SummaryWild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5Ј and 3Ј portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5Ј end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, PhetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

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The HetR protein has long been recognized as a key player in the regulation of heterocyst development. HetR is known to possess autoproteolytic and DNA-binding activities. During a search for mutants of Anabaena sp. PCC 7120 that can overcome heterocyst suppression caused by overexpression of the patS gene, which encodes a negative regulator of differentiation, a bypass mutant strain, S2-45, was isolated that produced a defective pattern (Pat phenotype) of irregularly spaced single and multiple contiguous heterocysts (Mch phenotype) in combined nitrogen-free medium. Analysis of the S2-45 mutant revealed a R223W mutation in HetR, and reconstruction in the wild-type background showed that this mutation was responsible for the Mch phenotype and resistance not only to overexpressed patS, but also to overexpressed hetN, another negative regulator of differentiation. Ectopic overexpression of the hetRR223W allele in the hetRR223W background resulted in a conditionally lethal (complete differentiation) phenotype. Analysis of the heterocyst pattern in the hetRR223W mutant revealed that heterocysts differentiate essentially randomly along filaments, indicating that this mutation results in an active protein that is insensitive to the major signals governing heterocyst pattern formation. These data provide genetic evidence that, apart from being an essential activator of differentiation, HetR plays a central role in the signaling pathway that controls the heterocyst pattern.patS ͉ pattern formation ͉ prokaryotic development ͉ signal pathway

Transposon-generated mutant C3 of Anabaena sp. strain PCC 7120 is unable to form heterocysts upon deprivation of combined nitrogen but forms a pattern of spaced, weakly fluorescent cells after 2 days of deprivation. Sequence analysis of chromosomal DNA adjacent to the ends of transposon Tn5-1058 in mutant C3 showed a 1,044-amino-acid open reading frame, designated hetC, whose predicted protein product throughout its C-terminal two-thirds has extensive similarity to the HlyB family of bacterial protein exporters. Its N-terminal third is unique and does not resemble any known protein. hetC lies 1,165 bp 5 from the previously described gene hetP. Reconstruction of the C3 mutation and its complementation in trans with a wild-type copy of hetC confirmed that hetC has an essential regulatory role early in heterocyst development. hetC is induced ca. 4 h after nitrogen stepdown, hours after induction of hetR. Expression of hetC depends on HetR and may depend on HetC. Highly similar sequences are present 5 from the initiation codons and in the 3 untranslated regions of hetC and of two heterocyst-specific genes, devA and hetP.Upon deprivation of a source of combined nitrogen, certain filamentous cyanobacteria initiate a sequence of morphological and physiological changes that result in the differentiation of specialized cells called heterocysts. These cells, which form at semiregular intervals along the filaments, provide a microaerobic environment and a supply of ATP and reductant for nitrogenase. During the course of differentiation of a vegetative cell into a heterocyst, a thick envelope is synthesized. This envelope consists of an external, polysaccharide layer and an internal, laminated layer of glycolipids that functions to limit gas permeation. To provide a route, unblocked by the glycolipid layer of the envelope, for nutrients to move between the heterocyst and an adjacent vegetative cell, a channel, or pore, is formed that contains a neck region of reduced diameter, at the end of which the plasma membranes of the heterocyst and vegetative cell are narrowly separated. Structures called microplasmodesmata appear to join the two plasma membranes. The inner, thylakoid membranes are rearranged within heterocysts, with formation of a honeycomb of membranes, rich in oxidative enzymes, near the poles of the developing heterocyst. This membrane system may function in the scavenging of oxygen that enters the heterocyst through the pore. Within the developing heterocyst, oxygen production is blocked by the inactivation of components of photosystem II; specific proteases are activated; and RuBP carboxylase, the principal CO 2 -fixing enzyme, disappears early during differentiation. Late in differentiation, several genomic rearrangements take place and nitrogenase proteins are abundantly synthesized (for recent reviews, see references 9, 51, and 53). How these processes are regulated remains unclear.We describe the transposon tagging, cloning, and analysis of a gene that appears to be involved specifically in the early regula...