In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N 2 . Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA in Anabaena sp. strain PCC 7120. In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomal hepA::luxAB fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation. In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resembles N-acetylmuramoyl-L-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction of hepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA. Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.

The DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of cyanobacteria contains a unique core component, -y, which is absent from the RNA polymerases of other eubacteria (G. J. Schneider, N. E. Tumer, C. Richaud, G. Borbely, and R. Haselkorn, J. Biol. Chem. 262:14633-14639, 1987). We present the complete nucleotide sequence of rpoCl, the gene encoding the -y subunit, from the heterocystous cyanobacterium Nostoc commune UTEX 584. The derived amino acid sequence of -y (621 residues) corresponds with the amino-terminal portion of the ,' polypeptide of Escherichia coli RNA polymerase. A second gene in N. commune UTEX 584, rpoC2, encodes a protein which shows correspondence with the carboxy-terminal portion of the E. coli f1' subunit. The rpoBCIC2 genes of N. commune UTEX 584 are present in single copies and are arranged in the order rpoBCIC2, and the coding regions are separated by short AT-rich spacer regions which have the potential to form very stable secondary structures. Our data indicate the occurrence of divergent evolution of structure in the eubacterial DNA-dependent RNA polymerase.The transcription of genes is directed through the activity of DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). In eubacteria, a single form of the core RNA polymerase, together with ancilliary sigma factors, is responsible for the synthesis of virtually all cellular RNAs (5). The RNA polymerase of Escherichia coli consists of at least four different subunits, 3, P', a, and ur, and is present in two main enzyme forms, core (W'%a2) and holoenzyme (core plus cr; 4). The two genes encoding the P (rpoB) and 3' (rpoC) subunits of this RNA polymerase are adjacent to one another and are cotranscribed from the major promoter PL10 (5). The basic (P3'a2) design has been found in the RNA polymerases purified from representatives of gram-positive and gramnegative eubacteria (17, 38). Recently, however, an additional core component, y, has been described for the RNA polymerase (Py,Y'a2ofC of the cyanobacterium Anabaena sp. strain PCC 7120 (32). The -y subunit is serologically unrelated to the other subunits of the cyanobacterial RNA polymerase, but anti-y serum cross-reacts with both E. coli pI' subunit protein and subunit A of the RNA polymerase from Sulfolobus acidocaldarius, an archaebacterium (31). The -y subunit has since been detected in the RNA polymerases of 15 out of 15 taxonomically diverse cyanobacteria, including two Nostoc species (31).Three different nuclear RNA polymerases are found in eucaryotes, each one responsible for the transcription of a different class of genes (17). Comparison of the amino acid sequences of the largest subunit, A, of RNA polymerases II and III from Saccharomyces cerevisiae and the P' subunit of the E. coli RNA polymerase revealed six regions (I to VI) of marked conservation (1).The RNA polymerases of archaebacteria appear to be more closely related to those of eucaryotes (6,38 whether archaebacteria, like...

Salt-induced genes in the cyanobacterium Anabaena sp. strain PCC 7120 were identified by use of a Tn5-based transposon bearing luxAB as a reporter. The genomic sequence adjacent to one site of insertion of the transposon was identical in part to the sequence of thelti2 gene, which was previously identified in a differential screen for cold-induced transcripts in Anabaena variabilis. The lti2-like gene was induced by sucrose and other osmotica and by low temperature, in addition to salt. Regulatory components necessary for the induction of this gene by osmotica were sought by a further round of transposon mutagenesis. One mutant that displayed reduced transcriptional activity of thelti2-like gene in response to exposure to osmotica had an insertion in an open reading frame, which was denoted orrA, whose predicted product showed sequence similarity to response regulators from two-component regulatory systems. The corresponding mutation was reconstructed and was shown, like the second-site transposon mutation, to result in reduced response to osmotic stress. Induction of the lux reporter gene by osmotica was restored by complementation with a genomic fragment containing the entire open reading frame for the presumptive response regulator, whereas a fragment containing a truncated copy of the open reading frame for the response regulator did not complement the mutation.

The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp. PCC 7942. A new high-CO2-requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5'-flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco). The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli. The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO2 for Rubisco. Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr O221, Rubisco was markedly associated with the carboxysomes. Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.

Element analysis using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) was performed in a symbiotic Nostoc sp. strain found in the upper stem tissue of Gunnera manicata, and in Nostoc PCC 9229, a free‐living heterocyst‐forming cyanobacterium able to enter into symbiosis with the angiosperm Gunnera in reconstitution experiments. ESI and EELS unequivocally identified the four elements nitrogen (N), sulphur (S), phosphorus (P) and oxygen (O) in different inclusion bodies of these biological specimens. High amounts of nitrogen were solely detected in huge cyanophycin granules in vegetative cells of the symbiotic Nostoc strain, whereas large polyphosphate bodies, containing high amounts of phosphorus, sulphur and oxygen, could be seen in the free‐living Nostoc PCC 9229. The latter were usually not present or, when found, very small in vegetative cells of the cyanobiont.