Restricted cellular differentiation in cyanobacterial filaments

Enrique Flores1 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, E-41092 Seville, Spain lthough commonly considered A duced by mature heterocysts inhibit the simple organisms that grow as differentiation of nearby cells in a process A single cells, many bacteria can that can be described as lateral inhibition, grow as filaments made of chains which likely involves an interaction with of cells. In cyanobacteria, prokaryotes that HetR (12, 13). The interplay between perform oxygen-evolving photosynthesis, positive and negative regulators appears to filamentous forms developed early in be widely used in the development of fi evolution (1). In the most complex la- B spatial patterns in metazoans (14), and it mentous cyanobacteria, as many as four also plays a key role in the development of cell types can be found: vegetative cells the diazotrophic cyanobacterial filament. that perform oxygenic photosynthesis and Investigating Nostoc punctiforme (Fig. fi concomitant CO2 xation; the cells of A fi fi 1 ), a lamentous cyanobacterium capa- hormogonia, which are small motile la- ble of producing the four different cell ments frequently made of small cells; types described earlier, Risser et al. (5) akinetes, which are resting cells; and het- Fig. 1. (A) Filaments of N. punctiforme containing identify a gene, patN, whose mutation in- erocysts, which are terminally differenti- fi photosynthetic vegetative cells and N2- xing het- creases the frequency of heterocysts. In ated cells specialized in the fixation of erocysts (arrowheads). (Micrograph courtesy of José the patN mutant, vegetative cell intervals atmospheric nitrogen (2, 3). In a cyano- E. Frías, Consejo Superior de Investigaciones Cien- between heterocysts are shorter than in bacterial filament, the heterocysts differ- tificas, Seville, Spain.) (B) Scheme of a N. punctifome filament showing the uneven distribution and bi- the wild type, but the distribution of het- entiate from some vegetative cells in fi response to nitrogen deficiency (2–4). In ased inheritance of PatN [after Risser et al. (5)]. PatN erocysts in the laments is still non- cyanobacteria of the genera Anabaena or location in the cytoplasmic membrane is noted in random. Thus, the PatS- and HetN- orange. Cells in the filament at top are not neces- Nostoc, heterocysts form intercalary along dependent lateral inhibition seems to op- fi sarily synchronous, but cells a and b are sister cells erate in the patN mutant, and inhibition of the lament, producing a nonrandom (one without PatN, the other with PatN) resulting pattern of one heterocyst to approximately differentiation by PatS has indeed been from a recent cell division. The patN gene is con- fl 10 to 15 vegetative cells. However, can any stitutive, and the PatN protein eventually localizes corroborated (5). Analyzing the uores- cell in a filament differentiate into a het- to the half of the cell adjacent to the most recently cence from a PatN-GFP fusion protein, erocyst? In PNAS, Risser et al. (5) present formed intercellular septum. After cell division 1, Risser et al. observe the protein in the evidence consistent with the idea that, at PatN is inherited in only one cell of each pair of periphery of the cell, consistent with a cy- ′ ′ a given time, only some cells in the fila- daughter cells (a from a; b from b), but patN is toplasmic membrane location, but with an ment have the capability to differentiate. expressed and PatN is localized close to the new uneven distribution between cells. Further, septa in cells a′ and a′′, as well as in cells b′ and b′′. Nitrogenase, the enzyme that catalyzes a dynamic pattern of localization was ap- fi After cell division 2, cells with and without PatN are N2 xation producing ammonia, is oxy- again produced. Cells transiently devoid of PatN parent (5). Before cell division, PatN-GFP

gen-labile, but the heterocysts provide (after cell division 2, a′′2,a′1,b′1, and b′′2) are poised localizes to one half of the cell, resulting in a microoxic environment for its function to differentiate if nitrogen starvation is sensed. inheritance of PatN-GFP in only one of (2–4). The process of heterocyst differ- the two daughter cells, but both cells entiation involves the execution of a spe- eventually accumulate PatN-GFP in the fi mote heterocyst differentiation, another ci c program of gene expression that regulator, HetR, is required that appears half of the cell proximal to the most re- includes the induction of regulatory genes, to be also a transcription factor (8). The cently formed intercellular septum. Thus, some of which act early in the process, induction of ntcA and hetR after nitrogen in each round of cell division, some cells and of genes encoding the proteins for the deprivation is mutually dependent pro- result that are transiently devoid of PatN- morphological and biochemical differen- fi GFP (Fig. 1B). It is postulated that PatN tiation of the heterocyst, including nitro- ducing an ampli cation loop of gene expression (9). Thus, after nitrogen dep- impedes heterocyst differentiation and genase (6). When a source of combined that the cells lacking (or having a low nitrogen is exhausted from the culture rivation, the cells (or at least some cells) fi concentration of) PatN are poised to dif- medium, the cells in the filament sense in the lament experience a positive re- ferentiate if nitrogen deficiency is sensed. nitrogen starvation as an increase in the sponse toward differentiation. However, fi Thus, biased inheritance of a cell fate cellular levels of 2-oxoglutarate, the me- because the lament cannot contain too tabolite in which ammonia is incorporated many heterocysts, which do not carry out determinant, likely a universal theme in fi biology (15), would also play a role in the through the glutamine synthetase–gluta- the photosynthetic xation of CO2, nega- mate synthase pathway to produce the tive elements counteracting the positive development of the diazotrophic cyano- fi nitrogen-containing organic material (2). regulators play a role. The patS and hetN bacterial lament. 2-Oxoglutarate is a positive effector of genes encode polypeptides containing a NtcA, a transcription factor that triggers conserved amino acid sequence (RGSGR) the expression of genes encoding proteins that inhibits heterocyst differentiation (10, Author contributions: E.F. wrote the paper. for the assimilation of sources of nitrogen 11). A PatS-derived peptide produced by The author declares no conflict of interest. alternative to ammonia, the preferred ni- some cells early in differentiation and See companion article on page 15342. trogen source (2, 7). To specifically pro- HetN or a HetN-related compound pro- 1E-mail: efl[email protected].

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Two important questions arise. First, differentiation (2, 4). By performing ge- termediary factors to influence gene how is the asymmetric distribution of PatN netic analysis, Risser et al. (5) find that expression. established? Second, how does PatN im- patA is epistatic to patN (i.e., in a patA A two-stage model of heterocyst differ- pede cell differentiation? First, analyzing mutant, the phenotype associated with entiation involving biased initiation fol- a transcriptional fusion of the gfp gene to lowed by competitive resolution has been the patN gene promoter, Risser et al. (5) Risser et al. now identify previously proposed (3). Whereas experi- show that the expression of patN takes mental support for competitive resolution place in all cells of the filament when they a factor putatively of cells simultaneously starting differenti- grow in the presence of ammonium, as well ation was available (10–13), Risser et al. as after nitrogen deprivation. This implies influencing biased (5) now identify a factor putatively influ- that the uneven distribution of PatN in encing biased initiation of heterocyst dif- the filaments results from posttranslational initiation of heterocyst ferentiation. Genes encoding homologues regulation rather than from differential to PatN are present in all heterocyst- gene expression. Therefore, a mechanism differentiation. forming cyanobacteria for which the for the dynamic localization of PatN re- complete genomic sequence is available, sulting in an asymmetric distribution in the indicating that PatN may generally par- cyanobacterial filament is there to be un- a patN mutation is not observed), which is ticipate in heterocyst differentiation. Evo- covered. Second, by using DNA microarray consistent with the hypothesis that PatN lution has worked producing complex analysis, the authors find that PatN affects acts by negatively affecting expression of organisms in the most distant phylogenetic the expression of some genes, including patA, although other targets of PatN may groups, but the evidence that general patA (5). Mutants of this gene form het- also exist (5). PatN bears a predicted principles govern the development of erocysts preferentially at the filaments transmembrane segment (consistent with biological patterns in distant organisms, termini (16), and PatA is postulated to a cytoplasmic membrane location, as from cyanobacteria to metazoans, illus- counteract the negative effects of PatS- noted earlier), but has no obvious DNA trates the unity that underlies diversity and HetN-related regulators on heterocyst binding motifs, which calls for further in- in biology.

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