end product. Given that ArsM homologs are widespread in cyanobacteria, we propose that they play an important role in As biogeochemistry.     

Carbohydrate, glycolipid, and lipid components from the photobiont (Scytonema sp.) of the lichen, Dictyomema glabratum

The photobiont of the lichen, Dictyonema glabratum (Scytonema sp.), was isolated and cultivated in a soil-extract medium and submitted to chemical analysis. Successive extractions with CHCl3-MeOH, aqueous MeOH, and H2O gave rise to solutions of lipids (25%), low-molecular-weight carbohydrates (22%), and polysaccharides (4%), respectively. TLC of the lipid extract showed the presence of glycolipids, which were further purified and examined by NMR spectroscopy and GC-MS. Monogalactosyldiacylglycerol (1%), digalactosyldiacylglycerol (0.8%), trigalactosyldiacylglycerol (0.4%), and sulfoquinovosyldiacylglycerol (0.5%) were identified. The most abundant fatty acid ester in each fraction was palmitic (C16:0), but a great variation of the ester composition from one to another was found. Others present were those of C12:0, C14:0, C15:0, C16:1, C17:0, C18:0, C18:1, C18:2, C18:3, C22:0, C22:2, and C24:0. The lipid extract was also subjected to acid methanolysis, which gave rise to dodecane, 2-Me-heptadecane, 2,6-Me2-octadecane, and 8-Me-octadecane, methyl esters of C14:0, C15:0, C16:0, C16:1, C17:0, C18:0, C18:1, C18:2, C20:0, and C24:0 fatty acids, and the dimethyl ester of decanedioic acid. The polysaccharide had mainly Glc, Gal, and Man, with small amounts of 3-O-methylrhamnose and 2-O-methylxylose, both found in plants, and unexpectedly, some of the units were beta-galactofuranose, typical of fungal, but not cyanobacterial polysaccharides. The low-molecular-weight carbohydrates showed mannose as the main free reducing sugar, which differs from Nostoc sp. and Trebouxia sp. photobionts.     

Structure of cyanoviridin RR,a toxin from the blue-green alga,Microcystis viridis

Mass culture of an axenic clone ofMicrocystis viridis (NIES-102) was carried out, and three toxins were isolated from the cell. Structure elucidation of one of the toxins, designated cyanoviridin RR (microcystin RR), was performed mainly by means of modern NMR techniques. Cyanoviridin RR was a cyclic heptapeptide consisting of seven amino acids, Adda, l-arginine, erthro-β-methyl-aspartic acid, l-arginine, d-alanine, N-methyldehydroalanine, and d-glutamic acid. Structurally, this toxin belongs to the cyanoginosins already isolated fromM. aeruginosa.     

Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids.

The activities of uptake of thirteen 14C-labeled amino acids were determined in nine cyanobacteria, including the unicellular strains Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803; the filamentous strain Pseudanabaena sp. strain PCC 6903, and the filamentous, heterocyst-forming strains Anabaena sp. strains PCC 7120 and PCC 7937; Nostoc sp. strains PCC 7413 and PCC 7107; Calothrix sp. strain PCC 7601 (which is a mutant unable to develop heterocysts); and Fischerella muscicola UTEX 1829. Amino acid transport mutants, selected as mutants resistant to some amino acid analogs, were isolated from the Anabaena, Nostoc, Calothrix, and Pseudanabaena strains. All of the tested cyanobacteria bear at least a neutral amino acid transport system, and some strains also bear transport systems specific for basic or acidic amino acids. Two genes, natA and natB, encoding elements (conserved component, NatA, and periplasmic binding protein, NatB) of an ABC-type permease for neutral amino acids were identified by insertional mutagenesis of strain PCC 6803 open reading frames from the recently published genomic DNA sequence of this cyanobacterium. DNA sequences homologous to natA and natB from strain PCC 6803 were detected by hybridization in eight cyanobacterial strains tested. Mutants unable to transport neutral amino acids, including natA and natB insertional mutants, accumulated in the extracellular medium a set of amino acids that always included Ala, Val, Phe, Ile, and Leu. A general role for a cyanobacterial neutral amino acid permease in recapture of hydrophobic amino acids leaked from the cells is suggested.     

Colorimetric Immuno-Protein Phosphatase Inhibition Assay for Specific Detection of Microcystins and Nodularins of Cyanobacteria

A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp³-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R² = 0.94, P < 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.     

营养型葛仙米果冻的制作工艺研究

研究营养型葛仙米果冻的制作工艺,为武陵山区葛仙米特色资源的开发提供新途径。将烫漂护色后的葛仙米打浆制得葛仙米汁,以葛仙米汁为主要原料,卡拉胶、魔芋胶为胶体基材,进行营养型葛仙米果冻生产工艺的研究。利用单因素试验和正交试验,通过感官评价和质构指标的综合分析,得到葛仙米果冻的最佳配方为:复合胶含量1%(m卡拉胶∶m魔芋胶=6∶4),柠檬酸含量0.3%,葛仙米汁含量30%,白糖含量14%和β-环糊精含量2%~4%。在此条件下制作的葛仙米果冻口感爽滑,绿色天然、酸甜适口、质地均匀,具有一定的保健功能。     

Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water

A na?ve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 microg liter(-1)) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 micro g of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.     

Age-dependent effects on biochemical variables and toxicity induced by cyclic peptide toxin microcystin-LR in mice

Microcystins are naturally occurring hepatotoxins produced by certain strains of Microcystis aeruginosa and microcystin-LR is the most toxic among the 60 microcystin variants isolated so far. These toxins have been implicated in both human and livestock mortality. In the present study we evaluated the age-dependent hepatotoxic effects of microcystin-LR (MC-LR) in mice after intraperitoneal and oral route of exposure. For acute toxicity studies by intraperitoneal route, 1 LD(50) dose of MC-LR (43.0 microg/kg) was administered to 6- to 36-week-old mice. Results showed that time to death in toxin treated animals decreased with age of mice. In comparison to control mice, treated animals of all age groups showed significant increases in liver body mass index and increases in serum enzymes (lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, sorbitol dehydrogenase). For acute oral toxicity studies, 1 LD(50) of microcystin-LR containing extracts (3.5 g of MCE/kg) was administered to 6- and 36-week-old mice. The effects on biochemical variables were similar to intraperitoneal route of exposure. Significant age-dependent effects that were observed in microcystin treated animals by intraperitoneal and oral routes of exposure include: time to death, hepatic lipid peroxidation, glutathione depletion and DNA fragmentation. The age-dependent effects observed in some of the biochemical variables may be due to difference in the amount of microcystin-LR up take and also the age-dependent ability to detoxify the toxin in mice.     

Polyadenylated RNA in two filamentous cyanobacteria.

Polyadenylated RNA was detected in the cyanobacteria Nostoc sp. strain MAC and Anabaena variabilis by oligodeoxythymidylic acid-cellulose chromatography and by hybridization to [3H]polyuridylic acid. Polyadenylate tracts from A. variabilis were located at the 3' end of RNA chains and had an estimated length of 15 to 22 nucleotides.     

Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR

Immunoassays are increasingly used to investigate the production, properties and fates of the cyanobacterial hepatotoxic microcystins in vitro and in vivo. Responses of an ELISA immunoassay to microcystins have been determined using the authentic toxin antigen, microcystin-LR, and conjugation products between the toxin and glutathione, cysteine-glycine and cysteine. The antibodies against microcystin-LR crossreacted with the toxin conjugation products with similar affinities (96-112%) to that of microcystin-LR, when assayed at a concentration of 1 microg l(-1). Toxicity assessment of the conjugates, in comparison to microcystin-LR, indicated a reduction according to mouse bioassay. In vitro protein phosphatase inhibition assay indicated that the conjugates possessed approximately 3-9-fold lower toxicity than microcystin-LR.     

Extended heme promiscuity in the cyanobacterial cytochrome c oxidase: characterization of native complexes containing hemes A, O, and D, respectively.

The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the S- and N-sources, respectively. In addition, Anabaena and Nostoc were grown under dinitrogen-fixing conditions in a medium free of combined nitrogen. Membranes were isolated from late-logarithmic cells (culture density corresponding to approximately 3 microliters packed cells per milliliter); cytoplasmic and thylakoid membranes were separated and purified according to established procedures. Acid-labile hemes were extracted from the membranes and subjected to reversed-phase high-performance liquid chromatography. Separated hemes were analyzed spectroscopically and identified by comparison with authentic standards. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally discernible heme D in the membranes and extracts therefrom. However, spectroscopic and kinetic investigation of the membrane-bound heme D rather disproved any reaction with oxygen or carbon monoxide. Kinetic measurements performed with the membrane-bound respiratory oxidase gave evidence for only two kinetically competent terminal oxidases, a3 and o3, both apparently associated with a single type of apoprotein, viz. subunit I of the known cyanobacterial aa3-type cytochrome c oxidase. The heme D, on the other hand, seems to form a spectrally distinguished, yet kinetically ill-defined hemoprotein complex which does not qualify as a fully functional d-type terminal oxidase on our (wild-type) cyanobacteria even after growth under semianaerobic pseudo-reducing conditions. Also growth (of Anabaena and Nostoc) under dinitrogen-fixing conditions did not change this situation. Thus, we are left with (wild-type) cyanobacteria forming an unbranched respiratory chain with only a single type of terminal oxidase protein, viz. the known aa3-type cytochrome c oxidase. This oxidase, however, may incorporate different prosthetic (heme) groups in the sense of "heme promiscuity." Biosynthesis of the different heme groups thereby seems to respond to the ambient redox environment. In particular, however, conditions for expression of the two quinol oxidases potentially and additionally coded for by the genome of, e. g., Synechocystis sp. PCC6803 (see http://www.kazusa.or.jp/cyano), have not yet been found.