Anabaena 7120 mutant 216 fails to differentiate heterocysts. We previously identified a 2.4-kb wild-type DNA fragment able to complement this mutant. We show here that the sequence of this fragment contains a single open reading frame [hetR), encoding a 299-amino-acid protein. Conjugation of deletion subclones of this fragment into strain 216 showed that the /ieti?-coding region is both necessary and sufficient for complementation of the Het~ phenotype. The mutation in 216 is located at nucleotide 535 in the betR gene, converting a serine at position 179 in the wild-type protein to an asparagine in the mutant. Interruption of the hetR gene in wild-type cells results in a mutant phenotype identical to that of 216. Both 216 and wild-type cells containing wild-type hetR on a plasmid display increased frequency of heterocysts, even on media containing fixed nitrogen. These results suggest that hetR encodes a product that is not only essential for but also controls heterocyst development. This putative regulatory protein lacks known structural motifs characteristic of transcription factors and probably acts at a level one or more steps removed from its target genes.

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Heterocyst differentiation in the filamentous cyanobacteriumAnabaena PCC 7120 requires a functional hetR gene. Increased expression of the hetR gene is seen in developing and mature heterocysts in response to fixed nitrogen limitation. We mapped four likely transcriptional start sites for hetR and identified a specific transcript that is positively autoregulated. By using the copper-responsive petE promoter from Anabaena PCC 7120 to drive hetR expression, we show that ectopic expression of hetR increases heterocyst frequency and induces heterocyst differentiation under fully repressing conditions. Coexpression of a reporter gene shows that expression from the petE promoter is smoothly induced depending on the amount of copper supplied. In the heterocyst pattern mutant PatA, where terminally positioned heterocysts are formed almost exclusively, expression of the petE::hetR fusion does not result in the formation of intercalary heterocysts. These results suggest that although the intracellular concentration of HetR has to be elevated for the differentiation decision, PatA plays a role as well. This role may be in the form of posttranslational modification of HetR, because PatA is a member of the response regulator family of proteins.

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper‐inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild‐type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild‐type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild‐type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.

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