Heterocyst development in Anabaena
Introduction
Biological nitrogen fixation requires nitrogenase to be protected from oxygen; several mechanisms for this protection have evolved in different nitrogen-fixing organisms 1., 2.. Many filamentous cyanobacteria, which produce oxygen as a byproduct of photosynthesis, exhibit a striking example of prokaryotic cellular differentiation, to form heterocysts — specialized cells that create a microoxic environment for nitrogen fixation (Figure 1). A one-dimensional developmental pattern of single heterocysts separated by approximately ten photosynthetic vegetative cells is established to form a multicellular organism composed of two interdependent cell types. Heterocyst differentiation must require global changes in gene expression 3., 4.. Heterocysts lack components of oxygen-evolving photosystem II, and have a unique structure and physiology 2., 3.; they obtain photosynthate, probably sucrose [5], from nearby vegetative cells and, in return, supply those cells with fixed nitrogen as amino acids. Metabolites and signals must be exchanged between cells along the filament to support growth and regulate the developmental pattern. The mechanism for transport of these molecules is not well understood, but it has been proposed that the continuous periplasm might function as a conduit along the filament. Interestingly, heterocyst pattern and the physiology of the cyanobacterial partner are significantly altered in symbioses with plants [6•].
Several cyanobacterial genomes have been fully or partially sequenced. For heterocystous strains, the Anabaena (Nostoc) PCC 7120 genome sequence is complete and annotated [7•], and a draft sequence of Nostoc punctiforme ATCC 29133 is available. It is expected that Anabaena variabilis ATCC 29413 will be sequenced soon.
The Anabaena PCC 7120 genome is composed of a 6.4 Mb chromosome and 6 plasmids, totaling 0.8 Mb. It contains many genes that are potentially involved in regulation and signaling. 211 genes contain homology to two-component regulatory systems, 66 encode putative Ser/Thr protein kinases and phosphatases, and at least 118 genes presumably encode transcriptional regulatory proteins 7.•, 8., 9.. Many of these regulatory proteins contain complex multiple-domain structures 8., 9., 10.. Other domains that are associated with signaling pathways and regulation are also found here, including 87 GAF domains, encoded by 62 genes and 140 PAS domains, encoded by 59 genes [8].
Heterocyst development and the genetic tools available for filamentous cyanobacteria have been explored recently, in several reviews 4., 6.•, 11., 12.. Meeks and Elhai, in particular, provide an extensive discussion of pattern formation and emphasize the role of biased initiation due to unknown intrinsic factors, which is then followed by regulative resolution of clusters to single heterocysts [6•]. This review will focus on the results obtained during the past few years only.
Section snippets
Regulation of early events
The regulation of heterocyst development involves a response to the external cue of nitrogen deprivation, uncharacterized internal cues related to physiology and possibly the cell cycle, and intercellular communication between cells along filaments 3., 6.•. Although global changes in gene expression must occur during heterocyst development, this regulation is not well understood and there is a conspicuous lack of identified transcription factors 3., 6.•; however, the expression pattern of
PatS influences the initial heterocyst pattern
The patS gene encodes a small peptide inhibitor of heterocyst differentiation and plays an important role in the control of pattern formation 36., 37.. It is thought that PatS works by lateral inhibition, in such a way that PatS produced by a differentiating cell inhibits the differentiation of its neighbors to establish a pattern of single heterocysts along chains of vegetative cells.
A patS–gfp reporter strain revealed pairs and small clusters of patS-expressing cells during the early stage of
Heterocyst-specific expression of patB
PatB contains an N-terminal domain with two putative 4Fe-4S centers and a C-terminal domain containing a DNA-binding motif. The original frameshift mutation in patB, which results in the truncation of the C-terminal domain, showed poor diazotrophic growth and caused filaments to accumulate more heterocysts than normal, resulting in an abnormal Mch pattern [40]. Recent work has shown that a patB deletion mutant is completely defective for diazotrophic growth and that a patB–gfp reporter fusion
Differentiation and maturation
The DevBCA exporter is essential for heterocyst envelope formation and mutants are blocked at an early stage of development, suggesting that completion of the envelope might be a developmental checkpoint [47]. Further support for this type of morphological checkpoint is provided by recent data, which demonstrate that heterocyst maturation requires HcwA, an autolysin, presumably required for restructuring the peptidoglycan layer [48]. It was also found that pbpB, which encodes a putative
Metabolism and nitrogen fixation
The relationship between nitrogen sufficiency and heterocyst development was examined in A. variabilis because of its ability to carry out nitrogen fixation via Nif2 nitrogenase in vegetative cells under anoxic conditions [53]. The authors conclude that heterocyst development may not be controlled by nitrogen insufficiency of individual cells because Nif2 nitrogenase supported the growth of a nif1 mutant but did not prevent heterocyst differentiation. They suggest that filaments might sense
Symbiosis
Heterocystous cyanobacteria form nitrogen-fixing symbiotic associations with a variety of fungi and plants [6•]. One model association is between N. punctiforme and the bryophyte Anthoceros punctatus 6.•, 57.. In symbiosis with plants, heterocyst frequencies increase several-fold and heterocyst differentiation proceeds independently of the nitrogen status of a cell, responding instead to signals that are produced by the plant. Wong and Meeks have shown that these plant signals do not bypass the
Conclusions
The past few years of research have produced an increased understanding of several important aspects of heterocyst development. However, an overall model of the regulatory network controlling development remains elusive. The signals influencing the initiation of development and the control of pattern are still unclear. Many questions are yet to be answered: what diffusible factors, other than PatS, influence pattern and how do these signals move along filaments? What is the relationship between
Update
Recent work provides genome-wide transcription profiling data for heterocyst development in Anabaena PCC 7120 [59••]. In addition to confirming the expression patterns of known genes, many previously uncharacterized genes were found to be upregulated after nitrogen step-down at early, middle, and late stages of development. The authors noted that many upregulated genes were present in ‘expressed islands’ on the chromosome and they suggest the possibility of global regulation involving
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
This work was supported by National Institutes of Health grant GM36890 and Department of Energy grant DE-FG03-98ER020309.
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