RESULTS:

Two neurotoxic alkaloids anatoxin-a and its homologue homoanatoxin-a were purified from the filamentous cyanobacteria Oscillatoria sp. strain 193 (PCC 9240) and Oscillatoria formosa NIVA CYA-92 (PCC 10111) respectively and characterized by mass spectrometry. Biological activity was determined by examining the capacity of the toxins to competitively inhibit the binding of radiolabelled bungarotoxin to acetylcholine receptors using post-synaptic membrane fractions of Torpedo electric tissue. Inhibition was concentration dependent with a K i of 5·4±1·1×10−8 M for anatoxin-a and 7·4±0·9×10−8 M for homoanatoxin-a. Their high affinities for the nicotinic cholinergic receptors were exploited to adapt the radioligand-binding assay for routine detection of this class of neurotoxins directly in low-molecular-mass cell extracts of cyanobacteria. Confirmation of the results and toxin identification were achieved by coupled gas chromatography-mass spectrometry (GC/MS). Seventy-six axenic strains representative of 13 genera were analysed. Five strains of the genus Oscillatoria hitherto unknown for their toxicity inhibited bungarotoxin binding. GC/MS revealed that Oscillatoria sp. strains PCC 6407 PCC 6412 and PCC 9107 synthesized exclusively anatoxin-a whereas both anatoxin-a and homoanatoxin-a were produced by strain PCC 9029. Oscillatoria sp. strain PCC 6506 an isolate co-identic with strain PCC 9029 also produced both neurotoxins but their respective presence depended upon growth conditions. The latter results suggest that regulatory differences in at least some of the cyanobacterial strains may account for the preferential synthesis of only one of the two neurotoxins or for their simultaneous occurrence.

The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The rrnS sequences were determined for five strains including representatives of Anabaena flos-aquae Aphanizomenon flos-aquae Nodularia sp. and two alkaliphilic planktonic members of the genera Anabaenopsis and Cyanospira whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that with the exception of members assigned to the genus Cylindrospermopsis all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings of Anabaena and Aphanizomenon strains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions and the high degree of sequence similarity between strains of Anabaenopsis and Cyanospira suggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiple rrn operons and ITS regions of variable size. Finally evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.

An in situ hybridization method was applied to the identification of marine cyanobacteria assignable to the genus Prochlorococcus using horseradish-peroxidase-labelled 16S rRNA-targeted oligonucleotide probes in combination with tyramide signal amplification (TSA). With this method very bright signals were obtained in contrast to hybridizations with oligonucleotides monolabelled with fluorochromes which failed to give positive signals. Genotype-specific oligonucleotides for high light (HL)- and low light (LL)-adapted members of this genus were identified by 16S rRNA sequence analyses and their specificities confirmed in whole-cell hybridizations with cultured strains of Prochlorococcus marinus Chisholm et al. 1992 R8 Prochlorococcus sp. and Synechococcus sp. In situ hybridization of these genotype-specific probes to field samples from stratified water bodies collected in the North Atlantic Ocean and the Red Sea allowed a rapid assessment of the abundance and spatial distribution of HL- and LL-adapted Prochlorococcus. In both oceanic regions the LL-adapted Prochlorococcus populations were localized in deeper water whereas the HL-adapted Prochlorococcus populations were not only distinct in each region but also exhibited strikingly different depth distributions HLI being confined to shallow water in the North Atlantic in contrast to HLII which was present throughout the water column in the Red Sea.

PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium Nostoc PCC 7120 gave three products. Two represented true ITS regions of different sizes while the third was a heteroduplex. The longer spacer (ITS-L) contained 512 nucleotides and carried tRNAIle and tRNAAla genes separated by a large stem–loop structure (V2) composed of short tandemly repeated repetitive sequences. Both tRNA genes and the 5′ half of the intervening stem were absent from the shorter spacer (ITS-S) of length 283 nucleotides which was otherwise almost completely identical to ITS-L. The two spacer regions of Nostoc PCC 7120 were aligned to published ITS sequences of cyanobacteria the cyanelle of Cyanophora paradoxa and Escherichia coli. Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNAIle and tRNAAla genes only the tRNAIle gene or neither there is no correlation between ITS size and coding capacity for tRNAs. Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E. coli. Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified and regions homologous to bacterial antiterminators (box B–box A) were located. The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels.

Protein kinase activities have been detected in cell-free extracts of the cyanobacterium Anabaena PCC 7120. At least 12 polypeptides in the soluble fraction were phosphorylated in vitro at the expense of [γ -32P]ATP and the pattern of phosphorylation was shown to be regulated by intermediary metabolites and other effectors at physiological concentrations. Glucose 6-phosphate exerted a regulatory effect on a phosphopolypeptide of M r 56000 (p56) by stimulating a protein phosphatase whereas ribulose 5-phosphate inhibited the corresponding protein kinase. In addition DTT and the calmodulin antagonist trifluoperazine influenced the phosphorylation state of several different polypeptides indicative of control by redox conditions and a calmodulin-like mediator respectively. Furthermore it was established that the phosphorylation of p56 required Mg2+ (> 100 μM) whereas that of a polypeptide of M r 16000 occurred in the absence of Mg2+ and was inhibited by high concentrations (> 1 mm) of this cation. Several of the phosphopolypeptides detected in vitro corresponded in mobility on SDS-PAGE to species phosphorylated in vivo.

Summary: On the basis of a comparative study of 178 strains of cyanobacteria representative of this group of prokaryotes revised definitions of many genera are proposed. Revisions are designed to permit the generic identification of cultures often difficult through use of the field-based system of phycological classification. The differential characters proposed are both constant and readily determinable in cultured material. The 22 genera recognized are placed in five sections each distinguished by a particular pattern of structure and development. Generic descriptions are accompanied by strain histories brief accounts of strain properties and illustrations; one or more reference strains are proposed for each genus. The collection on which this analysis was based has been deposited in the American Type Culture Collection where strains will be listed under the generic designations proposed here.