RESULTS:

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.

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.

SUMMARY: Following transfer to medium lacking a nitrogen source cells of Synechocystis PCC 6803 continued to divide giving a doubling of cell number after 40 h. Phycocyanin degradation commenced immediately after the transfer with a rapid phase lasting 5 h in which 50% of the phycocyanin disappeared and a slow second phase in which the phycocyanin content decreased to 10% of its initial value by 24 h. In the presence of glucose a utilizable carbon source for this facultatively heterotrophic cyanobacterium phycocyanin was degraded initially at a rate 60% of that observed in the absence of the sugar and proteolysis was almost completely inhibited after 6 h when only 27% of the phycocyanin had been lost; cell division ceased at this time. Photosynthetic O2 evolution decreased rapidly in the presence of glucose and the cells consumed O2 in the light after 7 h indicative of a switch to oxidative metabolism; net O2 uptake in the absence of glucose occurred only after approximately 25 h. Inhibition of phycocyanin degradation by glucose required metabolism of the sugar probably via the oxidative pentose phosphate cycle and appeared to result from irreversible inactivation of the protease.

Synchronized akinete differentiation occurred following the transition from exponential to non-exponential (linear) growth the major trigger being energy limitation. Young akinetes first accumulated cyanophycin then developed a multilayered extracellular envelope and a thickened wall. The dry weight chlorophyll a glycogen and carbon contents of mature akinetes were greater than those of vegetative cells while their contents of DNA RNA protein phycocyanin and nitrogen were similar to those of vegetative cells. Akinetes were resistant to desiccation and low temperatures but not to temperatures above the maximum for vegetative cell growth.

In N2-grown cultures heterocyst differentiation ceased at the end of exponential growth while cell division continued and akinetes first appeared in a regular pattern at a fixed distance (9 cells) from the nearest heterocyst. Exogenous NH4 + inhibited the differentiation of heterocysts and in their absence akinetes developed in irregular positions. The regular spatial pattern imposed on akinete differentiation by heterocysts was like the heterocyst spatial pattern itself independent of N2 fixation. Similar changes in both patterns induced by 7-azatryptophan suggested that they share a common mechanism of control.

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.